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adverse reaction to metal debris ( armd ) in arthroplasty surgery of the hip is a well - known problem and has resulted in several products being withdrawn from the marketplace .
complications related to the release of metal ions can be devastating for patients and represent a significant surgical challenge .
although the bearing surfaces are a causative factor for metal debris to accumulate , corrosion at the head neck or neck
stem junction in a modular prosthesis has been described as a significant problem . in the absence of any reciprocal articulating surface a hemiarthroplasty
however , case reports have surfaced which highlight the potential for such a complication to arise [ 35 ] .
we present the case of a unipolar hemiarthroplasty requiring revision within 18 months of insertion secondary to an adverse reaction to metal debris .
six months previously she had sustained a fragility fracture of her hip whilst on holiday in canada .
she was treated locally with an uncemented hemiarthroplasty ( zimmer press fit ml taper titanium alloy stem , zimmer versys cobalt - chrome femoral head and neck adapter ) .
the patient reported persistent groin pain and a leg length discrepancy since the time of surgery .
she had a normal white cell count , esr and crp . on aspiration a turbid , green ,
an mri scan revealed fluid around the femoral head and neck but no evidence of soft - tissue compromise or pseudotumour formation .
the injection of local anaesthetic afforded temporary relief and confirmed the hip as the source of pain .
subsequently , the decision was made to revise the hemiarthroplasty to a total hip replacement .
intraoperatively a large amount of the pea - soup like fluid was again noted together with some lysis of the proximal femur .
the femoral head was explanted and corrosion was seen at the head neck junction with black staining around the trunnion and on the reciprocal surface of the adapter sleeve .
the femoral stem was found to be well - fixed and after cleaning the trunnion was left in situ .
the area of lysis proximally was debrided and packed with reamings taken from the acetabulum .
biopsies taken intraoperatively confirmed an adverse metal reaction with an alval score of 8/10 ( loss of synovial lining with attached surface fibrin , numerous perivascular lymphohistiocystic aggregates , loss of normal tissue arrangement with the presence of acellular hyalinised zones ) ( fig .
1 ) .
figure 1:intra - operative biopsies confirmed an adverse metal reaction with an alval score of 8/10 ( loss of synovial lining with attached surface fibrin , numerous perivascular lymphohistiocystic aggregates , loss of normal tissue arrangement with the presence of acellular hyalinised zones ) intra - operative biopsies confirmed an adverse metal reaction with an alval score of 8/10 ( loss of synovial lining with attached surface fibrin , numerous perivascular lymphohistiocystic aggregates , loss of normal tissue arrangement with the presence of acellular hyalinised zones ) a year post - operatively our patient is clinically improving and her metal ions are undetectable .
the diagnosis was considered pre - operatively but only confirmed post - operatively with histological analysis .
reported a similar case involving a patient who required revision surgery for armd 3 years after insertion of a hemiarthroplasty with components analogous to ours .
the patient required two revisions and only improved after the cocr head was changed to ceramic .
this led the authors to conclude that the trunnion mismatch between the adapter sleeve and the titanium stem caused both the initial and the on - going metal sensitivity .
described two cases of patients experiencing severe trunnion corrosion and armd more than 10 years after insertion of a hemiarthroplasty . in one case
the corrosion of the trunnion was so severe that the head disengaged from the stem .
a further case of armd complicating hemiarthroplasty has been reported in the literature . in this case
, high levels of titanium metal debris in specimens taken from the femoral - bone stem interface led the authors to conclude that armd resulted from metal release from the femoral stem itself .
the prosthesis concerned consists of a titanium alloy stem ( zimmer ml taper tivanium ) and a cobalt chromium ( cocr ) femoral head and neck adapter ( zimmer versys ) .
it is our belief that there were two modifiable factors that contributed to armd in this case .
firstly , the torque produced by a large femoral head is sufficient to increase the risk of fretting corrosion at the head / neck junction similar to that seen in total hip replacement surgery .
secondly , the neck adapter interposing the femoral head and stem is an independent risk factor for both corrosion and metal ion release .
this is the first case in the literature where the patient required revision surgery within 18 months of the primary procedure .
we are familiar with the presentation of armd as there has been a large cohort of metal on metal prostheses in this hospital .
the fact that this was revised early meant there was less severe soft tissue damage at the time of surgery compared to similar cases in the literature [ 35 ] .
we feel that armd should be considered as part of your differential diagnosis for a painful hemiarthroplasty especially if a modular prosthesis is involved .
such cases should be investigated thoroughly and the pain should not be ignored even when infection has been excluded .
corrosion at the head neck junction is possible in hemiarthroplasty when a modular system is used and is exacerbated by the large diameter femoral head inherent to this type of prosthesis .
we feel that armd should be considered in all cases where pain and dysfunction in the presence of a hip prosthesis can not be explained by routine investigations . | adverse reaction to metal debris ( armd ) in total hip arthroplasty surgery is a well - known problem .
we present the case of a unipolar hemiarthroplasty requiring revision within 18 months of insertion secondary to an adverse reaction to metal debris .
this case demonstrates a rare cause for failure of a hemiarthroplasty following a fragility fracture .
we feel that armd should be considered in all cases where pain and dysfunction in the presence of any hip prosthesis can not be explained by routine investigations . |
type 2 diabetes ( t2 dm ) is a global public health problem and the number of affected individuals is increasing at an alarming rate [ 1 , 2 ] .
the pathogenesis of t2 dm is characterized by a progressive insulin resistance with eventual insulin deficiency .
both environmental and genetic factors are known to contribute to the development of the disease .
thus , insulin secretion increases to maintain normal glucose concentration in the face of insulin resistance and/or decreasing -cell mass [ 4 , 5 ] .
although exact prodiabetic mechanisms are still largely unknown , it has been implicated that increased serum level of free fatty acids ( ffas ) , alone or combined with hyperglycemia , may contribute to the pathophysiology of the disease .
accumulating evidences have demonstrated that chronic high levels of circulating ffas play a pivotal role in -cell failure characterized with impaired glucose - stimulated insulin secretion ( gsis ) and enhanced apoptotic level .
many signaling factors , such as sterol regulatory element - binding protein ( srebp)-1c , farnesoid x receptor ( fxr ) , and forkhead box - containing protein o ( foxo ) 1 , have been identified to be associated with ffas - induced cell dysfunction [ 9 , 10 ] .
thus , delineating the molecular mechanisms underlying ffa - triggered pancreatic -cell apoptosis would put new insights into our understanding of t2 dm and open new avenues for therapy .
( death receptor - induced ) and intrinsic ( bcl-2-regulated or mitochondrial ) pathways , have been demonstrated to be contributing significantly to apoptosis .
the b - cell lymphoma-2 ( bcl-2 ) family is closely related to the intrinsic pathway of apoptosis .
bcl-2 family proteins are a large family , which may either facilitate cell survival ( bcl-2 , bcl - xl , bcl - w , and others ) or promote cell death ( bax , bak , bad , and others ) .
bcl-2 , one of antiapoptosis genes , was reduced significantly when treated with fatty acid [ 13 , 14 ] .
accumulated evidence showed that ffa - induced pancreatic -cell apoptosis was accompanied by downregulating the apoptosis inhibitor bcl-2 expression [ 15 , 16 ] .
moreover , bcl-2 overexpression does prevent cell death induced by staurosporine , fasl , and some intrinsic signals .
many endeavors have been made to elucidate the mechanisms that participate in dysregulation of genes in response to palmitate in -cells . besides transcription factors , such as the forkhead box o ( foxo ) transcription factor foxo1 , micrornas ( mirnas )
have been shown to represent another layer of gene regulation in palmitate - induced alteration of the gene expression [ 4 , 17 ] .
mirnas , endogenous noncoding small rnas , bind to specific sites at the 3-untranslated region ( 3-utr ) of target genes , thereby suppressing translation and/or inducing degradation of target mrnas [ 18 , 19 ] .
deregulated expression of mirnas is correlated with various pathological processes including cancer and diabetes [ 18 , 20 ] .
recent studies by lovis et al . have showed that mir-34a is one of the significantly upregulated mirnas in the -cell treated with palmitate .
moreover , elevated expression of mir-34a enhanced the -cell apoptosis [ 4 , 21 , 22 ] .
however , the possible role of mir-34a in palmitate - induced -cell apoptosis is largely unknown . here , we found that downregulation of bcl-2 expression by mir-34a accounts for palmitate - induced increased apoptosis of pancreatic -cell
min6 pancreatic -cells were maintained in dmem medium ( gibco , carlsbad , ca ) containing 25 mm glucose supplemented with 10% fetal bovine serum ( fbs ) , 10 nm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) , 2 mm l - glutamine , 100 unit / ml penicillin , 100 g / ml streptomycin , and 50 m -mercaptoethanol at 37c in 5% co2 . a stock solution ( 100 nm ) of palmitate ( sigma , st .
louis , mo ) was prepared by dissolving the fatty acid in 50% ethanol and then diluted in culture medium with 0.5% fatty acid - free bsa ( sigma , st .
cloning of the mouse bcl-2 3-utr containing mir-34a binding site was performed by pcr using the following primers : forward : 5-gcgccgcggtcgacaaacctgccccaaac-3 ; reverse : 5-gcgctgcagacgtttcttggcaattcctg-3. the pcr product was then cloned into a modified pgl3 control vector where sacii and psti sites were introduced into the original xba i site downstream of the luciferase gene . to mutate the binding sites within the 3-utr , quikchange site - directed mutagenesis kit from stratagene ( la jolla , ca )
cloning of the mouse bcl-2 orf was performed by pcr using following primers : forward : 5-ataggtaccatggcgcaagccgggagaac-3 ; reverse : 5-cggtctagatcacttgtggcccaggtatg-3. the pcr product and the expression vector pcdna3 ( invitrogen , carlsbad , ca ) were cut with kpni and xbai and ligated to generate the construct pcdna3-m - bcl-2 .
real - time pcr was performed using sybr premix ex taq ( takara , dalian , china ) in an abi7500 system ( applied biosystems , carlsbad , ca , usa ) .
bulge - loop mirna qpcr primers for mir-34a were synthesized by ribobio ( guangzhou , china ) .
the following primary antibodies were used : anti - bcl-2 and anti - cleaved - caspase-3 ( cell signaling , beverly , ma ) , and anti--tubulin ( sigma - aldrich , st louis , mo ) .
mirna mimic , mirna inhibitor , and their corresponding control oligonucleotides ( purchased from ribobio , guangzhou , china ) were transfected using lipofectamine 2000 following the manufacturer 's instruction ( invitrogen , carlsbad , ca ) .
sense - strand sequences of bcl-2 sirnas are 5-aaguacauacauuauaagcug-3 and 5-guacauccauuauaagcug-3. the number of apoptotic cells was evaluated by scoring cells displaying pycnotic nucleus and/or fragmented nucleus visualized with hoechst 33342 as previously described .
a commercial caspase 3 activity assay kit ( roche , mannheim , germany ) was used to measure the intracellular caspase 3 activity according to manufacturer 's instruction . for the reporter assay
, cells were seeded in triplicate in 24-well plates and allowed to settle for 24 h. indicated plasmids and oligonucleotides were transfected into the cells using the lipofectamine 2000 reagent ( invitrogen , carlsbad , ca ) .
forty - eight hours after transfection , dual - luciferase reporter assays were performed according to the manufacturer 's instructions ( promega , madison , wi ) .
results are presented as means standard deviation ( sd ) of three independent experiments performed in triplicate .
all values represent at least three independent experiments and are expressed as the means sd .
the differences between two experimental conditions were compared on a one - to - one basis using student 's t - tests . statistical significance within groups
as shown in figure 1(a ) , the expression level of mir-34a was significantly elevated by palmitate treatment in min6 cells .
next , we investigated whether change in the level of mir-34a induced by palmitate was able to influence apoptotic rate of the cells . as shown in figure 1(b ) , ectopic expression of mir-34a increased the number of dying cells .
as the caspase-3 activity is a key marker of apoptosis , the expressions of cleaved caspase-3 and caspase-3 activity were investigated to confirm the proapoptotic role of mir-34a . as shown in figures 1(c ) and 1(d ) ,
together , these data suggested that upregulated mir-34a contributes to proapoptotic effects of palmitate on pancreatic cell .
bcl-2 , an antiapoptotic protein involved in cell apoptosis , is of specific interest as the computational tool ( targetscan http://www.targetscan.org/ ) predicted a mir-34a - binding site within the bcl-2 3-utr ( figure 2(a ) ) . to verify the potential targeting of bcl-2 by mir-34a , we measured the protein level of bcl-2 in mir-34a transfected min6 cells .
as shown in figure 2(b ) , the expression of bcl-2 was significantly suppressed by mir-34a transfection . to further confirm
that the suppression of bcl-2 expression is directly mir-34a - driven , we generated a luciferase construct containing the bcl-2 3-utr .
meanwhile , a construction with the mutation at the putative binding site ( figure 2(a ) ) was also generated as control .
cotransfection of the wild - type bcl-2 3-utr and mir-34a mimic led to a significant inhibition of luciferase activity ( figure 2(c ) ) .
in contrast , no significant alteration of luciferase activity was detected in cells transfected with mutated construct .
consistently , palmitate treatment also suppressed the luciferase activity of the wild - type bcl-2 3-utr and had no effect on mutated construct ( figure 2(d ) ) . taken together
, these data suggest that mir-34a suppresses bcl-2 expression via directly binding to its 3-utr . considering the apparent increase in the expression of mir-34a in response to palmitate
, it is expected that pretreatment of mir-34a inhibitor may prevent the palmitate imposed effects .
indeed , we showed that the increased apoptotic rate and decreased bcl-2 expression induced by palmitate in pancreatic cell could be counteracted by pretreatment with mir-34a inhibitor ( figures 3(a ) and 3(b ) ) .
therefore , upregulated mir-34a expression participates in the palmitate - induced effects on pancreatic cell . to confirm the functional role of bcl-2 in pancreatic cell , inhibition of bcl-2 expression by sirnas
wb assays were performed and confirmed that bcl-2 expression was significantly silenced ( figure 4(a ) ) .
as expected , data presented in figure 4(b ) showed that inhibition of bcl-2 caused increased min6 cell apoptosis .
moreover , reexpression of bcl-2 in mir-34a - expressing min6 cells ( figure 4(c ) ) partially antagonized the mir-34a - induced apoptosis . taken as a whole , these data indicate that bcl-2 is a functionally relevant target of mir-34a 's effect in response to palmitate treatment .
it has been previously demonstrated that bcl-2 expression is suppressed during palmitate - induced apoptosis and mir-34a expression was upregulated in response to palmitate treatment .
this raised the possibility that suppression of bcl-2 in apoptotic cells may be regulated by mir-34a .
we evaluated this hypothesis in min6 pancreatic cell , where we showed that bcl-2 is directly targeted by mir-34a .
mir-34a - induced suppression of bcl-2 is associated with palmitate - associated pancreatic cell apoptosis .
t2 dm is a metabolic disorder that is driven by the interaction between genetic and acquired factors .
the impact of ectopic fat and lipotoxicity on pancreatic -cell dysfunction and insulin resistance in humans has been paid great attention .
accumulated evidence shows that lipotoxic stress - induced -cell death ( lipotoxicity ) is a key contributor to the development of t2 dm by inducing insulin resistance .
lipotoxicity is caused by circulating free fatty acids increased , changes in lipoprotein profiles and body fat distribution .
it is widely accepted that a sustained exposure to fatty acids impairs glucose - stimulated insulin secretion ( gsis ) , promotes apoptosis , and alters insulin gene expression in vitro and in vivo .
bcl-2 , an antiapoptotic bcl-2 family member , is a crucial controller of the mitochondrial pathway of pancreatic -cell apoptosis induced by lipotoxicity .
although previous studies suggest that the suppression of bcl-2 is involved in lipotoxicity induced pancreatic cell apoptosis , the mechanisms involved in the decreased expression of bcl-2 in response to palmitate remains to be clarified . in researching the mechanism of palmitate - induced downregulated expression of bcl-2 , we noted that mir-34a , as palmitate , can dramatically induce its expression .
it has been demonstrated that mir-34a was involved in various cell apoptosis conditions , including pancreatic beta cells .
roggli et al . have demonstrated that anti - mir-34a can protect pancreatic cell from proinflammatory cytokines - induced cell death and mir-34a - triggered bcl-2 downregulation might be associated with cytokine - mediated cell apoptosis .
if bcl-2 was inhibited by mir-34a overexpression , it is likely that the effect of palmitate on cell apoptosis is mediated through suppression of bcl-2 by mir-34a . here
an in silico approach identified a potential mir-34a binding site within the 3-utr of bcl-2 gene .
furthermore , the protein level of bcl-2 is significantly suppressed by ectopic expression of mir-34a .
intriguingly , anti - mir-34a pretreatment was able to attenuate palmitate - induced bcl-2 downregulation , as well as cell apoptosis , strongly indicating the functional role of mir-34a in the cell in response to lipotoxicity .
we also investigated the importance of bcl-2 for palmitate - induced cell apoptosis in min6 pancreatic cell lines by loss- and gain - of - function experiments . given that one specific mirna can regulate a group of target genes , together with the fact that repressing bcl-2 could not fully recapitulate mir-34a phenotype in pancreatic cell , it is expected that other target genes regulated by mir-34a are involved in palmitate - induced cell dysfunction .
future work is still needed in the area of defining possible target genes of mir-34a that contribute to apoptosis of cell exposed to palmitate . in conclusion ,
suppression of bcl-2 by mir-34a accounts for palmitate - induced increased apoptotic rate in pancreatic cell .
our findings reveal a new mechanistic aspect of the proapoptotic effect of palmitate on pancreatic cell and raise the possibility of influencing mir-34a as a therapeutic strategy for diabetes . | recent studies have demonstrated that the expression of mir-34a is significantly upregulated and associated with cell apoptosis in pancreatic -cell treated with palmitate .
nevertheless , the underlying detailed mechanism is largely unknown . here
, we showed that mir-34a was significantly induced in min6 pancreatic -cell upon palmitate treatment .
elevated mir-34a promoted min6 cell apoptosis . intriguingly , ectopic expression of mir-34a lowered the expression of bcl-2 , an antiapoptotic protein .
luciferase reporter assay indicated the direct interaction of mir-34a with the bcl-2 3-utr .
moreover , downregulated expression of bcl-2 induced by palmitate could be restored by inhibition of mir-34a .
we conclude that direct suppression of bcl-2 by mir-34a accounts for palmitate - induced increased apoptosis rate in pancreatic -cell . |
neonatal diabetes mellitus ( ndm ) is a form of diabetes that is diagnosed under the age of 6 months .
the most common causes of neonatal diabetes are activating mutations in the adenosine triphosphate ( atp)-sensitive potassium ( katp ) channel .
this review discusses functional effects and therapy implications of ndm mutations in the regulatory subunit of the katp channel - sulphonylurea receptor-1 ( sur1 ) .
neonatal diabetes mellitus ( ndm ) is defined as hyperglycemia that presents within the first 6 months of life .
it is a rare disorder that can be either permanent throughout life ( permanent ndm , pndm ) or transient ( transient ndm , tndm ) , with a period of remission .
tndm accounts approximately for 50% cases of neonatal diabetes ( nd ) and affects approximately one in 100,000 live births .
the majority ( about 80% ) of cases of tndm are caused by abnormalities of an imprinted locus on chromosome 6q24 that results in the overexpression of a paternally expressed gene .
most of remaining cases are caused by mutations in either kcnj11 or abcc8 genes that encode the atp - sensitive potassium ( katp ) channel respectively .
the precise mechanism of tndm is unknown ; it has been proposed that it could be due to either a reduced insulin requirement at the time of remission or because of some compensation effects at the level of the -cell , pancreas , or whole body .
pnd may be either isolated or form part of a syndrome , such as wolcott - rallinson syndrome due to mutations in the eif2ak3 gene , pancreatic agenesis due to mutations in ipf-1 gene and nd with cerebellar agenesis due to mutations in the ptf-1a gene .
the most common cause of isolated pndm are mutations in the genes that encode insulin ( ins ) and the katp channel ( kcnj11 and abcc8 ) .
katp channels act as metabolic sensors , coupling the metabolism of a cell to its membrane potential and electrical excitability .
their activity is primarily regulated by intracellular adenosine nucleotides , with atp having an inhibitory and magnesium adenosine diphosphate ( mgadp ) or magnesium adenosine triphosphate ( mgatp ) a stimulatory effect on channel activity .
katp channels are expressed in many tissues including pancreas , skeletal and smooth muscle , and the brain . in these tissues ,
katp channels play a multitude of physiological roles ; however , their physiological role has been best characterized in the pancreatic -cell . the -cell katp channel links glucose metabolism and insulin release . at substimulatory glucose concentrations ( fig .
hence the cell membrane is hyperpolarised , voltage - gated calcium channels are closed and no insulin is released . when blood glucose concentration rises , glucose is transported into pancreatic -cells and metabolised , thereby increasing the ratio of atp : adenosine diphosphate ( adp ) concentrations ( fig .
this closes the katp channel , produces a membrane depolarisation that opens voltage - gated calcium channels ; resulting in influx of calcium into the -cell which triggers insulin exocytosis .
given the crucial role of the -cell katp channel in insulin secretion and glucose homeostasis , it is not surprising that mutations in this channel can lead to diseases of both hypoglycaemia and hyperglycaemia .
loss of function mutations in katp channels cause over - secretion of insulin and result in hyperinsulinaemia .
conversely , gain of function mutations cause undersecretion of insulin , hyperglycemia and result in nd .
the -cell katp channel is a hetero - octameric complex comprising four kir6.2 subunits ( encoded by the kcnj11 gene ) and four sulphonylurea receptor-1 ( sur1 ) subunits ( encoded by the abcc8 gene ) .
kir6.2 is an inwardly rectifying k - channel that forms the potassium - selective pore and possesses an inhibitory site for atp [ 8 - 10 ] .
it confers channel sensitivity to stimulation by mg - nucleotides , activation by k channel openers , such as diazoxide ; and inhibition by sulphonylureas .
in addition , sur1 also enhances the inhibitory effect of atp and stabilizes the open state of the channel in the absence of nucleotides .
the sur1 protein contains three transmembrane domains ( tmds ) linked by the cytosolic linker region and two nucleotide - binding domains ( nbds ) ( fig .
2 ) . tmd1 and tmd2 contain six transmembrane helices and tmd0 , sited at the hydrophobic n - terminus and important for interactions with kir6.2 , contains five transmembrane helices .
each of the nbds contains sequence motifs called walker a and walker b that are essential for binding the phosphate groups of nucleotides .
atp binding to sur1 causes head to tail dimerization of the nbds and formation of two nucleotide binding sites ( nbs1 and nbs2 ) within the dimer interface .
nbs2 possesses greater atpase activity than nbs1 and its occupancy by mgadp stimulates katp channel activity .
most of patients with sur1 mutations have isolated diabetes ; approximately 30% of patients have additional neurological features .
these include developmental delay , learning difficulties , epilepsy , minor dystonia , tonic posturing , and muscle weakness .
these features are consistent with the expression of katp channels containing the sur1 subunit in the central nervous system . to date , only two sur1 mutations ( f132l and i49f ) were identified that cause the most severe form of nd with developmental delay and epilepsy ( dend syndrome ) .
one mutation ( l213r ) caused nd with developmental delay but without epilepsy - intermediate dend ( i - dend ) syndrome .
unlike in some other cases of i - dend syndrome , mutation l213r did not cause muscle weakness .
nd mutations in sur1 are all missense mutations and account for more than 10% of pndm and a frequent cause of tndm .
in contrast to mutations in kir6.2 which are all dominant heterozygous , sur1 mutations can be either dominant or recessively inherited .
recessive mutations could be homozygous , mosaic due to segmental uniparental isodisomy or compound heterozygous for another activating mutation or if the second allele is inactivated .
approximately 50% of sur1 mutations are spontaneous , arising de novo during embryogenesis . to date , over 60 mutations in sur1
have been identified ; they are scattered throughout the protein sequence , but are particularly concentrated in tmd0 and their connecting loops , in the cl3 linker connecting nbd0 with nbd1 and in nbd2 ( fig .
the most commonly occurring mutations in sur1 are at positions r1183 and r1380 , both of which cause tndm .
not all gain of function mutations in sur1 result in nd , for example , mutation s1369a ( the most important polymorphism of the abcc8 gene ) merely increases risk for the type 2 diabetes .
gain of function mutations in the sur1 subunit increase katp current in the presence of mgatp . in general
, there is a good correlation between the magnitude of katp current and disease severity ; thus mutations that cause largest increase katp channel lead to the most severe form of nd with associated neurological complications - dend syndrome . to date , available functional studies show that increase in current produced by mutations in sur1 is smaller than that caused by kir6.2 mutations .
this may explain the relative high incidence of tndm than pndm among patients with abcc8 mutations as well as why most patients with dend syndrome ( > 90% ) have mutations in kir6.2 .
sur1 mutations mediate their effect on channel activity via two main mechanisms - by reducing the inhibition produced by atp binding at kir6.2 and by enhancing channel activation by mg - nucleotides .
mutations in sur1 that decrease the amount of inhibition at kir6.2 may do so in one of two ways .
it is well established that the presence of sur1 enhances atp inhibition at kir6.2 , which suggests that sur1 either contributes to the atp binding site itself , or influences it allosterically .
disruption of this could reduce atp binding directly ; mutations in sur1 that are likely to act via this mechanism include a30v in tmd0 and g296r in tmd1 which together form a compound heterozygous mutation that results in pndm .
these mutations stabilize the open state of the channel and thus increase its single channel open probability .
examples include f132l in tmd0 , l213r in the cl3 linker between tmd0 and tmd1 and v324 m in tmd1 . among the mutations that enhance the activatory effect of mg - nucleotides ,
only one sur1-pndm mutation ( r826w ) is found in nbd1 , nevertheless this mutation lies in the linker that is predicted to form part of nbs2 .
functional studies showed that these mutations enhance the time that nbs2 spends in the activating mgadp - bound state either directly by enhancing mgadp binding or indirectly by altering other reaction steps of the atpase . some nd mutations which enhance mg - nucleotide activation
these mutations may exert their effect via enhancing the transduction of the mg - nucleotide stimulation from the nbss of sur1 to the channel pore at kir6.2 ; alternatively , they may also allosterically affect nucleotide handling at the nbds .
in addition to affecting channel activity , some sur1 mutations can also affect channel expression at the plasma membrane .
an example of such mutation is v324 m , of which the activating effect ( enhanced mg - nucleotide activation , enhanced stability of channel open state ) is dampened by reduced channel expression at the cell surface .
the interplay between the two types of opposing defects may be responsible for the fact that the v324 m mutation results only in the transient form of the disease .
in general , there is no correlation between the position of the mutation in the sur1 and the clinical phenotype - mutations in the same residue may result in different types of disease .
for example , mutations of the conserved glutamate residue e1506 in nbd2 can result in either hyperinsulinism or nd .
it is important to point out that several sur1 mutations have been identified in patients with nd which were later found not to be responsible for the disease . in some cases ,
no functional effects have been found , in others the parents were nonsymptomatic carriers of the mutation .
this emphasizes the need of the functional study in order to confirm that the mutation is the cause of the disease .
prior to the discovery that nd can be caused by mutations in the katp channel , many patients were assumed to be suffering from early - onset type 1 diabetes .
recognition that these patients actually possess gain of function mutations in katp channel genes rapidly led to a switch to sulphonylurea treatment .
fortunately , since sulphonylureas had been used to safely treat patients with type 2 diabetes for many years , no clinical trials were required .
sulphonylurea drugs bind to both kir6.2 and sur1 subunits of the katp channel with very different affinities .
the low - affinity binding site , which lies on kir6.2 , is of no clinical relevance as it is occupied only at concentrations much higher than those found in the plasma of patients treated with sulphonylureas .
the primary effect of the drug is mediated via a high - affinity binding site on sur1 with associated ic50 values for block of wild - type katp channels being substantially lower than the usual therapeutic plasma concentrations of sulphonylureas .
depending on the type of sulphonylurea , the maximal extent of high - affinity block in the absence of nucleotides is only 50% to 80% . in the presence of nucleotides , binding of sulphonylureas to the high affinity site suppresses the activatory effect of mgadp and unmasks the inhibitory effect of nucleotides at kir6.2 .
this leads to an apparent increase in the high - affinity block of wild - type katp channels in the presence of mg - nucleotides , which becomes virtually complete inside pancreatic -cells .
thus , sulphonylureas exert dual action on katp channel activity : a direct block and indirect block via modulation of nucleotide - dependent channel gating .
given their molecular mechanism of action , many nd mutations in sur1 can be expected to affect sulphonylurea inhibition .
mutations that impair atp inhibition indirectly via stabilizing the open state of the channel will reduce both direct and indirect block of the drug .
mutations that reduce atp binding to the inhibitory site on kir6.2 directly will impair mg - nucleotide - induced enhancement of the high - affinity sulphonylurea block .
finally , mutations that enhance mgadp activation may potentially impair the ability of sulphonylureas to suppress it .
relatively little information is available at present about the efficacy of sulphonylurea block of katp channels with nd mutations in the sur1 subunit .
impaired direct and indirect block of sulphonylureas has been demonstrated for mutation l213r , which disrupts gating .
as expected , direct block was unaffected by sur1 mutations that enhance mg - nucleotide activation or directly reduce atp binding to kir6.2 .
functional studies further suggest that mutations that enhance mg - nucleotide activation may have either no effect or cause small reduction in the indirect block of sulphonylureas .
the latter is likely to be mediated via displacement of sulphonylureas from sur1 by mg - nucleotide binding to nbs2 . in support of this idea ,
two nd mutations that enhance atp affinity for nbs2 were found to enhance glibenclamide displacement from sur1 by the nucleotide . to date , most patients with nd caused by mutations in sur1 were successfully transferred to sulphonylureas [ 16,33,39 - 43 ] .
mutation f132l strongly impairs channel function via altering channel gating and causes the most severe form of the disease - dend syndrome ; as demonstrated by previous studies on nd mutations in kir6.2 , successful transfer to sulphonylurea therapy of patients with this syndrome is quite rare .
it is currently unclear why patients with f132v mutation that causes pndm alone are unable to transfer to sulphonylureas . even more puzzling
is that in the only other reported case of dend syndrome due to mutations in sur1 ( i49f ) , the patient was able to transfer to sulphonylurea therapy .
further studies are needed to gain insight into the functional effects of these two mutations .
an important contributing factor for successful transfer to sulphonylurea treatment is the age of the patient ; the sooner the transfer is carried out the better .
studies of mouse models of nd suggest that this could be due to gradual loss of -cell mass which can be prevented by therapy .
successful treatment of patients with nd demands higher doses of sulphonylureas compared to those with type 2 diabetes . as discussed above ,
this may be caused , at least in part , due to the fact that many nd mutations impair sulphonylurea block .
patients with mutations in sur1 require lower doses of sulphonylureas than those with mutations in kir6.2 , consistent with the idea that the former mutations cause in general milder impairment of channel function than the latter . in spite of relatively high doses ,
the only side effects reported include transitory diarrhea or tooth discoloration in a few patients .
transfer to sulphonylurea treatment does not only improve quality of life of patients with nd ; it also appears to enhance their blood glucose control .
fluctuations in blood glucose are reduced and there is also a decrease in the hemoglobin a1c ( hba1c ) levels .
the discovery that mutations in sur1 can cause nd has led to more insight into the role of this regulatory subunit in the katp channel function and transformed therapy for patients with these mutations .
more functional analysis as well as high resolution crystal structure of the whole katp channel complex are necessary in order obtain detail understanding how the sur1 atpase works and how it is coupled to katp channel gating .
finally , mouse models of nd will help us to understand why some mutations in sur1 result in remitting - relapsing form of nd and why others cause additional neurological problems . | adenosine triphosphate ( atp)-sensitive potassium ( katp ) channels in pancreatic -cells play a crucial role in insulin secretion and glucose homeostasis .
these channels are composed of two subunits : a pore - forming subunit ( kir6.2 ) and a regulatory subunit ( sulphonylurea receptor-1 ) .
recent studies identified large number of gain of function mutations in the regulatory subunit of the channel which cause neonatal diabetes .
majority of mutations cause neonatal diabetes alone , however some lead to a severe form of neonatal diabetes with associated neurological complications .
this review focuses on the functional effects of these mutations as well as the implications for treatment . |
mixed methods research uses both quantitative and qualitative data to improve understanding of a research problem beyond what is possible with either approach alone .
mixed methods studies are challenging for health researchers to plan and implement due to their design complexity and difficulties related to appropriate integration of data and results [ 24 ] .
there is acknowledgement among health researchers about the value of mixed methods , but concerns regarding a lack of formal education and skills in using mixed methods .
while many investigators have received formal graduate training in either quantitative methods or qualitative methods , few had been exposed to the specifics of mixed methods designs and the process for mixing and integration of data .
there continues to be a gap in our understanding of how mixed methods can be used in health research , especially when combining two methods in a single study . while mixing two methods
can offer a more comprehensive investigation of a research problem , there are unique challenges to integrating two methods .
our goal is to describe the process of implementing a sequential explanatory mixed methods study involving a randomized , controlled trial ( rct ) with a subanalysis of quantitative data and a qualitative grounded theory approach .
using an example study , we will explore how this mixed methods study was introduced after a large multisite rct was in progress .
the investigators felt that the mixed methods study would provide greater context and explanation for preliminary quantitative findings arising from the rct .
as a result of implementing a mixed methods study during the course of an rct , methodological considerations were made in order to maintain the integrity of the trial and the mixed methods component the quantitative subanalysis of emergency department site results from the rct and grounded theory approach .
the focus of this paper will be on describing the methodological modifications made to the grounded theory approach when used as an explanatory component for a rct and then providing design and analytic considerations for the conduct of similar studies .
the strengths and challenges of combining a rct with a grounded theory approach in a mixed methods study will also be discussed .
the philosophical underpinnings of mixed methods have been extensively debated including whether or not qualitative methods should be mixed with quantitative methods [ 610 ] .
these debates have focused on discussions about whether or not quantitative and qualitative research can be combined [ 6 , 8 , 1013 ] , the methodological challenges of combining quantitative and qualitative methods [ 9 , 1113 ] , and the inconsistent integration of both quantitative and qualitative methods , most often with more emphasis on maintaining the integrity of quantitative methods [ 10 , 11 , 1315 ] .
central to the philosophical debate is the concern about juxtaposing worldviews , such as combining positivist perspectives with that of social constructivist standpoints .
the rct is the gold standard in evaluating the effectiveness of an intervention on preidentified health outcomes [ 9 , 10 , 1214 , 16 ] .
the rct is grounded in the positivist worldview , where the goals are to ( 1 ) identify causal relationships leading to one empiric truth using deductive methods like hypothesis generation and prediction , ( 2 ) use control over the experimental environment to reduce the influence of potential extraneous variables , and ( 3 ) interpret the findings using statistical tests without consideration of the participant 's interpretation [ 15 , 17 ] .
qualitative methods , such as grounded theory , often follow the social constructivist worldview whereby the goal is to understand participant perspectives including the individual and multiple meanings allocated to phenomena , surroundings , behaviours , and social practices [ 15 , 17 ] .
qualitative research emphasizes understanding individual perspectives as multiple truths and aims to aggregate the beliefs , social behavior , and processes that arise from participant perspectives and do not use the same practices or methods as with quantitative research [ 15 , 17 ] .
because of these opposing philosophical stances , authors have described the debates about whether or not these two methods can be combined [ 517 ] .
teddlie and tashakkori stated that a focus on paradigmatic differences can halt the productivity of innovations in mixed methods research .
others have argued that a mixed methods design can help resolve the paradigm debate through its philosophical grounding in pragmatism which offers a utilitarian approach to research [ 18 , 19 ] .
mixed methods permit a deeper and richer understanding of the phenomenon under study through an emphasis on plurality of philosophical paradigms , theoretical assumptions , and methodological techniques .
many authors recognize the multiplicity of paradigms which can compete and give rise to contradictions , but which is a normative component of mixed methods research .
this means that methods are combined , regardless of their individual philosophies , in order to meet the goals of the overall research project .
many investigators now value the need for mixed methods studies that incorporate qualitative and quantitative methods to better understand phenomena or to provide a richer explanation of results [ 6 , 7 , 11 ] .
qualitative research used within a rct can explore the meaning that participants attribute to the intervention , which might serve to maximize its efficacy through greater understanding of context and patient experiences . finally , using a qualitative approach to better understand the process of events and actions could add insight into the feasibility of the intervention .
researchers have combined qualitative research with a rct , so that the shortcomings of either method could be overcome while comprehensively addressing a research question , such as the effectiveness of a complex intervention . for this study
, we acknowledge this debate but have adopted a pragmatic approach [ 1719 ] to the use of qualitative and quantitative methods together for study .
lewin et al . in their systematic review of rcts of complex interventions randomly retrieved 100 out of 492 eligible studies to explore the use of qualitative methods to understand rct results . in the 100 rcts ,
only 30 incorporated qualitative studies to explain their results . among the 30 rcts that did involve formal qualitative approaches
, the authors found that there was limited rationale provided for mixing methods , little discussion of the integration of methods , and few studies presented the contributions of both quantitative and qualitative methods to overall study interpretations .
the following methodological flaws were identified with the qualitative studies : limited explanation of the design / approach , lack of discussion regarding recruitment and sampling methods , failure to describe modifications to data collection methods , and poor description of qualitative data analysis and thematic development .
these methodological flaws in the qualitative studies accompanying intervention trials were attributed to a lack of expertise in qualitative methods and resource constraints to carry out both a rct and a qualitative study .
kinn and curzio conducted a systematic review that assessed the incorporation of quantitative and qualitative methods in research articles .
the authors searched medline , cinahl , and psycinfo bibliographic databases from 1982 to 2000 .
findings indicated that qualitative approaches were used for different purposes , ranging from preliminary exploratory work to validation of quantitative findings .
, kinn and curzio found that the qualitative component of the mixed methods studies were poorly described such as the qualitative design selected , sampling procedures , coding and analysis techniques , and the methods for the integration of both quantitative and qualitative findings .
other authors have confirmed these systematic review results and emphasized the need for greater cohesion of methods when incorporating qualitative approaches with a rct [ 14 , 21 ] . combining methods in a transparent manner can help overcome methodological weaknesses in both approaches and provide a comprehensive examination of the research question under study .
the most advantageous use of qualitative approaches with the randomized trial occurs when both hold equal importance and weighting and are not considered subservient to one another [ 14 , 21 ] .
sequential explanatory mixed methods designs aim to answer one type of question by collecting and analyzing two types of data ( qualitative and quantitative ) and drawing inferences using both data types usually during the interpretation stage of the study in two distinct phases [ 1 , 22 ] . with the sequential design one type of method ( quantitative or qualitative )
is timed to be completed first from data collection to analysis ( e.g. , phase 1 ) before beginning the second method ( e.g. , phase 2 ) .
the results from the first phase help to inform the second phase of data collection and analysis involving the second method .
the primary purpose of the sequential explanatory design is to explain and expand upon findings identified during the first phase of the study [ 1 , 23 ] . in our example , the purpose for this sequential mixed methods design was to enhance initial quantitative results by using followup qualitative methods .
this mixed methods study was initiated a few months after a multisite rct was underway .
the rct examined the effectiveness of routine screening for intimate partner violence ( ipv ) in health care settings compared with usual care in reducing violence and improving life quality ; its methods are reported elsewhere .
initial rct results from the emergency department sites indicated that some participants disclosed ipv to the emergency department health care provider , yet the specific nature of the decisions leading to this disclosure , including the processes that abused women used to disclose , could not be described . as a result ,
a mixed methods study was identified as the most ideal method for this study , as it could provide a more comprehensive analysis of the quantitative data from the rct emergency department sites ; a qualitative component could expand on these results to better understand abused women 's decision making regarding disclosure of violence in urban emergency department settings in on , canada .
the first phase of this mixed methods study was quantitative and involved a subanalysis of rct data from three emergency department sites including : demographic information ; two screening instruments to assess women 's report of ipv ( 1 ) the composite abuse scale ( cas ) and ( 2 ) the woman abuse scale tool ( wast ) ; and a verifying report from the health care provider that the subject disclosed ipv .
the quantitative subanalysis results helped to describe rates of ipv and the proportion of participants that disclosed violence to an emergency department health care provider .
however , these results did not explain why those women who scored positive for ipv decided not to disclose during their visit to the emergency department .
the quantitative results also did not describe the problems that women perceived with disclosure or the processes used by women who did disclose violence in the emergency department setting .
after review of these quantitative results , grounded theory was considered the best qualitative method to explain these findings due to its ability to both describe and explain a system of behaviour and seek , as an end result , a substantive midrange theory grounded in the data . grounded theory aims to build a midrange , substantive theory that portrays a central problem , its characteristics and contributing factors , the process and strategies used to resolve the problem , and the consequences if the problem was not resolved .
in this example study , we sought to understand what women saw as the central problem related to disclosure of ipv to an emergency department health care provider and the strategies and processes that they used to resolve the problem related to disclosure .
traditional grounded theory was selected to guide data collection , organization and data analysis [ 2830 ] ( phase 2 ) ( figure 1 ) .
sampling for mixed methods studies follows the general rules governing both quantitative and qualitative methods and involves selecting participants using both probability and purposive sampling techniques . according to collins et al .
, determining an appropriate sampling scheme is pivotal to the preservation of rigour and the overall integration of results for a mixed methods study .
macmillan et al . provided details of the overall rct randomization and recruitment process .
for this quantitative subanalysis phase of the mixed methods study , participants in the rct were selected from may to november 2006 , across three emergency department settings , and those who met the following eligibility criteria were recruited:18 years or older and previously enrolled in the rct at one of the three recruitment emergency departments;recruited into the rct and scored positive for ipv using the wast and the cas ( details of instruments in macmillan et al . ) ; able to disclose ipv to a health care provider during their emergency department visit;healthy enough to participate in a 6090 minute interview;able to speak , read , and write in english .
18 years or older and previously enrolled in the rct at one of the three recruitment emergency departments ; recruited into the rct and scored positive for ipv using the wast and the cas ( details of instruments in macmillan et al . ) ; able to disclose ipv to a health care provider during their emergency department visit ; healthy enough to participate in a 6090 minute interview ; able to speak , read , and write in english .
in addition to the wast and cas , an additional instrument called the clinical interactions instrument was completed by participants who disclosed ipv during their visit to the emergency department
. this instrument involved participants selecting the type of intervention that they were offered by the health care provider after disclosure of ipv .
data were analyzed to determine the rates of ipv exposure using the screening instruments from the rct and the rates of disclosure to an emergency department health care provider . after the analysis of quantitative data was completed for phase 1 , the second phase involving a qualitative method began .
the quantitative results were used to help inform the grounded theory study , and all participants were recruited from the rct subanalysis sample that met eligibility criteria ( n = 174 ) and interviewed from may 2006 to december 2007 . for the grounded theory component of the mixed methods study , an estimated goal to sample
2027 women was identified in keeping with other grounded theory studies involving participants who were either deemed vulnerable or who attended acute care settings [ 33 , 34 ] .
theoretical sampling and saturation guided sampling and data collection for the grounded theory phase . according to schwandt ,
theoretical sampling includes collecting data on activities and events as dictated by the evolving theory .
continuous comparison is an essential feature of grounded theory where comparisons are made between the developing theory and the raw data until no new findings or views emerge regarding a concept or category a key feature of saturation .
constant comparison is a key element of grounded theory where comparisons are made between the developing theory and the raw data until no new findings or views emerge regarding a concept or category . in order to begin the process of developing codes and categories to drive initial theoretical sampling ,
criteria were created to identify variation among participants and build codes that would drive future interviews prior to entering the theoretical sampling phase of the study .
once a preliminary set of codes and interview guide were developed , eligibility for inclusion continued to focus on theoretical sampling , which is an integral part of the analytic process for grounded theory .
data were simultaneously collected , coded , and analyzed using activities to saturate the evolving theory and increase its level of abstraction [ 29 , 36 ] . during theoretical sampling , our goal was to obtain conceptual density and a theory grounded in the data , despite a bounded sample of participants from phase 1 of the mixed methods study .
as a result , we used the constant comparison of findings was undertaken based on the incidents , events , or situations related to the disclosure of ipv in urban emergency departments [ 30 , 37 ] .
we called these ipv disclosure events , and this unit of analysis
approach allowed us to maximize potential for theoretical density using constant comparison across ipv disclosure events .
this meant that we analyzed over 100 intimate partner violence disclosure histories and events ( units ) involving emergency department health care providers , health care providers in other settings , and other professionals in order to identify features and components for developing concepts and categories .
the unit of analysis did not represent actual participants but rather events , incidents , or examples .
in addition to recruiting participants based on ipv disclosure events , as a means of achieving theoretical saturation , we also recruited participants who scored positive on the wast and negative on the cas measures used in phase 1 of the mixed methods study or those who did not disclose ipv to a health care provider during their visit to the emergency department . once the core phenomenon was defined and relationship linkages developed , two other sampling methods used to assess the developed categories and to raise the description of the theory to a higher level of abstraction .
maximum variation sampling , sometimes referred to as extreme case sampling , involves interviewing participants who hold different perspectives , sometimes at opposite ends of the spectrum in order to obtain a full understanding of the phenomenon under study [ 1 , 31 ] .
negative case sampling was also used as an approach to help assess the developing grounded theory .
the goal of negative case sampling is to test emerging theoretical hypotheses in order to achieve a higher level of theoretical abstraction beyond a description of relationships between concepts .
this type of sampling is typically performed after the development of the grounded theory in order to identify new participants whose experiences refute or deviate from the developing theory [ 31 , 36 ] .
deviant cases offer opportunity for comparison across other categories and are often selected outside of the sample used to generate the grounded theory .
data were collected in face - to - face , in - depth , and semistructured interviews lasting from 60 to 90 minutes from may 2006 to december 2007 .
all interviews were digitally recorded and conducted in a private location determined with the participant ; all participants were provided with a $ 25 honorarium per interview .
interview guides were used at each interview and were modified during analysis to capture the developing categories and themes and to enhance saturation .
three levels of coding were used for the grounded theory phase including in vivo , substantive , and theoretical coding . throughout the coding process , the constant comparison method was used to review data for fit and relevance , to evaluate codes with one another to generate categories , and to identify relationships between categories for the ultimate generation of a theory grounded in the data . during in vivo coding , codes were labelled , separated , and compiled to capture the main idea of the participant ( level 1 ) .
these codes were then grouped together to create clusters or families which were then classified under a label as concepts . these concepts were then organized together to form categories ( level 2 ) [ 36 , 37 ] . constant comparison identified relationships among codes and categories ( level 3 ) , properties and dimensions of the categories , and conceptual linkages within the data .
with grounded theory , the goal is to identify a core phenomenon and basic social psychological problem ( bsp ) .
once these were identified from the data , selective coding strategies were used to build categories and extend theoretical abstraction .
table 1 outlines the main demographic characteristics of participants . on average , participants were 2534 years old , in common - law relationships , had an average of 13.9 years of education , worked full or part time outside of the home , and had an annual household income in the $ 40,000$62,000 range .
analysis involving the wast and the cas instruments identified that 14.7% ( n = 174 ) women reported a true positive score for ipv exposure . for the remaining participants , 73.6% ( n = 870 ) indicated a true negative score for ipv , 10% ( n = 118 ) a false positive score , and 1.7% ( n = 20 ) a false negative score as seen in table 2
. the rate of mixed ipv exposure status ( i.e. , false positive or false negative score ) remained consistent across both groups . among the 174 participants with a true positive score for ipv exposure ,
only 1.9% ( n = 22 ) indicated that they had disclosed violence to an emergency department health care provider during their visit . among the women who disclosed ipv in the emergency department ,
the following interventions were reported most frequently as being offered to them : information regarding options to obtain help ( n = 15 ) , acknowledgement of ipv disclosure ( n = 13 ) , verbal information about ipv ( n = 13 ) , and assessment of immediate safety ( n = 13 )
. less frequently reported interventions offered to women were a discussion about the relationship situation and inquiry about the woman 's need for help ( n = 8) , a validation of the woman 's feelings ( n = 6 ) , and decision making about the type of health to obtain ( n = 1 ) .
the grounded theory phase began after the analysis of quantitative data was completed , and recruitment involved 20 participants from the quantitative subanalysis phase . early in this phase , data from one participant had to be removed when her responses raised concerns about the validity of the information , leaving a final sample of 19 participants .
the remaining 12 participants completed from one to three interviews . the data from 50 interviews identified over 100 ipv disclosure events involving emergency department nurses and health care professionals , social service professionals , police , family members , and friends .
using the wast , 16 participants scored positive ( 4 ) , and three scored negative ( < 4 ) ipv exposure ( the full range of scores from 0 to 16 points ) .
the mean score value across all participants was 8.03 . using the cas , 18 participants scored positive ( 7 ) for ipv with a mean score value of 31.9 ( the full range of scores from 0 to 150 points ) . using the subscales of the cas , 10 participants reported combined severe abuse ( mean = 4.12 ) ; 13 reported physical abuse ( mean = 5.80 ) ;
18 reported emotional abuse ( mean = 17.68 ) ; and 11 reported harassment ( mean = 4.33 ) ( figure 2 ) . for 15 participants the prevalence of emotional abuse was higher than other forms of abuse .
being found out was the basic social psychological problem among women exposed to ipv , as they feared that the health care provider would learn about the abuse during the provision of care at the emergency department .
women undertook a basic social psychological process of minimizing the risk of intrusion from health care providers when deciding whether to disclose ipv .
the process included three phases that women used to minimize their risk of intrusion from health care providers including : deciding how and when to seek emergency care , evaluating how well they trusted emergency department health care providers , and establishing a personal readiness to disclose .
consequences of this process were a self - initiated disclosure of ipv in the emergency department , perceived forced disclosure of ipv when women felt invaded , and a failure to disclose ipv because women were not ready to disclose or could not trust the health care provider . more information and detail regarding the grounded theory results can be found in catallo et al . .
mixing of the quantitative and qualitative data occurred at various points and two key mixing techniques were used in this study , embedding and connecting data . mixing began with connecting data which involves the analysis of one type leading to the need for another type of data . connecting the quantitative subanalysis data to the developing qualitative data through review of the rct subanalysis results helped to develop the first interview guide and initial coding structure .
the data obtained from the wast , cas , and clinical interactions instrument were used to inform participant selection , initial research questions , and the development of original interview guide .
for example , the wast and cas were used to identify ipv exposure and recruit potential participants from each of the following groups : true positive score , true negative score , and mixed scores ( i.e. , false positive and false negative ) .
quantitative results from all of the instruments were used to develop initial qualitative interview questions and an interview guide .
for example , results from the clinical interactions instrument indicated that a low percentage of women who were exposed to ipv disclosed in the emergency department ( i.e. , 1.9% ) , and interview questions were developed to understand why women either chose to disclose ipv or not and the nature of the interventions offered to women .
example questions from the interview guide that were developed through the integration of quantitative subanalysis data can be found in table 3 .
in addition to this data - connecting strategy , quantitative subanalysis results were embedded to support the analysis and interpretation of the qualitative data .
embedding data can involve mixing at the design level where one form of data is embedded in another form and can occur sequentially .
embedding quantitative data during qualitative analysis helped to support theoretical coding and this drove decisions about continued sampling of participants .
for example quantitative results were embedded into the grounded theory approach by analyzing categories of ipv exposure as defined by the cas .
these data were then used during theoretical coding to identify potential relationships between emerging categories and to create constructs in order to generate meaning for the developing theory .
the quantitative subanalysis results were embedded in the qualitative analysis in two ways : ( 1 ) to compare participants according to low , medium , and high scores for violence exposure and ( 2 ) to compare participants according to different types of ipv using the measures ' subscales .
participants were then grouped according to their scores : low ( 04 points wast , 029 points cas),moderate ( 59 points wast , 3059 points cas),high ( 1016 points wast , 60150 cas ) .
low ( 04 points wast , 029 points cas ) , moderate ( 59 points wast , 3059 points cas ) , high ( 1016 points wast , 60150 cas ) .
grouping participants according to severity of ipv exposure aided in qualitative analysis and interpretation of the data when identifying codes and categories .
participants with high scores were most concerned with being judged by health care providers for remaining in an abusive relationship .
we used this result to focus our qualitative analysis on this subgroup , so that we could assess unique features to enhance the understanding of the properties of the developing categories , especially those central to grounded theory such as the core phenomenon and basic social problem .
after identification of the basic social problem , participants were compared across low , moderate , and high levels of violence severity to verify existing categories and relationships or identify any new features or conditions not seen previously . in the second mixing strategy ,
cas results were used to organize qualitative participants according to the type of abuse including combined , severe abuse , physical abuse , emotional abuse , and harassment .
participants with high cas scores for emotional abuse had more difficulty identifying their relationship as abusive and seeking care for violence , unless it was for an immediate physical injury . participants who scored high for more than one type of abuse , like severe combined and emotional abuse , avoided health care as this group perceived the risks of exposure , and were too great . embedding the quantitative data during qualitative analysis permitted exploration of these relationships and categories to provide additional explanation
typically , when this type of sampling is used in grounded theory , new participants outside of the study are sought to test the full spectrum of a category 's properties and dimensions , including that of the core variable phenomenon as well as the developing relationship between categories .
our purpose was to understand violence disclosure from the perspective of the rct participants , so we did not recruit participants from outside of the trial .
we addressed maximum variation sampling by examining variants of intimate partner violence ipv disclosure events at opposing spectrums .
this was used when identifying the properties and dimensions of a concept , testing developing relationships and theoretical hypotheses .
this resulted in recruiting participants with mixed scores and negative scores on the intimate partner violence ipv screening measures .
for example , maximum variation sampling was used in this study to identify the dimensions of the category building trust with a health a care provider . disclosure events describing a participant 's ability to trust health care providers with extreme ease
studying these units of analysis allowed for further development of the category by identifying subcategories and relationships .
one example was to compare the units of analysis for participants who could immediately trust health care providers ( implicit trust ) , participants who trusted clinicians after a demonstration of trustworthiness ( distrust ) , and those participants having extreme difficulty trusting health care providers ( mistrust ) .
when more information was required , the unit of analysis was linked back to the original participant , and that participant was sought for further interviews .
for this mixed methods study , we modified the negative case sampling method used in grounded theory .
the goal of negative case sampling is to test emerging theoretical hypotheses in order to achieve a higher level of theoretical abstraction beyond a description of relationships between concepts .
this type of sampling is typically performed after the development of the grounded theory in order to identify new participants whose experiences refute or deviate from the developing theory [ 31 , 36 ] .
deviant cases offer opportunity for comparison across other categories and are often selected outside of the sample used to generate the grounded theory .
because we recruited participants only from the rct subanalysis ( phase 1 ) , we had to modify negative case sampling for use with the unit of analysis approach . instead of sampling for negative cases , disconfirming evidence related to ipv disclosure events
disclosure events were reviewed when theoretical hypotheses were generated in order to determine potential contradictory relationships that could test or challenge the developing theoretical hypotheses .
this is referred to as negative case analysis and was deliberately completed during the different stages of coding and analysis , not just after a full theory was developed .
for example , we explored ipv disclosure events where participants perceived a positive outcome after disclosure such as obtaining a referral , support , or other interventions to support their safety .
we then looked for disconfirming ipv disclosure events , such as an ipv disclosure where participants perceived a negative outcome after disclosure such as feeling intruded upon by the health care provider , feeling forced to involve police services , or being investigated by child protective services . during our negative case analysis we examined these ipv disclosure events that disconfirmed what we were seeing in the data . in this example
, we sought to recruit or offer additional interviews to participants who had perceived negative disclosure outcomes , so that we could better understand disconfirming cases and add conceptual density to our developing theory .
another example of negative case analysis occurred during the analysis of the basic social psychological process used by abused women to minimize the intrusion that they perceived in the emergency department .
the first phase of these process involved decisions about whether or not to seek care at the emergency department when ipv might be found out by the health care provider . through negative case analysis we sampled ipv disclosure events where women avoided treatment at the emergency department at all costs , even at risk of death . as a result , we theoretically sampled events where participants actively engaged in avoiding health care and the meaning of these actions .
in addition to sampling for disclosure events , we sought to recruit participants who actively avoided the emergency department for fear of ipv being found out by the health care provider .
because many diverse units of analysis were available for review , negative case analysis could be conducted .
when more information was required , the unit of analysis was linked back to the participant who underwent additional interviews to provide greater insight . however , this approach would not have been possible had there been a small sample of disclosure events or if the events from which to draw on had been very similar in nature . while negative case sampling which in its truest form tests the full theory after completion among new participants could not be performed , the principles underlying this approach were maintained .
negative case analysis using disclosure events as the unit of analysis provided the researcher with an opportunity to evaluate contradictory developments , as the grounded theory was developed and refined .
unique decisions about data collection were made in this mixed methods study . at the time of ethics approval , initial recruitment for the rct had been completed or was near completion for certain settings ( e.g. , primary care and specialty clinics ) .
recruitment among the acute care settings for the rct was still at an early stage .
this was fortuitous for the mixed methods study , as it allowed the most time for ongoing analysis and data collection for both the quantitative subanalysis and the qualitative explanatory phase .
there were occasions when participants were sought for the grounded theory phase but were no longer being followed by the rct .
when this sampling need was identified , the acute care recruitment was completed for the rct .
this created challenges for the grounded theory phase , as participants who had completed the trial were no longer interested in participating in an additional study , extending the time of recruitment .
this study provides new insights into the use of a grounded theory approach within the context of an rct .
this study provides a unique description of how a grounded theory study was used with rct data to provide greater explanation for quantitative results .
we address a current gap in nursing research by outlining explicit strategies for mixing quantitative and qualitative methods and describing how these were integrated to inform the overall study , typically an area that is overlooked when using a qualitative approach with a rct .
mixed methods research can make an important contribution to nursing where many clinical issues require a comprehensive and holistic approach which requires data from various perspectives .
a unique contribution to nursing research is this study 's use of grounded theory as an equally weighted approach alongside the rct to improve the depth and richness of results when examining a complex intervention .
more typically we identified sequential explanatory mixed methods studies with heavier weighting in the quantitative component [ 10 , 13 , 14 , 16 , 17 , 21 ] .
other research areas in nursing can benefit from this type of mixed methods design such as the evaluation of nursing interventions , exploration of patient centred care , in - depth exploration of complex phenomena , and instrument development and testing .
our study 's weighting between quantitative and qualitative phases enabled a more thorough understand of a complex phenomenon like ipv disclosure in emergency departments that would not have occurred using a rct alone .
more common is the use of qualitative types of data collection , such as open - ended questions , to help explore rct results or to understand implementation issues specific to the rct [ 7 , 11 ] .
we identified that it is possible to integrate a qualitative approach with an rct if modifications are made , in this case to the grounded theory sampling strategies of maximum variation and negative case sampling . using the unit of analysis
approach during the grounded theory phase enabled us to maintain the principles of theoretical sampling in accordance with traditional grounded theory methods and offer pragmatic adaptations that could be used to strengthen the sampling when using grounded theory in a mixed methods study . despite adaptations to the grounded theory , we still maintained methodological rigour which made it possible to combine with a rct .
because our quantitative subanalysis originated from strict principles governing a rct , namely , randomization with predetermined inclusion and exclusion criteria , participants had an equal and unbiased opportunity to be randomly allocated to either the experimental group ( i.e. , routine screening ) or the control group ( i.e. , usual care ) . this added strength to the recruitment strategy for the study , as random selection and allocation are important ways of reducing error and selection bias .
it is important to outline the key limitations associated with incorporating a grounded theory approach with a rct .
the most significant limitation of this study was beginning this mixed methods study after the overall rct was underway .
creswell and plano clark recommend deliberately planning a mixed methods study as study in and of itself .
the rct was highly complex and involved multiple health researchers , so it was difficult to estimate the types of mixed methods research questions that would arise .
it was challenging to predict the need for a mixed methods study at the outset of the rct .
the pragmatic approach , in keeping with the philosophical principles of mixed methods design , was to incorporate a mixed methods study into this developing research program .
an additional challenge that arose in incorporating a qualitative approach with a rct was the large amount of time needed to complete both phases of the study . during the grounded theory phase , it was necessary to recruit participants who had previously completed the rct .
because these participants were no longer involved , it was more difficult to reengage their interest for the qualitative component . as a result , it is uncertain what the overall impact was for having fewer participants from the group who were no longer followed by the rct on the developed grounded theory .
the following recommendations should be considered when incorporating a qualitative approach such as grounded theory with a rct as part of a mixed methods study . for this study
, we adopted a pragmatic approach to design and implement a mixed methods study after the commencement of a multisetting rct .
the mixed methods design was chosen as a result of secondary research questions that arose from preliminary trial data .
ideally mixed methods studies should be planned for during proposal development and include specific research questions driving the mixed methods study .
future research teams need to create environments where the norm is to integrate qualitative results into trials examining complex interventions .
there is a growing consensus that combining quantitative and qualitative research methods can provide a more comprehensive understanding of complex interventions , especially to address the varied information needs of policymakers , decision makers , and stakeholders [ 4244 ] .
in addition , the use of both types of data can contribute to the evaluation of the intervention including a broader understanding of its context and justification for its use in patient populations [ 6 , 42 , 44 ] . in the early stages of a mixed methods study involving a randomized trial , early planning to include
a qualitative component can help examine trial implementation such as challenges to study recruitment , attrition rates of participants , and patient preferences regarding the intervention .
planning for a mixed methods design at the outset of a study will enable consideration of the design needs including the implementation of the quantitative and qualitative components and opportunities for mixing the two data sets . while this study involved both the embedding and connection of quantitative and qualitative data as mixing strategies , future mixed methods studies should seek and incorporate a greater number of opportunities for data mixing using both rct results and qualitative results [ 5 , 42 , 43 ] . at minimum ,
data mixing needs to support the explanatory phase of the sequential mixed methods study , in our case the grounded theory phase .
while we used data mixing to support the earlier stages of the grounded theory phase ( e.g. , initial recruitment , development of an early interview guide , coding , and analysis ) an improvement would be to plan for multiple data mixing opportunities such as the early and later stages of the explanatory phase to aid in recruitment , data collection , and analysis .
for our study , in order to use more of the quantitative data in the later stages of the theory development , we would have needed additional quantitative measures beyond what was used in our quantitative subanalysis phase .
for example , an instrument that explored participant decision making regarding ipv disclosure in the emergency department setting could have produced results that would aid in conceptual density of the developing theory on minimizing intrusion from health care providers for women seeking to disclose ipv .
this suggests that those undertaking this type of study in the future need to incorporate multiple types of measures , so that the data can be better used at different stages of the qualitative phase .
this study found that the timing of both the quantitative phase and the qualitative phase was crucial in terms of recruitment , reducing attrition , and analysis . when incorporating a grounded theory approach with a rct , adequate time
should be allotted , so that the initial interviews for the grounded theory approach can be completed during the data collection phase of the trial .
because grounded theory requires simultaneous data collection , coding , and analysis , it may take longer time periods to identify the participant characteristics required for ongoing sampling . for this study
, we required 18 months and up to four interviews per participant to carry out theoretical sampling and build conceptual density of the developing theory . because the subanalysis of data had been completed earlier , it was more challenging to maintain contact with participants for the length of time needed in the grounded theory phase of the study .
those planning such a study in the future need to consider additional time needed such as extending rct recruitment time or maintaining participant engagement during periods when they were not being interviewed in order to reduce attrition . implementing a grounded theory with a rct
requires human resources support , especially when studying a highly vulnerable and transient sector of the population .
proposal planning for a mixed methods study should take into consideration the human resources need to carry out both the quantitative and qualitative components of the study .
when using grounded theory with an rct , it is necessary to have a research team with expertise in both trial and qualitative methodologies . a team with expertise in both methods will help to identify challenges to study implementation and effective strategies to address these while maintaining the overall rigour of both methods .
in conclusion we described a mixed methods study , which involved the mixing and interpretation of a quantitative and qualitative data sets in order to explain how women decided to disclose ipv in emergency departments .
we have strived to address some of the methodological gaps in the literature by providing a rationale for the mixing of methods , describing how qualitative methods were modified in order to maintain the rigour of the overall mixed methods study and outlining the methods used for qualitative data analysis and thematic development .
using grounded theory enabled us to understand results from an rct quantitative subanalysis and provided a comprehensive examination of the problems that women identify with ipv disclosure and the strategies that they use to address these problems . in order to incorporate a grounded theory approach with a rct , key modifications to sampling and data analysis
incorporating a qualitative approach with a rct can enhance the study 's overall findings and provide better explanations regarding incongruent findings .
key recommendations for use of a qualitative approach with a randomized trial include attention to timing , mixing , and the incorporation of appropriate expertise to carry out both approaches . | little is known about how to systematically integrate complex qualitative studies within the context of randomized controlled trials .
a two - phase sequential explanatory mixed methods study was conducted in canada to understand how women decide to disclose intimate partner violence in emergency department settings . mixing a rct ( with a subanalysis of data ) with a grounded theory approach required methodological modifications to maintain the overall rigour of this mixed methods study .
modifications were made to the following areas of the grounded theory approach to support the overall integrity of the mixed methods study design : recruitment of participants , maximum variation and negative case sampling , data collection , and analysis methods .
recommendations for future studies include : ( 1 ) planning at the outset to incorporate a qualitative approach with a rct and to determine logical points during the rct to integrate the qualitative component and ( 2 ) consideration for the time needed to carry out a rct and a grounded theory approach , especially to support recruitment , data collection , and analysis .
data mixing strategies should be considered during early stages of the study , so that appropriate measures can be developed and used in the rct to support initial coding structures and data analysis needs of the grounded theory phase . |
cross - sectional studies of community - residing samples revealed that 19%50% of elderly who developed dementia experienced disruptions in their sleep.13 moreover , sleep disruption appears to worsen as the disease progresses;4 it has been estimated that elderly in the later stages of dementia spend as much as 40% of their time in bed awake.5 sleep disturbance is generally defined as having trouble falling asleep and maintaining sleep at any given time .
particularly , the sleep pattern of dementia patients is often marked by increased wakefulness at night ( increases in time awake and number of awakenings ) ; reduced slow - wave sleep ( sws ) and rapid eye movement ( rem ) sleep ; and increased daytime sleepiness.68 research has shown that poor sleep quality can diminish the well - being of seniors with dementia , particularly in aspects of cognitive impairment , incidence of physical illness , and development of psychological disorders.5 moreover , the high prevalence of sleep problems can be a significant source of physical and psychological distress to caregivers,6 and thus causes negative impact on the quality of life of both caregivers and care recipients . from the public health perspective
, the common occurrence of sleep disturbance has become a pressing concern among the dementia population . in response to the negative consequences of poor sleep , studies7,1113,15 have investigated the effectiveness of various interventions to enhance sleep quality of people with dementia .
traditional sedating medications such as benzodiazepine and antihistamines are common prescriptions for dementia patients with sleep problems .
some of these medications , however , can cause detrimental side effects , including excessive daytime sleepiness and impaired motor performance.7 in addition , pharmacological treatments also give rise to the issue of polypharmacy in elderly patients with dementia , which has been shown to be associated with functional decline.8 in this respect , non - pharmacological interventions have emerged as a viable alternative for treating sleep disturbance in this group of patients .
it is a form of traditional chinese medicine ( tcm ) that essentially corrects health problems by the use of sterilized needles inserted into meridian points of the human body . according to tcm theory , pain or diseases are a result of an imbalance in the yin and yang forces in the body or of excess or deficiency of qi ( air ) , blood , or body fluids .
the mechanisms of acupuncture work by recreating the balance in the yin and yang forces and restoring qi , blood , and body fluids to normalcy through stimulation of specific meridian points that govern different organs and their interactions.9 the procedure is considered safe , provided that it is handled by well - trained therapists and that necessary safety precautions are followed.10 as summarized from various literature , the common meridian points for sleep promotion are shenmen ( ht7 ) , baihui ( du20 ) , anmian ii ( ex - hn9 ) , yintang ( ex - hn3 ) , zusanli ( st36 ) , and sanyinjiao ( sp6 ) ; the treatment duration generally spans from 3 to 6 weeks.1115 empirical evidence has shown that acupuncture produces positive outcomes in alleviating sleep disturbances in patients with hiv disease and renal diseases.11,13 the therapy is also used as intervention in the elderly for diverse aging conditions , such as agitated behavior and cognitive impairment .
one study showed that the use of acupuncture could reduce agitated behaviors exhibited by dementia patients , thus resulting in better sleep quality.14 another trial demonstrated that a 5-week acupuncture intervention improved the sleep quality of anxious adult subjects by causing an increase in melatonin secretion,16 a hormone that plays a role in regulating the sleep
studies suggest that melatonin might help restore the circadian modulation of rem sleep in people with rem sleep behavior disorder , and thereby normalize rem sleep.17,18 it has been shown that melatonin improved the rem sleep quality of patients with schizophrenia , while hormone levels can be increased by acupuncture treatment.19 deficiency of melatonin is associated with insomnia in elderly people.20 the positive evidence for the therapeutic value of acupuncture warranted further investigation . the present research aimed to develop an acupuncture program for enhancing sleep quality in seniors with dementia and to evaluate its effectiveness in reducing sleep disturbances by adopting a within - subjects design .
it was expected that subjects would exhibit less sleep disturbances and show improvements in sleep quality after the acupuncture intervention .
twenty - two elders ( aged between 5590 years old ) diagnosed with dementia who had sleep disturbances were recruited by convenience sampling from elderly community centers , dementia - related health talks , and the memory clinic of a dementia service center .
inclusion criteria included ( 1 ) clinical diagnosis of dementia , ( 2 ) subjective complaints of sleep problems , and ( 3 ) stable drug regime or no new drug intake in the 3 months prior to the study .
exclusion criteria were skin problems around the application area , psychiatric disorder , or aggressive behavior . informed consent to participate was obtained from family caregivers . the treatment procedure was explained orally to the subjects .
three subjects were unable to complete the treatment due to illness unrelated to their sleep problems ; 19 subjects completed the treatment ( completion rate being 86% ) .
the study took place between march 2011 and august 2012 using a within - subjects design .
subjects were assigned to batches according to the dates they enrolled in the study , so that each batch consisted of three to five elders .
each batch of subjects went through a 6-week wait - list control period and then a 6-week treatment period , one batch followed by another .
assessments on sleep quality and cognitive functioning were carried out immediately before and after control and treatment periods . during the treatment period , one - on - one acupuncture treatment
the treatment took place at a dementia service center and was carried out by a registered chinese medicine practitioner with considerable clinical experience in acupuncture .
frequency of treatment was twice a week for 6 weeks ( a total of 12 sessions was offered to each subject ) , similar to that in a previous trial.16 each treatment session lasted approximately 30 minutes .
treatment was applied on six meridian points , namely , shenmen ( ht7 ) , baihui ( du20 ) , anmian ii ( ex - hn9 ) , yintang ( ex - hn3 ) , zusanli ( st36 ) , and sanyinjiao ( sp6 ) . for better experimental control , acupuncture
was only applied to these six meridian points for each subject throughout all treatment sessions .
sleep disturbance was measured by part k ( sleep and nighttime behavior disorders ) of the chinese version of the neuropsychiatric inventory ( npi).21,22 caregivers reported the presence of nighttime behaviors of the elders on eight items ( eg , getting up during the night , dressing and undressing repeatedly ) and rated the overall sleep problem in terms of frequency ( from 1 = occasionally or less than once a week to 4 = very frequently , once or more per day , or continuously ) and severity ( 1 = mild , 2 = moderate , 3 = severe ) . a domain score was formed by multiplying the frequency and severity scores .
sleep parameters were measured by wrist actigraphy ( actiwatch 2 , respironics , inc . , murrysville , pa , usa ) , which was a sufficiently sensitive method by which to detect the effects of intervention targeting insomnia in elderly subjects.23,24 the device detected the wrist movement of the wearer and recorded data for a number of sleep parameters . resting time was the period of time ( in minutes ) between the subject going to bed at night and rising from bed in the morning .
this information was recorded by the caregivers by pressing a button on the actiwatch at the beginning and end of the period . total sleep time
( movement of the subject as detected by actiwatch ) of individual subject within the resting time .
onset latency was the period of time ( in minutes ) between the start of resting time and sleep time .
( waso ) was the amount of waking minutes within total sleep time . awake
sleep statistics were recorded for three consecutive nights at each assessment time point for more reliable measurement.25 measures of each sleep parameter were yielded by taking the average of each sleep statistic across three nights .
cognitive function was measured by the chinese version of the alzheimer s disease assessment scale cognitive ( adas - cog).25,26 the adas - cog is a widely adopted pen - and - paper test designed for the evaluation of cognitive deficits in persons with dementia .
subscales of the adas - cog examined were list - learning , delayed recall , naming , ideational apraxia , construction , and recognition .
the test was administered by trained research assistants , and had a maximum possible score of 70 and minimum of 0 , with increasing scores indicating more severe cognitive impairment .
statistical analyses were carried out using spss software ( version 20 ; ibm corporation , armonk , ny , usa ) .
on the first level , differences between pretests and posttests in treatment and control periods were compared using the wilcoxon signed - rank test . on the second ,
thus , changes between pretest and posttest scores were first calculated then compared between the treatment and control periods using the wilcoxon signed - rank test .
twenty - two elders ( aged between 5590 years old ) diagnosed with dementia who had sleep disturbances were recruited by convenience sampling from elderly community centers , dementia - related health talks , and the memory clinic of a dementia service center .
inclusion criteria included ( 1 ) clinical diagnosis of dementia , ( 2 ) subjective complaints of sleep problems , and ( 3 ) stable drug regime or no new drug intake in the 3 months prior to the study .
exclusion criteria were skin problems around the application area , psychiatric disorder , or aggressive behavior . informed consent to participate was obtained from family caregivers . the treatment procedure was explained orally to the subjects .
three subjects were unable to complete the treatment due to illness unrelated to their sleep problems ; 19 subjects completed the treatment ( completion rate being 86% ) .
the study took place between march 2011 and august 2012 using a within - subjects design .
subjects were assigned to batches according to the dates they enrolled in the study , so that each batch consisted of three to five elders .
each batch of subjects went through a 6-week wait - list control period and then a 6-week treatment period , one batch followed by another .
assessments on sleep quality and cognitive functioning were carried out immediately before and after control and treatment periods . during the treatment period ,
the treatment took place at a dementia service center and was carried out by a registered chinese medicine practitioner with considerable clinical experience in acupuncture .
frequency of treatment was twice a week for 6 weeks ( a total of 12 sessions was offered to each subject ) , similar to that in a previous trial.16 each treatment session lasted approximately 30 minutes .
treatment was applied on six meridian points , namely , shenmen ( ht7 ) , baihui ( du20 ) , anmian ii ( ex - hn9 ) , yintang ( ex - hn3 ) , zusanli ( st36 ) , and sanyinjiao ( sp6 ) . for better experimental control ,
acupuncture was only applied to these six meridian points for each subject throughout all treatment sessions .
sleep disturbance was measured by part k ( sleep and nighttime behavior disorders ) of the chinese version of the neuropsychiatric inventory ( npi).21,22 caregivers reported the presence of nighttime behaviors of the elders on eight items ( eg , getting up during the night , dressing and undressing repeatedly ) and rated the overall sleep problem in terms of frequency ( from 1 = occasionally or less than once a week to 4 = very frequently , once or more per day , or continuously ) and severity ( 1 = mild , 2 = moderate , 3 = severe ) .
sleep parameters were measured by wrist actigraphy ( actiwatch 2 , respironics , inc . , murrysville , pa , usa ) , which was a sufficiently sensitive method by which to detect the effects of intervention targeting insomnia in elderly subjects.23,24 the device detected the wrist movement of the wearer and recorded data for a number of sleep parameters . resting time was the period of time ( in minutes ) between the subject going to bed at night and rising from bed in the morning .
this information was recorded by the caregivers by pressing a button on the actiwatch at the beginning and end of the period .
total sleep time was the total time ( in minutes ) of stage of falling asleep
( movement of the subject as detected by actiwatch ) of individual subject within the resting time .
onset latency was the period of time ( in minutes ) between the start of resting time and sleep time .
sleep efficiency was the percentage of total sleep time over resting time .
( waso ) was the amount of waking minutes within total sleep time . awake number was the number of continuous blocks of wake within total sleep time .
sleep statistics were recorded for three consecutive nights at each assessment time point for more reliable measurement.25 measures of each sleep parameter were yielded by taking the average of each sleep statistic across three nights .
cognitive function was measured by the chinese version of the alzheimer s disease assessment scale cognitive ( adas - cog).25,26 the adas - cog is a widely adopted pen - and - paper test designed for the evaluation of cognitive deficits in persons with dementia .
subscales of the adas - cog examined were list - learning , delayed recall , naming , ideational apraxia , construction , and recognition .
the test was administered by trained research assistants , and had a maximum possible score of 70 and minimum of 0 , with increasing scores indicating more severe cognitive impairment .
statistical analyses were carried out using spss software ( version 20 ; ibm corporation , armonk , ny , usa ) .
on the first level , differences between pretests and posttests in treatment and control periods were compared using the wilcoxon signed - rank test . on the second ,
thus , changes between pretest and posttest scores were first calculated then compared between the treatment and control periods using the wilcoxon signed - rank test .
the majority of subjects were aged between 75 to 84 years ( 63.2% ) at baseline .
most subjects were female ( 73.7% ) ; more than half were widowed ( 57.9% ) and 42.1% were married .
most subjects had graduated from primary school ( 36.8% ) and secondary school or above ( 31.6% ) .
all but one subject ( 94.7% ) was cared for by family members or a domestic helper .
according to the classification of diagnostic and statistical manual of mental disorders , fourth edition , text revision ( dsm - iv - tr),27 five of the subjects ( 26.3% ) had dementia of the alzheimer s type ; five ( 26.3% ) , dementia due to other general medical conditions ; three ( 15.8% ) , dementia due to multiple etiologies ; one ( 5.3% ) , vascular dementia ; and one ( 5.3% ) , dementia due to parkinson s disease ( 5.3% ) , while four subjects did not provide their clinical diagnosis on dementia .
four subjects ( 21.1% ) were under a stable prescription of drugs related to sleep regulation . at baseline , the subjects had a mean resting time of 521.04 minutes ( standard deviation [ sd ] 81.23 ) and sleeping time of 390.73 minutes ( sd 121.21 ) at night ; average sleep efficiency was 69.9% ( sd 18.4 ) .
mean adas - cog score was 27.28 ( sd 10.93 ) , and mean npi - part k domain score was 3.92 ( sd 3.65 ) .
frequency of sleep disturbances and nighttime behaviors reported by caregivers are summarized in table 1 .
after recruitment , there were 19 of 22 ( 86% success rate ) subjects who completed the intervention .
the reasons for withdrawal were intercurrent illness , perceived lack of response , and fear of needles . throughout the intervention , all but one subject attended and completed all sessions on schedule .
one subject did not attend the last session due to illness not related to the intervention .
wilcoxon signed - rank test showed that , during the treatment period , increase in total sleep time and resting time was statistically significant ( z = 2.13 , p < 0.05 and z = 1.97 , p < 0.05 , respectively )
. changes in other sleep and cognitive parameters ( sleep efficiency , onset latency , waso , awake number , and adas - cog ) during the treatment period were not statistically significant . as for control period
, no significant difference was observed between pretests and posttests in any of the sleep and cognitive parameters .
results of changes of sleep parameters and cognitive ability of the treatment period against the control period indicated that gain in resting time of the subjects during treatment period ( m = 42.05 , sd = 86.61 ) was significantly greater than that during the control period ( m = 45.21 , sd = 98.10 ;
similarly , gain in total sleep time of the subjects during the treatment period ( m = 42.02 , sd = 80.85 ) was significantly greater than during the control period ( m = 32.95 , sd = 96.61 ; z = 2.21 , p < 0.05 ; see figure 2 ) .
gain in sleep efficiency during the treatment period ( m = 2.60 , sd = 13.58 ) was also greater than that during the control period ( m = 2.27 , sd = 10.89 ) , although the difference was nonsignificant ( z = 1.57 , p = 0.12 ) .
no significant differences were observed in changes in adas - cog score and other sleep parameters ( onset latency , waso , and awake number ) between the treatment and control periods ( table 2 ) .
the majority of subjects were aged between 75 to 84 years ( 63.2% ) at baseline .
most subjects were female ( 73.7% ) ; more than half were widowed ( 57.9% ) and 42.1% were married .
most subjects had graduated from primary school ( 36.8% ) and secondary school or above ( 31.6% ) .
all but one subject ( 94.7% ) was cared for by family members or a domestic helper .
according to the classification of diagnostic and statistical manual of mental disorders , fourth edition , text revision ( dsm - iv - tr),27 five of the subjects ( 26.3% ) had dementia of the alzheimer s type ; five ( 26.3% ) , dementia due to other general medical conditions ; three ( 15.8% ) , dementia due to multiple etiologies ; one ( 5.3% ) , vascular dementia ; and one ( 5.3% ) , dementia due to parkinson s disease ( 5.3% ) , while four subjects did not provide their clinical diagnosis on dementia .
four subjects ( 21.1% ) were under a stable prescription of drugs related to sleep regulation . at baseline , the subjects had a mean resting time of 521.04 minutes ( standard deviation [ sd ] 81.23 ) and sleeping time of 390.73 minutes ( sd 121.21 ) at night ; average sleep efficiency was 69.9% ( sd 18.4 ) .
mean adas - cog score was 27.28 ( sd 10.93 ) , and mean npi - part k domain score was 3.92 ( sd 3.65 ) .
frequency of sleep disturbances and nighttime behaviors reported by caregivers are summarized in table 1 .
, there were 19 of 22 ( 86% success rate ) subjects who completed the intervention .
the reasons for withdrawal were intercurrent illness , perceived lack of response , and fear of needles . throughout the intervention , all but one subject attended and completed all sessions on schedule .
one subject did not attend the last session due to illness not related to the intervention .
wilcoxon signed - rank test showed that , during the treatment period , increase in total sleep time and resting time was statistically significant ( z = 2.13 , p < 0.05 and z = 1.97 , p < 0.05 , respectively ) . changes in other sleep and cognitive parameters ( sleep efficiency , onset latency , waso , awake number , and adas - cog ) during the treatment period were not statistically significant .
as for control period , no significant difference was observed between pretests and posttests in any of the sleep and cognitive parameters .
results of changes of sleep parameters and cognitive ability of the treatment period against the control period indicated that gain in resting time of the subjects during treatment period ( m = 42.05 , sd = 86.61 ) was significantly greater than that during the control period ( m = 45.21 , sd = 98.10 ; z = 2.05 , p < 0.05 ; see figure 1 ) . similarly ,
gain in total sleep time of the subjects during the treatment period ( m = 42.02 , sd = 80.85 ) was significantly greater than during the control period ( m = 32.95 , sd = 96.61 ; z = 2.21 , p < 0.05 ; see figure 2 ) .
gain in sleep efficiency during the treatment period ( m = 2.60 , sd = 13.58 ) was also greater than that during the control period ( m = 2.27 , sd = 10.89 ) , although the difference was nonsignificant ( z = 1.57 , p = 0.12 ) .
no significant differences were observed in changes in adas - cog score and other sleep parameters ( onset latency , waso , and awake number ) between the treatment and control periods ( table 2 ) .
using a within - subjects design , the present study investigated the effectiveness of acupuncture on treating sleep disturbance of elderly with dementia .
results show that the intervention elicited significant improvement in sleep quality of elders with dementia in terms of significant gain in resting time as well as sleep time in the treatment period over the control period .
the acupuncture treatment resulted in significant improvement in total sleep time ( time in bed that subjects actually spent sleeping ) .
this result is consistent with the finding of a previous study that acupuncture treatment led to increased release of melatonin,16 which functioned to regulate the sleep - wake cycle .
interestingly , increase in sleep time in our subjects did not result in significantly better sleep efficiency . as sleep efficiency
was calculated as the percentage of sleep time over resting time , the absence of significant findings could be due to the fact that resting time was also lengthened hence balanced out
two meridian points ( baihui , du20 ; yintang , ex - hn3 ) were included in the intervention protocol specifically for relaxation and calmness . in general
, subjects felt more relaxed during the intervention ; this might have resulted in an increase in resting time .
in addition , since the caregivers were not blinded to the acupuncture treatment , the caregivers might have adjusted the daily life of the subjects , eg , by arranging a longer resting time for the sake of the subjects .
while it is implausible to blind the caregivers or subjects to acupuncture treatment , future trials might include a sham acupuncture group or sham acupuncture period to rule out this confounding factor .
measures such as onset latency , waso , and awake number did not show significant changes compared to control period .
this could be due to the relatively less intense treatment schedule compared to previous studies .
for instance , a trial conducted on institutionalized residents in taiwan provided acupressure treatment on five consecutive days per week for 3 weeks and found significant improvements in latency and sleep efficiency , in addition to sleep time.28 in the present study , almost all the subjects ( 95% ) were community residing ; to implement such an intensive treatment schedule would be impractical and create a heavy burden on the caregivers , as they needed to accompany the elders in travel to and from the service center .
future studies are needed to further investigate the optimal duration and intensity of an acupuncture treatment schedule .
the figures related to recruitment and intervention progress refect that the subjects accepted the intervention and could manage to tolerate it . only one subject in the study dropped out of the intervention because of fear of acupuncture .
a study on acceptability of acupuncture on low back pain concluded that discomfort towards the intervention could be mediated by a good therapeutic relationship and clear explanations of the anticipated treatment outcome.29 the american medical association also suggests that information such as treatment options , expected outcomes , and relative risks should be given to the patients before treatment in a culturally and linguistically appropriate manner.30 a recent cochrane review highlighted the small number of rigorous efficacy studies currently available in the literature on the effectiveness of acupuncture in treating sleep disturbance.10 due to various limitations , few trials with rigorous research design on dementia patients have been conducted .
for instance , a previous trial that found melatonin increase following acupuncture treatment did not include a control group.20 by using a within - subjects design with control and treatment conditions , the subjects in this study served as their own control .
we were therefore able to determine the effects of acupuncture on sleep quality while reducing undesirable variance due to individual differences , which might be a potential shortcoming in other between - subjects trials with a relatively small sample size .
first , there was no significant difference observed on the outcome measures across demographic characteristics such as dementia type or drug intake ; given the small sample size , the present study might lack sufficient statistical power to detect significant findings .
second , although this study compared subjects sleep quality during the control period versus the treatment period , it did not introduce a sham acupuncture period or contain a sham control group , hence the possibility of placebo effect could not be ruled out .
third , there lacked posttreatment follow - up assessment , thus the long - term effect of the acupuncture intervention was not investigated in the present study .
a recent cochrane review highlighted the small number of rigorous efficacy studies currently available in the literature on the effectiveness of acupuncture in treating sleep disturbance.10 due to various limitations , few trials with rigorous research design on dementia patients have been conducted .
for instance , a previous trial that found melatonin increase following acupuncture treatment did not include a control group.20 by using a within - subjects design with control and treatment conditions , the subjects in this study served as their own control .
we were therefore able to determine the effects of acupuncture on sleep quality while reducing undesirable variance due to individual differences , which might be a potential shortcoming in other between - subjects trials with a relatively small sample size .
first , there was no significant difference observed on the outcome measures across demographic characteristics such as dementia type or drug intake ; given the small sample size , the present study might lack sufficient statistical power to detect significant findings .
second , although this study compared subjects sleep quality during the control period versus the treatment period , it did not introduce a sham acupuncture period or contain a sham control group , hence the possibility of placebo effect could not be ruled out .
third , there lacked posttreatment follow - up assessment , thus the long - term effect of the acupuncture intervention was not investigated in the present study .
using a within - subjects design , this pilot study provided preliminary but promising evidence for the favorable effect of acupuncture on the sleep quality of elderly with dementia .
taking into consideration the strengths and limitations of this pilot study , acupuncture is suggested to be a viable alternative to traditional sleep mediation for treating sleep disturbances in this group of patients , while further studies are suggested to investigate the effects of various intensities in treatment schedule and the lasting effect of acupuncture intervention with a larger sample size and by including follow - up assessments . a proper sham acupuncture control group or sham acupuncture period ( if not a randomized , controlled trial ) | purposeelderly with dementia are often afflicted with sleep problems .
recent studies have suggested that acupuncture may be a feasible alternative to traditional sleep medicine for treating sleep disturbance .
this study investigated the effectiveness of acupuncture on sleep quality of elderly with dementia.patients and methodsnineteen elders with dementia were followed through a control period and an acupuncture treatment period , each lasting 6 weeks .
outcome measures were subjects sleep quality and cognitive function .
sleep parameters were recorded by wrist actigraphy .
cognitive function was assessed by the alzheimer s disease assessment scale cognitive ( adas - cog ) .
pretests and posttests were conducted immediately before and after the control and treatment periods .
changes in the outcome measures between control and treatment periods were compared.resultswilcoxon signed rank tests revealed that the subjects gained significantly more resting time and total sleep time in the treatment period than in the control period ( p < 0.05 ) . a nonsignificant trend for improvement in sleep efficiency was observed .
improvement in cognitive function was not statistically significant .
a total of 86% of the subjects completed the treatment regime.conclusionresults reveal that acupuncture was effective in improving some domains of sleep quality of elderly with dementia , and the subjects showed acceptance towards the intervention .
strengths and limitations of the present study as well as suggestions for further studies were considered . |
ventriculostomy catheters , also known as external ventricular drains ( evds ) , are frequently used in neurosurgery to monitor and relief intracranial pressure .
complications arising from evds include hemorrhage , misplacement , dislodgement , blockage , and , most significantly , infection , which may be complicated by ventriculitis , meningitis , brain abscess , or subdural empyema .
evd - related infections significantly prolong hospital stay , increase costs , and often negatively affect the overall prognosis .
reported rates of evd - related infections range from < 5% up to 23% , most commonly close to 10% .
an increased risk of infection has been observed in patients with subarachnoid or intraventricular hemorrhage , in patients with concurrent systemic infections as well as with longer duration of catheterization , cerebrospinal ( csf ) leakage , and frequent manipulation of the evd system [ 24 ] .
in addition we hypothesized that multibed accommodation of patients with evd in a mixed surgical intensive care setting may constitute a risk factor for evd - related infections .
the objective of the present study was to assess the incidence and outcome of evd - related infections and to identify new and confirm already known risk factors .
for this single - center retrospective study we included all patients over 18 years of age that underwent evd placement during a 30-month period ( january 2010 to june 2012 ) at our 1.500 bed tertiary center ( jena university hospital , germany ) . for drainage conventional ( promedics ) and silver - impregnated catheter ( ventriguard ) were used .
all catheters were inserted under sterile conditions in the operating theatre using a tunneled procedure technique and a closed system for drainage .
there was no policy of routine csf sampling or replacement of catheters during the whole study period .
the third - generation cephalosporin ceftriaxone was given at 2 g once daily to all patients from insertion until evd removal .
patients that were not immediately transferred to the icu after evd insertion ( n = 5 ) , patients presenting with open skull fracture and csf leakage ( n = 4 ) , and patients with active infection of the central nervous system ( cns ) at first evd implantation ( n = 18 ) were excluded from the study .
the 26-bed icu is a mixed icu treating patients of all surgical disciplines in four single - bed rooms , seven two - bed , and two four - bed rooms .
the charts of all patients were retrospectively reviewed and demographics , asa - score ( physical status classification of preoperative patients for anaesthetic risk assessment from the american society of anaesthesiologists ) , evd - related data , type of accommodation , and underlying or arising healthcare - associated infections during ventricular drainage were documented .
we defined evd - related infection as ( 1 ) positive csf culture result plus clinical symptoms or cns pleocytosis / cell count increase , or ( 2 ) in the case of negative csf culture , clinical symptoms , and cns pleocytosis / cell count increase [ 5 , 6 ] .
continuous values were expressed as median ( range ) and compared using the mann - whitney u test .
categorical data were presented as frequencies and percentages and compared using the chi - square test or fisher 's exact test where appropriate .
cox regression analysis and the kaplan - maier method were used to determine predictors of evd - related infection . on the basis of poisson distribution and wilson - score method ,
external drainage was continued in median for 7 days ( range 144 ) resulting in 1.725 catheter days ( table 1 ) .
the first and second evd remained each in place for a median of 6 days ( 1st : range 119 days , 2nd : range 117 days ) , the third evd for 12 days ( range 916 ) .
one patient required a fourth and fifth evd which remained in situ for 11 and 12 days , respectively .
indications for extraventricular drainage were nontraumatic subarachnoid and/or intraventricular hemorrhage in 133 ( 61% ) patients , traumatic brain injury with subarachnoid and/or intraventricular hemorrhage in 38 ( 17% ) patients , intracranial tumors in 25 ( 12% ) patients ( n = 8 benign ; n = 17 malignant ) , and others in 22 ( 10% ) patients ( n = 16 contusion / edema ; n = 6 hydrocephalus ) .
eighteen patients developed an evd - related infection resulting in a cumulative incidence of 8.3% ( 95% ci , 5.312.7 ) .
all patients experienced only one episode of infection . in 14 patients the first evd was infected , in four patients the second one .
six patients with an infection of the first evd received a second evd , one of those a third , and one patient five evds in total .
reasons for a change were the underlying infection and/or clogging with still necessity of ventricular drainage .
thirteen patients without an evd - related infection received a second evd ; the reasons were displacement or clogging .
the device - associated infection rate was 10.4 per 1.000 ( 95% ci , 6.216.5 ) evd days considering the total time of evds in place ( 1.725 days ) and 11.8 per 1000 ( 95% ci , 6.918.6 ) evd days considering catheter days without a previous evd - related infection ( 1.572 days ) . on average an evd - related infection became in mean
seven evd - related infections were diagnosed within three days after evd placement ( figure 1 ) . in 88 evd procedures a conventional catheter was used and in 122 procedures a silver - impregnated catheter was inserted . in 26 procedures the type of the used catheter was not documented .
no significant difference of evd - related infection rate was found between patients with conventional evd or silver - impregnated evd ( 2% versus 9% ; p = 0.08 ) . of 18 evd infections , 16 ( 89% )
the pathogens most commonly identified were coagulase - negative staphylococcus ( cns ) ( 62% ) followed by enterococcus spp .
( 19% ) and other pathogens ( 19% ) including klebsiella pneumoniae , escherichia coli , and micrococcus luteus ( table 2 ) .
no association of evd - related infections with demographical parameters , indication for evd placement , and the type of accommodation could be detected ( table 3 ) .
furthermore , there was no difference in mortality ( single - bed 14% versus multibed 18% , p = 1.0 ) or in the occurrence of healthcare - associated infections other than evd - related infection between patients accommodated in single - bed rooms and multiple - bed rooms , respectively ( 21% versus 25% , p = 1.0 ) .
concomitant healthcare - associated infections ( hais ) were found significantly more often in patients with evd - related infections ( 44% versus 23% , p < 0.01 ) , with a trend towards more surgical site infections ( ssi ) , urinary tract infections ( uti ) , and central line - associated bloodstream infections ( clabsi ) in patients with evd - related infections . no coherence between the organisms responsible for hais and those responsible for the evd - related infection was found .
patients with an evd - related infection had a significantly longer icu ( 11 versus 21 days , p < 0.01 ) and hospital stay ( 20 versus 28.5 days , p < 0.01 ) than patients without .
median total duration of external drainage was twice as long in patients with evd - related infection ( 6 versus 12 days , p < 0.01 ) .
however , there was no significant difference in the duration between first evd placement and the occurrence of evd - related infection , respectively , evd removal in patients without evd - related infection ( 6 versus 7 days , p = 0.87 ) .
when controlled for the duration of the infection - free evd drainage as a continuous variable , the occurrence of any hai remained a statistically significant risk factor for the development of an evd - related infection in a multivariate logistic regression model ( univariate or 4.3 , 95% ci 1.611.6 ; adjusted or 7.1 , 95% ci 2.222.9 ) , whereas the duration of drainage until infection or removal was not .
cox regression analysis did not identify a significant association between any further studied parameter and evd - related infection .
a change of an evd before infection or removal of the evd was also not associated with an increased risk for a subsequent evd - related infection ( p = 0,47 ) .
evd - related infection was not associated with increased in - hospital mortality ( 17% versus 18% ; p = 1.0 ) the only parameter associated with adverse outcome was asa - score classification with an odds ratio of 2.7 per 1-class increase ( p < 0.01 ) .
the cumulative incidence of evd - related infection in our study was 8.3% and hence comparable with previous published studies [ 13 ] .
the device - associated infection rate observed in this study ( 10.4 per 1000 evd - days ) is higher than the rate reported by scheithauer et al .
( 6.3 per 1000 device days ) , which has been the only device - associated infection rate reported until now .
noticeably , the mean duration of drainage time in patients without evd - related infection in that study was considerably longer compared to our cohort ( 11 versus 8 days ) .
the majority of published series reported a statistically significant higher incidence of evd - related infections in patients with subarachnoid and/or intraventricular hemorrhage when compared with patients with nonhemorrhagic pathologies [ 1 , 4 ] . according to two previous studies [ 6 , 7 ]
we however could not confirm subarachnoid and/or intraventricular hemorrhage as an independent risk factor for evd - related infection in logistic regression analysis .
several studies examined the relationship between concurrent systemic infections and evd - related infection , showing that concurrent systemic infections are a risk factor for evd - related infection .
these results could not be confirmed in our study , in which the presence of a healthcare - associated infection during external ventricular drainage was not a risk factor for evd - related infection .
however , patients with an evd - associated infection had significantly more often concurrent healthcare - associated infections , which might be a consequence of the prolonged length of stay in the intensive care unit .
as in previous studies there was no coherence between the organism responsible for the healthcare - associated infection and the organism responsible for the evd - related infection in the individual patient .
multiple studies examined the duration of catheterization as a risk factor for evd - related infection .
interpretation of these studies is complicated because some investigators used cumulative infection rates , either uncorrected or censored by the use of life - table analysis , while others used daily infection rates . even though there is some controversy regarding the actual daily infection rate , lozier et al .
could show that the hazard rate varies over time , suggesting daily changing infection risks .
although the median total duration of external drainage was twice as long in patients with evd - related infection than in patients without infection , there was no difference between insertion of the first evd and occurrence of evd - related infection or evd removal in patients without evd - related infection .
this underlines the fact that prolongation of total drainage time in patients with evd infection is a result of evd infection and not vice versa . in line with these findings
seven evd - related infections were diagnosed within three days after placement of the infected evd .
four of the seven evd - related early infections were caused by coagulase - negative staphylococci ( cns ) which may have arisen from initial inoculations , which develop in detectable infections after variable incubation periods of around five days as previously suggested .
this emphasizes the necessity of evd placement under fastidious aseptic conditions . in accordance with previous studies ,
coagulase - negative staphylococci were the bacteria most commonly isolated in patients with evd - related infections accounting for 62% of cases .
other common organisms include enterococcus spp . , enterobacter spp . , and staphylococcus aureus .
this pattern coincides with that of the usual skin flora and hospital environment . concerning our additional research question , whether multibed accommodation of patients in the icu setting poses a risk factor for evd - related infections we were not able to show any difference in the infection rate between patients who were placed in single- or multibed rooms , respectively .
the current notion is that evd - related infections result from either inoculation of pathogens during evd placement and/or contamination and colonization of the evd system during the postoperative period .
postoperative colonization can either arise from endogenous organisms present on the skin , which spread along the intracutaneous tract or by exogenous organisms introduced into the evd system during manipulation at the evd system by healthcare workers .
endogenous infections might be prevented by using antimicrobial coated evd catheters which may decrease bacterial colonization and thus prevent infection .
performed a meta - analysis to assess the efficacy of antimicrobial - impregnated catheters in preventing catheter - related infections during external ventricular drainage . compared with standard catheters , a significantly lower rate of csf infection
was noticed for clindamycin / rifampin - impregnated catheters ( or 0.27 , 95% ci , 0.100.73 , p < 0.05 ) and for minocycline / rifampin - impregnated catheters ( or 0.11 , 95% ci , 0.060.21 , p < 0.05 ) .
no statistical significance was found when standard catheters were compared with silver - impregnated catheters ( or 0.33 , 95% ci = 0.07 to 1.69 , p = 0.18 ) . in the meantime
however several additional studies were published which assessed the efficacy of silver - impregnated catheters .
winkler et al . compared in their prospective randomized trial the rate of evd - related infections of 61 evd placements with either antibiotic - coated ( n = 32 ) or silver - bearing catheters ( n = 29 ) in 40 patients .
regarding csf infection rate and dysfunction , no statistical significant differences between the two evd catheters bactiseal versus ventriguard were found .
the rate of infections by catheter type was 7.6% ( 11/145 ) and 13.8% ( 4/29 ) for two different types of noncoated polyurethane catheters .
silver - impregnated polyurethane catheters became infected in 6.1% ( 14 out of 228 ) .
there was a significant difference in infection risk between the two study arms : 21.4% ( 30/140 ) for plain catheters versus 12.3% ( 17/138 ) for silver catheters ( p = 0.04 ) .
in contrast to these studies , however , we could show a trend towards a higher infection rate in patients with a silver - coated evd catheter when compared to patients with nonimpregnated catheters ( 9% versus 2.3% ; p = 0.08 ) .
selection bias can be excluded as the catheters were not available in our institution at the same time .
because of the retrospective nature of the present study , various limitations must be mentioned .
furthermore our findings might be limited due to the number of patients with evd - related infection which was smaller than infected and the small number of patients who were accommodated in single - bed rooms .
many studies have been conducted to identify risk factors of evd - related infections . however , none of these risk factors could be confirmed in our cohort of patients .
furthermore we could not show any difference in infection rates between patients who were placed in single- or multibed rooms , respectively . | external ventricular drainage ( evd ) is frequently used in neurosurgery to drain cerebrospinal fluid in patients with raised intracranial pressure .
we performed a retrospective single center study in order to evaluate the incidence of evd - related infections and to identify underlying risk factors .
246 evds were placed in 218 patients over a 30-month period .
evd was continued in median for 7 days ( range 144 ) .
the cumulative incidence of evd - related infections was 8.3% ( 95% ci , 5.312.7 ) with a device - associated infection rate of 10.4 per 1000 drainage days ( 95% ci , 6.216.5 ) .
the pathogens most commonly identified were coagulase - negative staphylococcus ( 62% ) followed by enterococcus spp .
( 19% ) .
patients with an evd - related infection had a significantly longer icu ( 11 versus 21 days , p < 0.01 ) and hospital stay ( 20 versus 28.5 days , p < 0.01 ) than patients without .
median total duration of external drainage was twice as long in patients with evd - related infection ( 6 versus 12 days , p < 0.01 ) .
however , there was no significant difference in the duration between first evd placement and the occurrence of evd - related infection and evd removal in patients without evd - related infection ( 6 versus 7 days , p = 0.87 ) , respectively .
interestingly no risk factor for evd - related infection could be identified in our cohort of patients . |
malignant peripheral nerve sheath tumors ( mpnsts ) are very rare high - grade sarcomas with a poor prognosis at childhood and adolescence , and only 10%20% of mpnsts are seen in these periods.14 mpnsts often present with pain associated with mass lesions or neuronal involvement in the head neck , abdomen , chest wall , and other anatomic regions , but extremity involvement is uncommon , especially in children or adolescents.3,5,6 the most important risk factor for mpnst development is the presence of neurofibromatosis type 1 ( nf1 ) , and half of the mpnst cases occur in patients with nf1 history.2,7,8 mpnsts may be confused with neurofibromas and/or plexiform neurofibromas in patients with nf1.7,9,10 we present a case of giant mpnst located at the right thigh of a patient with nf1 taking into account the available literature . in our study , electronic searches for reports of similar cases and other rare manifestations using the keywords neurofibroma , neural sheath tumors ,
soft tissue sarcomas in children with neurofibromatosis , neurofibroma and adolescents , and neurofibromatosis and malignancy were conducted in google , pubmed , springerlink , embase , elsevier , and ovid databases for all english articles with abstracts .
only 30 reports focusing on neurofibroma and mpnsts in the patient with or without neurofibromatosis or those that provided important theoretical contributions are referenced in the current work .
a 15-year - old male presented to our clinic with a swelling and light pain at the right thigh for a year . on physical examination , a mass with indeterminate borders was palpated at the anteromedial area of the right thigh .
there were typical nf1 skin findings such as caf au lait spots and peripheral neurofibromas all over the body ( figure 1 ) .
magnetic resonance imaging ( mri ) revealed a 139 cm mass at the anteromedial thigh with posterior extension , which was isointense in t1 weighted ( t1w ) images , heterogeneously iso- to hyperintense in t2 weighted ( t2w ) images , and diffusely contrast enhancing except the central cystic areas in the postcontrast images ( figure 2 ) . with a suspicion of malignancy ,
histopathological evaluation led to an initial diagnosis of benign neurofibroma , and total surgical excision was planned .
ethical approval for this study was not taken , but we informed both the patient and the patient s parents about this study and obtained written informed consent .
the mass was excised under general anesthesia . during surgery , the mass was observed to have a hard , fixed , and large capsule .
histopathological evaluation revealed a smooth - surfaced , grayish - brown 1399 cm solid mass weighing 610 g , which appears nodular and heterogeneous ( figure 3 ) . a malign mesenchymal tumor involving diffuse necrotic areas was observed .
there were occasional atypical mesenchymal cells with clear cytoplasms and vesicular nuclei , as well as diffuse pleomorphic multinucleated giant cells .
diffuse atypical mitosis ( 42 mitoses per 10 hpf [ high - power field ] ) was striking .
immunohistochemical analysis revealed strong ( + ) staining with s-100 , vimentin , cd-56 , cd-99 , and p-53 , as well as 80% positivity for ki-67 staining ( figure 4 ) . considering these data ,
the lesion was diagnosed as mpnst ( grade-3 ; federation nationale des centres de lutte contre le cancer ) .
they may behave aggressively and make up 5%10% of all soft tissue sarcomas.2,4,7,1113 mpnsts may arise from all peripheral nerves and frequently reported localizations are the extremities , trunk , head , and neck.1,2,5,9,11,13,14 in patients with neurofibromatosis , mpnst most frequently occurs at ages of 3040 years .
when compared with benign tendon sheath tumors , mpnsts more frequently occur at extremities.15 mpnsts are rare in childhood and have an poor prognosis.5,10 although mpnst can be found in any part of the body , extremity involvement is observed less frequently in pediatric cases.3,5,6,16 patients with mpnst frequently are admitted with complaints of a palpable mass , pain , and/or other neurological symptoms due to neuronal involvement.11 solitary and/or plexiform neurofibromas in teenagers or children can be confused with mpnsts , but signs of atypia or significant mitotic activity are not present.1721 malignant transformation and recurrence of the solitary neurofibromas without neurofibromatosis are unusual.20 other cases of solitary and/or plexiform neurofibroma in pediatric patients without neurofibromatosis have been reported in body regions with slightly less frequency .
these include the mons pubis,17 eyelid dermis,18 intrascrotal and extratesticular sites,19 abdominal wall,20 and parotid region.17,21 mpnsts can resemble benign tumors , both histologically and radiologically.4 demir et al5 suggested that the diagnosis is easily made in patients without nf1 who present with a palpable mass and pain , but diagnosis is frequently delayed in patients with nf1 due to confusion of these lesions as neurofibroma and/or plexiform neurofibroma . to make an early diagnosis of mpnst
, mri evaluation and biopsy should be performed immediately when malignancy is suspected.22,23 however , both plexiform neurofibroma and mpnsts have similar mri findings , which demonstrates areas of low - to - intermediate signal intensity on t1w sequence and high signal intensity on t2w sequence with heterogeneous enhancement.21 wasa et al24 reported that patients with mpnst generally have giant masses , and presence of peripheral enhancement pattern , perilesional edema - like zone , and intratumoral cystic lesions are observed more frequently .
moreover , they reported that heterogeneity in t1-weighted images support mpnst in patients with nf1 .
in addition , presence of markedly increased nodularity also suggests malignancy.25 recent studies suggest that positron emission tomography computed tomography ( ct ) scanning is especially helpful to differentiate mpnst from neurofibroma and/or plexiform neurofibroma.16,2225 hemorrhagic or necrotic heterogeneity in mri or ct may suggest malignancy , but this finding may also be observed in benign peripheral nerve sheath tumors.10,21 garg et al17 reported that radiological investigations and fine - needle - aspiration cytology may not be very helpful for diagnosis .
the rarity of positive cytology may be secondary to the adhesive nature of cells in such tumors .
microscopy reveals proliferation of all elements of nerves , including axons , schwann cells , and fibroblasts .
immunoreactivity for s-100 protein , nse , and vim is characteristic.17 our case had findings of nf1 .
after mri , adequate sample was obtained with fine - needle - aspiration biopsy due to suspicion of malignancy .
histopathological evaluation was consistent with benign neurofibroma , and therefore total surgical excision was planned .
but histopathological evaluation of the excised material was interpreted as mpnst with malignant cells at the surgical border .
we suggest that malignancy should be suspected in nf1 cases that show masses with heterogeneous contrast involvement and occasional cystic and necrotic places .
histopathological evaluation of the needle biopsy material may yield a false diagnosis , but larger samples that are obtained after surgery show marked nuclear polymorphism , mitosis , vascular invasion , necrosis , hypercellularity , a high proliferative index ( immunopositivity to ki67 ) , and many more atypical spindle cells.3,12,25,26 the minimum amount of histological examination to establish diagnosis includes immunohistochemical stains for des , myog , vim , s-100 , proliferative activity marker mib-1 , potential expression of p53 , cerbb2 , p27 , p16 oncogenes , or tumor suppressor genes .
expression of p53 plays an important role in the evolution of mpnst.24,27,28 our case had histopathological and immunochemical findings similar to the earlier findings , and diagnosis of mpnst ( grade-3 ; fnclcc ) was made .
despite the fact that mpnsts have high local recurrence rates and are generally associated with poor prognosis , little is known about prognostic factors or effective clinical management for this tumor type.4,10,13,16 the most significant factors affecting the prognosis of mpnst in the pediatric age group are surgical resectability , local invasiveness , tumor size ( > 5 cm ) , extent of surgery , intergroup rhabdomyosarcoma study grade , tumor location ( central vs peripheral ) , presence of nf1 , and adjuvant treatment.2,5,7,9,15,22 recurrence is seen in 75% of the cases , and 5-year survival ranges between 16% and 53% .
poor prognosis is associated with a tumor size > 5 cm , subtotal resection of tumor , and preexisting nf1.7,29,30 because mpnst is an aggressive tumor , complete surgical resection is necessary for a successful treatment . however , complete surgical resection is rarely possible , and the most important reason of treatment failure is the difficulty in achieving local control .
information about clinical management of this tumor is sparse because there are very few reports in the literature , and this tumor is rarely seen in the pediatric population.2 although response to radiotherapy and chemotherapy is weak , adjuvant radiotherapy and chemotherapy are frequently required for mpnst patients.2,4,14,22 carli et al2 reported that postoperative radiotherapy may help to achieve local control in patients with minimal residual tumors .
in addition , they reported that chemotherapy may be effective in tumors thought to be unresectable.2,12 because mpnst has greater chance for recurrence than other sarcomas , current treatment is radical surgical resection .
recurrence may occur years after the surgery ; therefore , long period of follow - up is warranted.2,3,7,25
in conclusion , mpnst is a rare , soft tissue sarcoma of childhood and adolescence , which may be misdiagnosed as neurofibroma or plexiform neurofibroma in patients with nf1 .
especially , a giant mass and mri features of heterogeneous involvement , along with a cystic and nodular mass , should suggest malignancy .
the main target of surgical treatment is complete surgical excision . in young patients with mpnst ,
the most important risk factor is the presence of nf , and in such cases , total excision of the mass is very difficult . | malignant peripheral nerve sheath tumors ( mpnsts ) are rare sarcomas of children and adolescents , and they are aggressive tumors with a high rate of local recurrence .
we present a 15-year - old boy with neurofibromatosis type 1 ( nf1 ) , who had a giant mpnst on the right thigh taking into account the available literature .
diagnosis of mpnst may be delayed in nf1 patients due to confusion with a neurofibroma and/or a plexiform neurofibroma .
malignancy should be considered , especially in cases with big masses , with heterogeneous involvement , or in the presence of cysts or necrotic nodules .
the aim of surgical treatment is complete surgical excision . |
it was a pleasure to read the report from dunham and coworkers concerning the relationship between hypocholesterolemia and outcome in patients following severe trauma .
although their study involved only 28 patients , these patients were severely injured ( injury severity score 31 9 ) and they required at least 7 days of mechanical ventilation . upon admission to the surgical intensive care unit the mean cholesterol level was only 119 44 mg / dl , as compared with the expected normal cholesterol level ( taken from a database ) of 201 17 mg / dl ( p < 0.001 ) . in the patients who died ( n = 3 ) , the final cholesterol values were 33% lower than their initial postinjury levels , whereas in patients who survived the final cholesterol levels had risen by 28% .
interestingly , the initial postinjury levels were considerably higher in the three patients who died than in those who survived ( 175 62 mg / dl versus 112 37 mg / dl ) .
although these results are impressive , it is unknown whether the expected ( preadmission ) cholesterol values used as control , which were acquired from the general population , are consistent with those observed in the trauma population .
it would have been interesting to know the actual preadmission cholesterol levels ( obtained either retrospectively or through follow - up evaluation ) ; complete lipid profiles ( e.g. triglycerides , very - low - density lipoprotein , low - density lipoprotein [ ldl ] , high - density lipoprotein [ hdl ] ) , including how each was individually affected ; cytokine data ( especially tumor necrosis factor- , il-6 ) to correlate with cholesterol levels ; and nutritional status ( especially lipid intake ) of these patients .
cholesterol levels either remained low or fell in 90% of patients presenting with an infection .
this response was more often associated with infection than with traditional markers such as leukocyte response , which was positive in only 61% of patients .
cholesterol levels also decreased with the onset of each organ system dysfunction ( ratio of arterial oxygen tension to fractional inspired oxygen < 350 , creatinine > 2.0 mg / dl , glucose > 120 mg / dl , bilirubin > 2.5 mg / dl , arterial bicarbonate 28 or 23 mmol / l ; p < 0.01 ) .
hypocholesterolemia was first reported in 1911 , when chauffard and coworkers reported decreased cholesterol levels in patients who were in ' very bad general condition ' during the febrile phase of tuberculosis . in 1920 , kipp noted a relationship between the degree of hypocholesterolemia and the severity of infection .
we were unable to identify other articles about hypocholesterolemia following injury or infection in the literature until 1980 , when an article by coombes and coworkers was published that described the changes in lipoproteins after burn injury .
those investigators observed a profound decrease in cholesterol levels within a few days of the burn , with the lowest values occurring between the days 6 and 10 .
they noted that both ldl and hdl , which carry over 80% of the total cholesterol in humans , were both decreased .
in contrast , triglyceride - rich very - low - density lipoproteins increased in the acute phase . of the various reasons offered for the hypocholesterolemia seen in critically ill and injured patients , especially those with sepsis , one that seems especially important is related to the ability of lipids and lipoproteins to bind to and neutralize bacterial endotoxin ( lipopolysaccharide [ lps ] ) .
it has been noted that lps in blood binds to lps binding protein , activating the cell surface cd14 receptor .
this stimulates the release of a cascade of proinflammatory cytokines , including tumor necrosis factor- , il-1 , and il-6 .
if lps binds to lipoproteins ( e.g. cholesterol ) , then cytokine release is decreased .
for example , transgenic mice with elevated hdl or ldl concentrations are protected against lethal endotoxemia and severe gram - negative infections .
indeed , infusion of hdl blocked lps - induced cytokine production in rabbits and protected against lethal doses of endotoxin in mice .
the administration of hdl to human volunteers also blocks many of the effects seen with infusion of lps .
clinical support was provided by gordon and coworkers , who recently ( 2001 ) reported low cholesterol and lipoprotein concentrations in 111 critically ill surgical patients .
they noted that these levels correlated inversely with concentrations of il-6 , soluble il-2 receptor , and il-10 .
those investigators suggested that hypolipidemia ( hypocholesterolemia ) is an independent predictor of clinical outcome in critically ill patients .
they also implied that a vicious cycle is initiated in many critically ill patients with acute sepsis with an increased production of inflammatory cytokines , which then decreases lipid and lipoprotein concentrations and increases the susceptibility to lps .
dunham and coworkers should be congratulated for their contribution to this growing body of evidence and its implications for future treatments .
if there were a way to increase the low lipid concentrations in these patients , then this could be an important therapeutic option for preventing and treating sepsis .
hdl = high - density lipoprotein ; il = interleukin ; ldl = low - density lipoprotein ; lps = lipopolysaccharide . | hypocholesterolemia is an important observation following trauma . in a study of critically ill trauma patients ,
mean cholesterol levels were significantly lower ( 119 44 mg / dl ) than expected values ( 201 17 mg / dl ) . in patients who died ,
final cholesterol levels fell by 33% versus a 28% increase in survivors .
cholesterol levels were also adversely affected by infection or organ system dysfunction .
other studies have illustrated the clinical significance of hypocholesterolemia . because lipoproteins can bind and neutralize lipopolysaccharide , hypocholesterolemia can negatively impact outcome .
new therapies directed at increasing low cholesterol levels may become important options for the treatment of sepsis . |
cryptosporidium , a spore - forming protozoon , has been recognized as a human pathogen since 1976 .
the species most frequently involved in human infections are cryptosporidium hominis , which primarily infects humans , and cryptosporidium parvum , which infects humans and animals such as cattle , although infection with unusual species and genotypes occurs in both immunocompetent and immunocompromised populations . in immunocompetent individuals ,
infection causes self - limited watery diarrhea . while , in patients with immune deficiencies , cryptosporidiosis may present as chronic or severe life - threatening diarrhea . in children , mainly those living in developing countries
are known to cause traveler 's diarrhea and they are also responsible for outbreaks of diarrhea , including a memorable outbreak in milwaukee , wisconsin , in 1993 during which nearly 403,000 people developed cryptosporidiosis due to contamination of drinking water .
antimotility drugs play a key role in the treatment of cryptosporidiosis in both immunocompetent and immunocompromised individuals , while the efficacy of antiparasitic drugs in cryptosporidiosis remains controversial , particularly in immunocompromised individuals .
a recent meta - analysis of trials of antiparasitic drugs in cryptosporidiosis revealed significant improvement of non - aids patients with nitazoxanide , but no clear evidence of efficacy for other antiparasitic drugs in cryptosporidiosis or for nitazoxanide in aids patients .
we describe the clinical and parasitological course of a child with cryptosporidiosis , who was treated successfully with nitazoxanide ( alinia , romark laboratories ) .
a 16-month - old child was admitted to the pediatrics department of amiens university hospital ( france ) for diarrheal syndrome associated with rhinitis .
he was born in france to french parents living in a rural area who had never been outside of france .
the child 's father was a dairy farmer and his mother did not work on the farm .
the child did not have any particular family history , but had a personal history of acute otitis media and rhinopharyngitis .
his immune status was not investigated , but on the basis of this history of frequent rhinitis , a respiratory allergy was suspected and treated with antihistamine .
the child presented to our institution with a six - day history of fever , rhinitis , vomiting , and profuse diarrhea .
he was moderately dehydrated with very minor signs of dehydration such as weight loss , deep - set eyes , but no disorders of consciousness .
on initial physical examination , the young patient was afebrile but presented tachycardia of 124 bpm , a respiratory rate of 18/min , and blood pressure of 82/52 mmhg .
chest , cardiac , and abdominal examinations were normal . on ear - nose - throat examination
, the tympana could not be seen due to earwax and the throat was erythematous .
one day after admission , after removal of earwax , ear examination revealed bilateral acute otitis media .
the patient was placed on intravenous fluids and oral cefpodoxime was started while waiting for laboratory results .
the laboratory work - up revealed white blood cell count : 10,200 cells / mm with 46% neutrophils , 41% lymphocytes , 10% monocytes , 3% eosinophils ; hemoglobin : 13.3 g / dl ; platelets : 477,000/mm .
blood biochemistry showed hyponatremia ( 130 mmol / l ) and decreased alkaline reserve ( 19 mmol / l ) .
stool specimens were also sent for routine bacterial culture , rotavirus / adenovirus antigen , giardia antigen , clostridium difficile antigen , and all were negative .
routine stool examination for enteric parasites including direct saline wet mount examination and two concentration techniques : bailenger 's method and mif ( merthiolate iodine formaldehyde ) with both a fixative and a stain was negative .
cryptosporidium antigen was detected in stool by the immunochromatographic method ( rida quick cryptosporidium , r - biopharm diagnostic ) .
modified ziehl - nielsen staining of a stool smear showed several cryptosporidium oocysts , up to 2,400 per gram of stool .
polymerase chain reaction - restriction fragment length polymorphism ( pcr / rflp ) identified the species as cryptosporidium parvum .
three - day treatment ( 100 mg twice daily ) with nitazoxanide suspension ( alinia , romark laboratories , fl , usa ) was then initiated .
oocysts were no longer detected in stool using the modified ziehl - nielsen stain and immunochromatographic methods , while pcr detection was still weakly positive .
eradication of oocyst excretion was observed on the day-7 stool sample and pcr detection was also negative ( figure 1 ) .
cryptosporidium infections obviously do not represent a major public health threat in developed countries , although recurrence of gastrointestinal symptoms due to cryptosporidium is frequently reported , for example , in the milwaukee outbreak in 1993 and in sporadic cases in europe . in most sporadic cases ,
the source of infection is difficult to ascertain as many risk factors are commonly encountered in everyday life .
several factors facilitate transmission of cryptosporidium and account for the propensity to cause large - scale outbreaks of diarrhea .
it is frequently identified in farm animals , particularly calves , and in domestic animals ; ( ii ) the oocyst is very resistant and can conserve its infectivity in moist environments for a long time ; ( iii ) cryptosporidium genus is composed of a large number of species , several of which can infect humans ; ( iv ) the infectious dose is very low , and infected individuals excrete large numbers of oocysts , up to 10 in a single day .
public health surveys of cases must investigate all forms of exposure during the two weeks prior to onset of the illness in order to identify the source of contamination . in the present case ,
the child 's father was a dairy farmer and the family lived 3 km from the farm .
two years prior to onset of the child 's illness , the father reported cryptosporidium - related loss of calves in his livestock .
the child had never been on the farm , and therefore had never had any contact with the animals .
parasitological stool examinations including specific methods for cryptosporidium oocyst detection of other family members were negative , although the father reported occasional gastrointestinal disorders .
cryptosporidium parvum identified in the child and the documented presence of cryptosporidium in the father 's livestock are both in favour of these animals as the source of contamination , with transmission via the father .
[ 11 , 12 ] , this case report confirms the efficacy of nitazoxanide for the treatment of cryptosporidiosis in immunocompetent children .
however , the role of the child 's immune system in this rapid clearance of the parasite remains unknown .
the negative result of cryptosporidium antigen detection in stool observed immediately after the 3-day course of nitazoxanide is an interesting finding , suggesting that the rapid immunochromatographic method could be used for posttreatment stool tests instead of more time - consuming staining or pcr methods .
this case highlights the need to consider spore - forming protozoa as potential causes of diarrhea in children .
parasite - related diarrhea in both immunocompetent adults and children is probably underestimated due to underdiagnosis .
in contrast with bacterial and viral agents , parasites are less frequently considered by physicians as a potential cause of diarrhea .
clinical pathologists should therefore systematically perform screening for spore - forming protozoa in all patients with persistent or acute watery stools . | cryptosporidium has emerged as an important cause of diarrheal illness worldwide , especially amongst young children and patients with infectious or iatrogenic immune deficiencies .
the authors describe a case of mild cryptosporidiosis in a well - nourished , immunocompetent , one - year - old child .
rapid clinical and parasitological improvement was observed after a 3-day course of nitazoxanide . |
in recent years , sedentary living and physical activity have become an important topic of research .
sedentary lifestyle presents a significant risk factor for human health ( 13 ) . on the other hand ,
it was proven that habitual physical activity or exercise causes not only a reduction of the incidence of obesity , cardiovascular diseases and diabetes , but also improves daily functioning of elderly people ( 46 ) . moreover
, even moderate - intensity activity , such as half an hour of walking , can reduce post - prandial glucose and insulin levels in overweight and obese patients ( 7 ) .
researches of sedentary lifestyle are well documented and available in developed countries but data for developing countries are scarce ( 810 ) .
according to data for 2006 , more than two - thirds of our country s adult population is physically inactive ( 11 ) .
although national data are valuable for public health interventions , it does not provide enough information about the social and economic specificities of particular communities and their combined effect on sedentary lifestyle over time . educational level ,
recognizing relationships of a full range of sedentary lifestyle in members of the predominantly working class community is necessary to develop efficient preventive intervention procedures in order to reduce sedentary behavior and health risk it represents .
public health systems in developing countries are often limited in terms of personnel and funding . reducing the number of patients who have health problems related to lack of regular physical activity and reduction of total time they spent in leisure - time sedentary activities ( such as tv viewing , computer use , reading , sitting or sleeping )
therefore , we hypothesized that sedentary lifestyle in suburb , working class local community were associated with socioeconomic determinants .
we aimed to narrow focus and to characterize populations most appropriate to target where we expect sedentary behavior .
a total of 1126 consecutive attenders at a general practice were enrolled into this cross - sectional study .
they were independently functioning adults who regularly attended annual medical check - ups at rakovica health center in belgrade .
there are four branches of community health center in the municipality of rakovica in which medical examinations , interviews and measurements were performed .
we restricted our analysis to adult patients aged 18 yr or older and excluded patients who were hospitalized in last twelve months .
patients whose general condition did not allow any kind of physical activity and patients who were taking chemotherapy were also excluded from the study .
the study protocol included a complete clinical and biochemical investigation revealing age , gender , lipid status , height , weight and blood pressure . the questionnaires were used for collection of the following data : income status , educational level , occupation and level of physical activity .
the questionnaires were based on the standard questionnaires , which were used in similar types of surveys but adapted for this study ( 12 , 13 ) .
interviewers collected information from patients about current state of chronic diseases ( diabetes mellitus , arterial hypertension ) smoking , medication and other socioeconomic data .
smoking habits and diabetes were reported and dichotomized before the variables were entered into the analysis ( smoker vs. non - smoker , diabetic vs. non - diabetic respectively ) .
educational attainment was categorized into three groups : elementary school , high school and college or faculty .
patients who stated that they have excessive amount of daily sitting , watching television , computer use or reading were categorized as group 1 - patients with sedentary lifestyle . patients who reported to have some kind of exercise during the day such as light walking , stretching or non - exercise physical activities such as standing , washing dishes , doing laundry or cooking , were designated as group 2- low light - intensity physical activity .
moderate - to - vigorous physical activity includes exercise such as brisk walking , biking on level ground and sports involving catch and throw ( such as volleyball ) and activities such as stair climbing , mopping the floor , car washing or gardening .
the variables reflecting individual socioeconomic status were income status , occupation and education level of the respondent . to determine income status ,
respondents were asked to report real income they had during the past year : responses were coded as : low income : 320 usd or lower , average income : 320 590 usd and high income : higher than 590 usd ( statistical office of the republic of serbia , 2013 ) .
educational attainment was categorised into three groups : elementary school , high school and college or faculty .
information about the occupation are obtained by asking respondents : what is your occupation ?
the answer included : workers , entrepreneurs , retirees , unemployed , students or other . because of the small sample size , participants whose answer was other (
n = 4 ) were included in the category of workers ; this did not cause any significant changes in the statistical analysis . at the point of study entry
all patients underwent height and weight measurement for determination of body - mass index [ bmi measured as weight ( kg)/squared height ( m ) ] and waist and hip measurements for waist - to - hip ratio ( whr ) .
patients height was measured by using anthropometer ( sieber hegner , switzerland ) , with an accuracy of 0.1 cm .
body mass was recorded using f 150 s02-a balance ( sartorius , germany ) , with an accuracy of 0.1 kg .
waist circuimference was measured to the nearest centimetre at the level of the umbilicus and hip circumference was measured to the nearest centimeter at the level of the iliac crest .
the whole study was planned according to the ethical standards detailed in the declaration of helsinki ( as revised in 1983 ) and according to local institutional guidelines .
all patients enrolled into the study gave written informed consent to participate in the study .
this study was approved by the ethical committee of the community health center rakovica in belgrade .
blood sampling took place under standard conditions between 7 and 8 am after a 12-h overnight fast .
venous blood was collected into evacuated tubes ( vacutainer , becton dickinson , city , usa ) from the antecubital vein with minimal stasis .
total cholesterol , high - density lipoprotein ( hdl - c ) , low - density lipoprotein ( ldl - c ) and triglycerides were assayed by standard laboratory procedures using an ilab 600 analyzer ( instrumentation laboratory , milan , italy ) .
all data were nonparametrically distributed and the kruskal - wallis h test was employed to compare differences in medians between patients in four experimental groups of patients .
we used multivariate logistic regression analyses to calculate the odd ratios ( or ) and 95% confident intervals ( 95% ci ) of having sedentary lifestyle for education , ocupation and income status as covariates .
physical activity was therefore dichotomised into two groups ( patients who were engaged in sedentary lifestyle ( group 1 ) and patients who were engaged in any type of physical activity ( groups 2 , 3 and 4 ) ) before entered into the regression analysis .
a power calculation showed that a sample of 297 participants was necessary to detect effect size w = 0.3 with 99 percent power and with alpha of 0.05 . to ensure representative sample for our study , stratification with equal allocation
was applied and number of patients was selected geographically to correspond with the population data for four community areas ( in each area there is a health center branch ) . to ensure that sufficient participants were recruited from all socioeconomic groups , the number of patients included in the study was significantly higher than the minimum required .
, chicago , il , usa ) , and results were considered statistically significant where two - tailed p value was < 0.05 .
a total of 1126 consecutive attenders at a general practice were enrolled into this cross - sectional study .
they were independently functioning adults who regularly attended annual medical check - ups at rakovica health center in belgrade .
there are four branches of community health center in the municipality of rakovica in which medical examinations , interviews and measurements were performed .
we restricted our analysis to adult patients aged 18 yr or older and excluded patients who were hospitalized in last twelve months .
patients whose general condition did not allow any kind of physical activity and patients who were taking chemotherapy were also excluded from the study .
the study protocol included a complete clinical and biochemical investigation revealing age , gender , lipid status , height , weight and blood pressure . the questionnaires were used for collection of the following data : income status , educational level , occupation and level of physical activity .
the questionnaires were based on the standard questionnaires , which were used in similar types of surveys but adapted for this study ( 12 , 13 ) .
interviewers collected information from patients about current state of chronic diseases ( diabetes mellitus , arterial hypertension ) smoking , medication and other socioeconomic data .
smoking habits and diabetes were reported and dichotomized before the variables were entered into the analysis ( smoker vs. non - smoker , diabetic vs. non - diabetic respectively ) .
educational attainment was categorized into three groups : elementary school , high school and college or faculty .
patients who stated that they have excessive amount of daily sitting , watching television , computer use or reading were categorized as group 1 - patients with sedentary lifestyle . patients who reported to have some kind of exercise during the day such as light walking , stretching or non - exercise physical activities such as standing , washing dishes , doing laundry or cooking , were designated as group 2- low light - intensity physical activity .
moderate - to - vigorous physical activity includes exercise such as brisk walking , biking on level ground and sports involving catch and throw ( such as volleyball ) and activities such as stair climbing , mopping the floor , car washing or gardening .
the variables reflecting individual socioeconomic status were income status , occupation and education level of the respondent . to determine income status ,
respondents were asked to report real income they had during the past year : responses were coded as : low income : 320 usd or lower , average income : 320 590 usd and high income : higher than 590 usd ( statistical office of the republic of serbia , 2013 ) .
educational attainment was categorised into three groups : elementary school , high school and college or faculty .
information about the occupation are obtained by asking respondents : what is your occupation ?
the answer included : workers , entrepreneurs , retirees , unemployed , students or other . because of the small sample size , participants whose answer was other (
n = 4 ) were included in the category of workers ; this did not cause any significant changes in the statistical analysis . at the point of study entry
all patients underwent height and weight measurement for determination of body - mass index [ bmi measured as weight ( kg)/squared height ( m ) ] and waist and hip measurements for waist - to - hip ratio ( whr ) .
patients height was measured by using anthropometer ( sieber hegner , switzerland ) , with an accuracy of 0.1 cm .
body mass was recorded using f 150 s02-a balance ( sartorius , germany ) , with an accuracy of 0.1 kg .
waist circuimference was measured to the nearest centimetre at the level of the umbilicus and hip circumference was measured to the nearest centimeter at the level of the iliac crest .
the whole study was planned according to the ethical standards detailed in the declaration of helsinki ( as revised in 1983 ) and according to local institutional guidelines .
all patients enrolled into the study gave written informed consent to participate in the study .
this study was approved by the ethical committee of the community health center rakovica in belgrade .
blood sampling took place under standard conditions between 7 and 8 am after a 12-h overnight fast .
venous blood was collected into evacuated tubes ( vacutainer , becton dickinson , city , usa ) from the antecubital vein with minimal stasis .
total cholesterol , high - density lipoprotein ( hdl - c ) , low - density lipoprotein ( ldl - c ) and triglycerides were assayed by standard laboratory procedures using an ilab 600 analyzer ( instrumentation laboratory , milan , italy ) .
all data were nonparametrically distributed and the kruskal - wallis h test was employed to compare differences in medians between patients in four experimental groups of patients .
we used multivariate logistic regression analyses to calculate the odd ratios ( or ) and 95% confident intervals ( 95% ci ) of having sedentary lifestyle for education , ocupation and income status as covariates .
physical activity was therefore dichotomised into two groups ( patients who were engaged in sedentary lifestyle ( group 1 ) and patients who were engaged in any type of physical activity ( groups 2 , 3 and 4 ) ) before entered into the regression analysis .
a power calculation showed that a sample of 297 participants was necessary to detect effect size w = 0.3 with 99 percent power and with alpha of 0.05 . to ensure representative sample for our study , stratification with equal allocation
was applied and number of patients was selected geographically to correspond with the population data for four community areas ( in each area there is a health center branch ) . to ensure that sufficient participants were recruited from all socioeconomic groups , the number of patients included in the study was significantly higher than the minimum required .
, chicago , il , usa ) , and results were considered statistically significant where two - tailed p value was < 0.05 .
classification and general characteristics of the patients are shown in table 1 . classification and general characteristics of the study groups
continuous variables are shown as median ( 25th75th percentiles ) , categorical variables are shown as absolute and relative frequencies .
p<0.01 group 1 vs. group 2 ; p<0.01 group 1 vs. group 3 ; p<0.01 group 1 vs. group 4 ; p<0.01 group 2 vs. group 3 ; p<0.01 group 2 vs. group 4 ; p<0.01 group 3 vs. group 4 ; significant differences according to chi - square test ( men vs. women , smokers vs. non smokers , diabetic vs. non - diabetic ) to determine whether there was a statistically significant difference between the four groups of patients we used kruskal - wallis h test .
this test indicated significant differences between four experimental groups ( p<0.001 ) . to investigate particular differences between groups we employed mann - whitney u test .
patients with sedentary lifestyle were significantly older than members of other three groups , especially when compared to group 3 and group 4 .
patients in group 1 had a significantly higher body weight , body mass index , waist , hips , systolic and diastolic blood pressure , cholesterol , triglycerides and ldl cholesterol levels compared with group 2 ( p<0.001 ) . moreover , in group 3 and group 4 all aforementioned parameters were significantly lower compared with parameters measured in group of patients with sedentary lifestyle ( p<0.001 ) .
the hdl- c was significantly lower in group with sedentary lifestyle compared to patients in other three groups ( p<0.001 ) .
in addition , the results obtained with patients in group 2 were significantly different compared to group 3 and group 4 .
patients in group 2 had a significantly higher body weight , body mass index , waist , hips , diastolic blood pressure , cholesterol , triglycerides and ldl- cholesterol levels compared with group 3 and group 4 ( p<0.001 ) .
the hdl- c was significantly lower in patients who were members of group 2 compared with patients in group 3 and group 4 ( p<0.001 ) .
systolic blood pressure in group 2 was significantly higher only when compared to patients who were members of group 4 .
in addition , patients in group 2 were significantly older when compared to group 3 and group 4 .
when we compared data obtained in group 3 and group 4 , there were no significant differences between these two groups , with the exception of body weight , systolic blood pressure and total cholesterol levels , which were all higher in group 3 ( p<0.001 ) .
there was a significant difference between gender distribution in the four groups of patients with different habits in exercise or physical activity ( chi - square=20.180 ; p<0.001 ) .
additionally , significant differences were found between distribution of smokers and non - smokers ( chi - square=62.945 ; p<0.001 ) and diabetic and non - diabetic ( chi - square=21.931 ; p<0.001 ) in the four experimental groups . in order to define causes of the high number of patients with sedentary lifestyle , we further investigated factors such as education , occupation and income status .
educated patients had sedentary lifestyle than patients of college or faculty level of education ( che - square=57.965 ; p<0.001 ) .
sedentary lifestyle was most prevalent in retirees and workers in contrast to the unemployed , students and entrepreneurs when compared according to occupation ( che - square=126.387 ; p<0.001 ) .
patients who have high or average incomes were more physically active when compared to patients who had low income ( che - square=103.165 ; p<0.001 ) .
we used multiple binary logistic regressions to determine whether the socioeconomic indicators ( educational level , occupation and income status ) had potential to predict risk of sedentary behavior ( table 2 ) .
logistic regression analysis for the association of sedentary lifestyle and socioeconomic status of patients or , odds ratio ; ref , reference category ; ors were calculated with multivariate logistic regression analysis . all variables presented in the table were included in the models for education , the reference category was college or faculty degree , for occupation students and for income status patients with high - income status ( categories with the lowest number of sedentary patients ) .
patients with elementary school level of education were seven times more likely to be classified in the category with sedentary lifestyle ( or=7.00 ; 95% ci=4.1211.90 ; p<0.001 ) compared to patients with college or faculty degree .
being retired ( or=71.39 ; 95% ci=21.64235.38 ; p<0.001 ) and reporting low income ( or=5.53 ; 95% ci=3.528.69 ; p<0.001 ) were significantly associated with higher odds of sedentary behavior when compared with students and patients with high - income status , respectively .
considering multi - morbidity that occurs later in life , elderly were the most common patients in general practice on the primary care level .
patients in group 2 were significantly older when compared to group 3 and group 4 ( p<0.01 ) .
when the group 3 and 4 were compared , statistically significant differences were not found .
furthermore , the distribution of men and women differed significantly between the four experimental groups ( p<0.01 ) .
moreover , patients with high school degree were 2.16 times more likely to be classified in the category with sedentary lifestyle ( or=2.16 ; 95% ci=1.56 2.99 ; p<0.001 ) when compared to patients with college or faculty degree .
higher odds of sedentary behavior also were found in workers ( or=25.22 ; 95% ci=7.8680.90 ; p<0.001 ) , entrepreneurs ( or=17.42 ; 95% ci=4.9161.88 ; p<0.001 ) and unemployed ( or=13.80 ; 95% ci=3.9148.74 ; p<0.001 ) when compared with students .
sedentary lifestyle is a term used to describe activities commonly associated with sitting , sleeping , lying down and the other actions that are performed in these states of low energy consumption .
recent studies have revealed a clear role of sedentarism in development and progression of chronic diseases and disabling conditions ( 15 , 16 ) .
taking into consideration that understanding the health risks of sedentary lifestyle is important for general population health , our challenge was to investigate the impact of social determinants to this kind of behavior .
old age has been commonly recognized as a time of inevitable increase in chronic diseases and conditions .
in addition , sedentary lifestyles are more prevalent in elderly . over the past 32 years
, the older adult population has significantly increased in the republic of serbia . in 1981 , only 10.8% of the population was aged 65 or older and by 2013 , the percent had increased to 17.8% ( statistical office of the republic of serbia , 2013 ) .
sedentary lifestyle has been considered a factor that severely impacts human health and our findings were consistent with studies that have addressed this issue ( 17,18 ) .
therefore , when we compared the four experimental groups , the differences were as we had expected .
patients with sedentary lifestyle were significantly older than members of the other three groups were .
when compared to group 2 , patients in group 1 had a significantly higher body weight , body mass index , waist , hips , systolic and diastolic blood pressure , cholesterol , triglycerides and ldl cholesterol levels . and if the patients in group 2 were engaged in low light - intensity physical activity ( such as light walking ) , it was enough to affect the anthropomorphological and biochemical parameters , which were better in group 2 when compared to sedentary patients . the lipid status , anthropomorphological characteristics and blood pressure of the patients who were engaged immoderate or vigorous physical activity were within the physiological range and significantly better than in group 1 and group 2
. however , devastating result of our investigation refers to the fact that almost half of the study participants were engaged in sedentary lifestyle .
our findings further extend the understanding of the impact of socioeconomic indicators on a significant incidence of sedentary behavior in this suburb working class area . knowing the current prevalence of a sedentary lifestyle in adult population and the main characteristics of sedentary patients in certain areas is necessary for strategic promotion of physical activity ( 19 ) . considering that the high incidence of sedentary lifestyle and severity of the consequences that such behavior may have , our particular concern was the association of educational level and sedentarism .
our results showed that higher educational level ( college or faculty degree ) was significantly associated with a lower incidence of sedentary lifestyle .
even 78.5% of the patients who have elementary school were engaged in sedentary lifestyle , while in patients with secondary education , the percentage was 53.4% .
well educated patients tended to be less sedentary when compared to patients with elementary or high school .
therefore , our study confirms the well - known relationship between a sedentary lifestyle and lack of education , as crespo et al
transitioning to retirement leads to significant changes on older person s life ( 21 , 22 ) .
as older people no longer need to spend time at work , they tend to reduce their involvement in physical activities compared to younger individuals . in contrast
, students have proven to be extremely physically active . in other groups , 52.3% of workers ,
43.1% of entrepreneurs and 37.5% of unemployed subjects were engaged in a sedentary lifestyle . based on the obtained results
it could be concluded that the occupational sedentarism may be one of the most important factors that influence the physical activity of people .
finally , the objective of our research was related to the impact of income level on sedentary behavior .
concluded that people with higher income spend more time using the computer ( internet ) and reading ( 23 ) .
however , the percentage of people with low income who engaged in leisure - time physical activity were significantly lower when compared to total adult population and our results were in accordance with this study ( 24 ) .
the percentage of patients with high or average incomes who were less engaged in sedentary lifestyle , were lower when compared to patients who had low income .
the differences in study results indicate that there is a need for a specific strategy in reducing sedentary lifestyle and that it should be adapted to the characteristics of the local population . while we think that the findings obtained in this study can be useful for better understanding of the causes of sedentary lifestyle , some limitations of this study need to be considered .
first is concerning the fact that analysis assumed that all the experimental groups used the public services equally
. a limited number of patients use private health sector so we were not able to analyze engagement in sedentary lifestyle among these people . besides
, we can only assume that people who use private health sector have higher incomes and educational level , but we have no evidence for such a claim .
second , the income status , educational level , occupation and level of physical activity were self - reported and might be measured with errors .
community based prediction of risk of sedentary behavior could be benefited twofold : first , increase of the total time spent in some kind of physical activity and health improvement , and second , decrease of health care resources consumption . in order to identify a segment of the population , which is more likely to be engaged in sedentary lifestyle , we have tried to determine whether the socioeconomic indicators had potential to predict risk of sedentary behavior .
our research has shown that special attention should be paid to individuals who are retired , with low income and less educated because they have higher odds of spending less time in leisure - time physical activity . in old age , retired individuals with different educational and financial status may exert advice provided by health professionals differently .
therefore , it is very important that groups with higher odds for sedentary behavior get detailed advice and information about sedentarism as a significant health risk factor .
each community has its own specificities in terms of social and economic backgrounds . in conclusion ,
although our results identify older , retired people who are less educated and have lower incomes , as a risk group for sedentary lifestyle , further research on the local , community level should provide an answer which model of counseling and control provided by health professionals could be best in reducing sedentary lifestyle .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | background : sedentary lifestyle represents a growing health problem and considering that there is already a range of unhealthy habits that are marked as health risk factors and the increasing prevalence of sedentary lifestyle worldwide , we aimed to investigate association of sedentary way of living in suburb , working class local community with socioeconomic determinants such as educational level , occupation and income status.methods:in this community - based cross - sectional study , 1126 independently functioning adults were enrolled into the study .
the study protocol included a complete clinical and biochemical investigation revealing age , gender , lipid status , height , weight and blood pressure . trained interviewers ( nurses ) collected information from patients about current state of chronic diseases ( diabetes mellitus , arterial hypertension ) smoking , medication and other socioeconomic data .
descriptive analysis , chi - square and logistic regression were performed as statistical calculations.results:patients with elementary school were seven times more likely to be classified in category with sedentary lifestyle compared to patients with college or faculty degree . being retired and reporting low income were significantly associated with higher odds of sedentary behavior when compared with students and patients with high - income status , respectively.conclusions:the significance of this study
lies in the fact that our results may help to easier identification of patients who may have a tendency towards a sedentary lifestyle . |
lung parenchymal calcifications are uncommon , but may occur in numerous conditions so their main differential diagnosis should be known by the radiologist .
we demonstrate a rare etiology for lung calcifications in a 87 year - old male that came to our emergency department complaining of dyspnea and was diagnosed with diffuse pulmonary ossification secondary to a long - standing mitral stenosis .
radiographic and ct findindings of the patient are described and the most important imaging features of this pathology are here highlighted .
a 87 year - old male was admitted to the emergency department of our hospital referring progressively increased dyspnea over the last two days , together with anorexia in the last months .
he had a history of long known moderate to severe mitral stenosis , but due to his age was not considered for valvuloplasty .
the chest radiography done at arrival ( fig.1 ) revealed the presence of bilateral micronodular opacities that were more prominent on the right lung , where they were associated with coarse calcifications .
, there were exuberant confluent microcalcifications , more numerous in the lung bases . despite being bilateral , they were rather assymetric , being more conspicuous on the right side and rare on the left side .
a diagnosis of diffuse pulmonary ossification secondary to mitral stenosis was suggested based on both the imaging and clinical findings . despite the medical therapy instituted ,
dyspnea of the patient increased over the following days , leading to his death from cardio - respiratory arrest two weeks after this exam .
diffuse pulmonary ossification ( dpo ) is a rare diagnosis defined by the deposition of mature heterotopic bone in the lung parenchyma .
it either can be idiopathic or secondary , most commonly due to pulmonary or cardiac pathologies , particularly mitral stenosis .
the association of pulmonary ossification and mitral stenosis was firstly described by salinger in 1932 .
estimates establish it to occur in about 313% of patients with long - standing mitral stenosis at autopsies , but proper medical or surgical treatment of mitral stenosis and eventual underdiagnosis of dpo make it a rarely reported diesease in the literature .
a male predominance has been reported with patients being diagnosed more commonly between the 2nd to the 4th decades of life , , although there have been reports in individuals over 60 years - old .
a history of repeated insults to the lung , such as recurrent pneumonia , might be present .
several theories have been proposed for explaining the physiopathology of this disease , focusing on different factors : pulmonary congestion , connective tissue proliferation , hemosiderin deposition or recurrent small pulmonary hemorrhages .
patients may be asymptomatic . when symptoms and physical signs are present , they are not different from the ones of mitral stenosis .
dyspnea and fatigue are common and lower limb edema might also be present due to congestive cardiac failure .
chest radiography is the first - line imaging technique and might reveal signs of enlarged left atrium , such as : cardiomegaly , double right heart border , prominent left atrium and increase of subcarinal angle .
it also displays characteristic features such as micronodular calcified opacities that are asymmetrical , usually more conspicuous on the right lung and distributed unhomogenously , sparing the apices and being more numerous on lung bases .
the chest ct , particularly with high - resolution algorithm , is more sensitive in the identification of these features .
it may reveal enlarged left atrium and dilated pulmonary arteries , signs of pulmonary hypertension .
it can also depict the small calcifications , in the same distribution seen in the chest radiograph , often concentrated near the pleura .
pathologic findings include the presence of round or mulberry - shaped bone nodules that originally form in the alveoli but may then occupy interstitial air spaces .
diffuse pulmonary ossification is commonly associated with mitral stenosis , with high - resolution chest ct being the most accurate imaging technique for this diagnosis .
characteristic features include small calcified opacities more numerous on subpleural locations , especially on the right lung base and eventual signs of pulmonar hypertension .
the authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no financial support for this work that could have influenced its outcome . | diffuse pulmonary ossification ( dpo ) is a rarely diagnosed entity that may present with characteristic imaging features .
it is listed in the differential diagnosis of lung parenchymal calcifications and should be considered by the radiologist if the appropriate findings are identified .
we report a case of dpo secondary to mitral stenosis in a patient whose severe cardiac pathology lead to death few weeks after a chest ct was done . to date , there are no specific treatments with proved benefit in this pathology . |
it is well known that in type 1 diabetes mellitus , optimal glycemic control is extremely important to prevent or postpone long - term complications . in general , glycemic control in children with type 1 diabetes is monitored by measuring glycated hemoglobin ( hba1c ) and self - monitoring of blood glucose ( smbg ) . as demonstrated in various studies , hba1c correlates well with long - term complications .
however , hba1c is only a measure of mean blood glucose over the previous 23 months and not a representation of diurnal patterns of glucose variability .
research has demonstrated that in patients with diabetes , glycemic variability is an independent risk factor for developing long - term complications even when hba1c is in normal range .
when smbg is used to understand glycemic patterns , ideally it should be recorded at pre- and post - meals and at midnight every day which comes to about seven pricks per day .
this is not only stressful for a young child with diabetes but also incurs a huge cost to the family . as a result , many patients resort to a compromised smbg recording where they check blood glucose 34 times / day .
this may lead to erroneous recognition of patterns based on which insulin doses are adjusted .
there are various studies demonstrating the fact that smbg recording may miss significant hypoglycemic episodes , particularly nocturnal hypoglycemia as well as postprandial hyperglycemia which can be captured by continuous glucose monitoring ( cgm ) .
cgm helps to identify glycemic patterns which may not be evident on smbg record and assists patients achieve their goal of optimum glycemic control .
however , the data generated by cgm is enormous , and this sometimes poses difficulty for a clinician to interpret it .
therefore , a need for simpler and easily comprehensible summary of continuous glucose data which would reveal the important patterns of glycemic variability was recognized . ambulatory glucose profile ( agp )
( using freestyle libre flash glucose monitoring ( fgm ) system , abbott diabetes care , alameda , ca , usa ) combines all the data from cgm over a period of 14 days and gives a summarized visual display of glycemic patterns .
this system is sometimes referred to as fgm system as it measures glucose in the interstitial fluid every 15 min .
accuracy and reproducibility of cgm in adults as well as in children have been established in various studies .
earlier versions of cgm sensors used capillary blood glucose ( cbg ) for calibration of the sensor .
typically , cbg and random blood sugar ( rbs ) are used as references to measure the accuracy of the sensor devices .
there are differences in cbg ( measured using glucometer ) and rbs ( plasma glucose measured by laboratory analyses ) as glucose concentration in capillary and vein varies .
therefore , if cbg is used to calibrate cgm , it may affect the accuracy assessment of the sensor device .
newer sensor devices as used in our study ( freestyle libre fgm system , abbott diabetes care , alameda , ca , usa ) are factory calibrated and do not require calibration using cbg . the accuracy of this system has been documented in adults .
it is being used in several countries in adults , but its use in children is yet to be explored .
the aim of this study is to evaluate the accuracy of agp using freestyle libre fgm system in comparison to rbs as well as cbg ( monitored at hospital and home ) in children .
a total of 51 subjects were enrolled after written informed consent was obtained from them .
those with known allergy to medical grade adhesive or isopropyl alcohol used to disinfect skin , those with extensive skin diseases at the proposed application sites and with associated diseases such as untreated hypothyroidism were excluded from the study .
simultaneous capillary and venous blood glucose were measured on the day 1 and day 14 in the hospital .
venous blood glucose was analyzed in the laboratory using turbo chem 100 ( awareness technology inc .
day 14 samples could not be obtained in whom the sensor dislodged before 14 days .
the subjects were also asked to continue checking smbg at home during the sensor wear period using their own glucometer , 24 times a day ( before meals and 2 h after meals ) . during this time
, insulin doses were adjusted by the study participants or their parents based on their smbg readings .
the accuracy of the agp data was also evaluated over four different smbg ranges : < 75 mg / dl , 76140 mg / dl , 141200 mg / dl , and > 200 mg / dl to determine if there were differences in accuracy at various glucose concentrations
. clarke error grid analysis ( ega ) was used to evaluate the point accuracy for agp versus rbs and agp versus cbg .
accuracy measures such as mean absolute deviation , mean absolute relative difference ( mard ) , and coefficient of linear regression of agp on rbs , and cbg were calculated .
accuracy measures such as mean absolute deviation , mean absolute relative difference ( mard ) , and coefficient of linear regression of agp on rbs , and cbg were calculated .
seventy paired rbs , cbg , and agp data and 362 paired home - monitored smbg , and agp data were available .
two paired readings were excluded because the sensor readings were out of system 's recordable range ( < 40 mg / dl or more than 500 mg / dl ) . demographic characteristic of study participants the linear regression coefficient of agp over rbs was 0.93 , and that of agp over cbg was 0.89 ( p < 0.001 ) .
average mard of agp over rbs was 9.6% , and that of agp over cbg was 15.07% .
a detailed comparison of rbs , cbg , and agp data are illustrated in table 2 .
comparison of rbs , agp and cbg data the difference between agp and home - monitored smbg values were compared across different smbg ranges as illustrated in table 3 .
when smbg was < 75 mg / dl , only 65% of the agp readings were within 20% of smbg readings .
whereas , when smbg was more than 200 mg / dl more than 77% of the agp readings were within 20% of smbg readings .
the linear regression coefficient of agp over smbg was 0.93 ( p < 0.001 ) .
comparison of agp data across various smbg ranges the clarke ega for paired values of agp versus rbs yielded 75.9% , 22.5% , 1.6% , 0.0% , and 0.0% results in zones a , b , c , d , and e , respectively .
corresponding findings for agp versus cbg included 54.9% , 43.5% , 0.0% , 0.0% , and 1.6% results in zones a , b , c , d , and e , respectively .
clinically , acceptable values are represented by dots in zone a and b of clarke ega in figure 1a and b. ( a ) clarke ega of paired agp and rbs ( used as reference ) data in clinically significant zones a and b. ( b ) clarke ega of paired agp and cbg ( used as reference ) data in clinically significant zones a and b major adverse reaction was not noticed in any of the study participants who wore the fgm sensor .
mild pain and irritation at the sensor insertion site were complained by five of the study participants .
this study was undertaken to evaluate the accuracy of agp using freestyle libre fgm system in comparison to plasma glucose and cbg measured at the hospital as well as at home environment , in children with diabetes .
feasibility and acceptability of agp in our study subjects have been reported in a separate paper which is being submitted for publication .
the accuracy of cgm and fgm sensors have been studied against frequently sampled venous and capillary blood . in this study ,
the linear regression coefficient of agp over rbs was 0.93 and that of agp over cbg was 0.89 which is comparable to a recently published study evaluating the performance and usability of fgm system involving adults . in our study ,
mard for agp over rbs was 9.56% and that of agp over cbg was 15.07% .
earlier studies by andelin et al . , comparing cgm system ( using cbg for calibration ) have quoted mard of 11.7% using capillary values as a reference and 13.7% using venous samples which is similar to the findings in this study .
mard was lower when agp was compared to rbs than when it was compared to cbg . in this study ,
three different references ( rbs , cbg , and smbg ) have been used to assess the accuracy of agp . in the hospital
setting , the reference method used was the plasma glucose analysis , whereas agp sensor measures interstitial glucose .
many studies have compared interstitial glucose and plasma glucose and have found a good correlation between the two .
second , we compared capillary glucose checked at the hospital with a single glucometer with agp glucose data and found a similar correlation .
finally , testing the accuracy of agp at home by the patients reflects the performance of agp sensor in routine practical setting .
hence , the accuracy of agp was assessed in reference to smbg done at home by the patients .
this method also facilitates more number of paired glucose data to be obtained at different time of the day which is difficult to obtain in the hospital without admitting the study subjects .
however , in the home setting , since there is no supervision , the validity of the data is questionable . the linear regression coefficient for agp versus smbg was 0.93 .
in this study , glucose data obtained by agp and smbg were compared across various glucose ranges . in the lower range (
smbg < 75 mg / dl ) , only 65% of the agp data were within 20% of corresponding smbg data , whereas more than 70% of the agp data were within 20% of smbg data when glucose levels were higher ( > 75 mg / dl ) .
this is in line with other studies on cgm where it has been found that the accuracy may be compromised at lower glucose levels .
however , it is to be noted that this does not indicate the absolute accuracy , but the relative accuracy of agp device in comparison with smbg measurement by glucometer .
the main limitation of this study includes a small number of paired venous and capillary samples .
this is because of practical difficulties as well as ethical issues involved with the procedure of multiple venous sampling in children .
the study was performed in outpatient clinical setting and not in a research setting making it difficult to obtain multiple samples from the study participants .
smbg data obtained at home was not directly supervised but was counter checked and confirmed from individual glucometer records . in addition , smbg at home was monitored using different glucometers used by individual patients which could have affected accuracy results .
we could not check reliability over different periods in 2 weeks due to less number of samples .
earlier studies have mentioned that there is inconsistency in the results when the accuracy of cgm sensors are assessed at hospital and home environment . in contrary to this , in our study , the accuracy of the agp sensor was found to be similar both at hospital setting as well as at home environment ( r = 0.89 for agp vs. cbg and 0.93 for agp vs. smbg ) .
the results on agp from this study provide a novel and painless modality of studying glycemic trends in children with diabetes .
however , at lower glucose levels ( < 75 mg / dl ) , agp values need to be interpreted with caution and should be confirmed by smbg before clinical intervention .
the findings of the study revealed that agp glucose data correlates well with corresponding rbs as well as cbg . even at home setting , there is agreement between agp glucose data and corresponding smbg .
however , a large number of samples in a research setting would help to document reproducibility of our results .
partial funding received from medical education research society ( mert ) , bengaluru , karnataka , india .
partial funding received from medical education research society ( mert ) , bengaluru , karnataka , india .
| background : in children with type 1 diabetes , intensive diabetes management has been demonstrated to reduce long - term microvascular complications . at present , self - monitoring of blood glucose ( smbg ) by patients at home and glycated hemoglobin estimation
every 3 months are used to monitor glycemic control in children . recently , ambulatory glucose profile ( agp ) is increasingly being used to study the glycemic patterns in adults .
however , accuracy and reliability of agp in children have not been evaluated yet.objectives:to assess the accuracy of agp data in children with type 1 diabetes mellitus when compared with laboratory random blood sugar ( rbs ) levels , capillary blood glucose ( cbg ) measured by glucometer in the hospital , and smbg monitored at home.methods:paired rbs , cbg , and agp data were analyzed for 51 patients who wore agp sensors for 2 weeks .
simultaneous venous and cbg samples were collected on day 1 and day 14 .
smbg at home was checked and recorded by the patients for optimizing insulin doses .
accuracy measures ( mean absolute deviation , mean absolute relative difference ( mard ) , and coefficient of linear regression of agp on rbs , cbg , and home - monitored smbg were calculated.results:seventy paired rbs , cbg , and agp data and 362 paired home - monitored smbg and agp data were available .
the mard was 9.56% for agp over rbs and 15.07% for agp over cbg .
the linear regression coefficient of agp over rbs was 0.93 and that of agp over cbg was 0.89 ( p < 0.001 ) . the accuracy of agp over smbg was evaluated over four ranges : < 75 , 76140 , 141200 , and > 200 mg / dl.conclusion : in this study , agp data significantly correlate with rbs and cbg data in children with type 1 diabetes .
however , a large number of samples in a research setting would help to document reproducibility of our results . |
enzymes , of which structures and functions are prescribed in genes , basically mediate all metabolisms needed for the development and functions of living organisms . among the many enzymes , protein tyrosine kinases ( ptks )
are known to play key roles in intracellular signaling for the development and functions of cells .
the mechanisms for regulation of the catalytic activities of ptks seem to be principally prescribed in genes .
these mechanisms include phosphorylation and dephosphorylation of specific tyrosine residues on the kinase protein by other ptks or protein tyrosine phosphatases ( ptps ) , the structures and functions of which are all under genetic control [ 28 ] . for example , the catalytic activity of src , a representative ptk , is known to be downregulated through phosphorylation of tyr527 located in the tail of src protein by csk , another ptk , which leads to binding of the tail to the specific structure on the sh2 domain of src to make the catalytic domain of src closed [ 2 , 3 ] .
additionally , it is known to be upregulated by dephosphorylation of phosphorylated tyr527 , which allows the catalytic domain to open , inducing phosphorylation of tyr416 as the major autophosphorylation site and upregulation of the kinetic activity to phosphorylate exogenous specific substrates [ 24 , 7 , 8 ] . on the other hand , dephosphorylation of phosphorylated tyr416 by ptps downregulates the kinase activity [ 5 , 6 , 8 ] .
results of several early studies have , however , suggested that the catalytic activities of ptks are modulated by exposure of cells to environmental chemicals or oxidative agents [ 913 ] .
it could be that the catalytic activities of ptks are upregulated through redox mechanism - based inactivation of ptps which otherwise dephosphorylate phosphorylated tyrosines at the major autophosphorylation sites of ptks [ 1114 ] .
recent evidence has , however , also suggested that environment - linked chemical or redox reactions directly attack ptks for modification of their catalytic activities and subsequent signaling for cell activation [ 1519 ]
. this may occur through oxidization of sh groups of specific cysteines on ptk molecules by ros that are generated intracellularly after clustering of cell surface receptors in association with membrane lipid rafts by environment - linked chemical or redox reactions [ 15 , 2024 ] .
chemical or redox reaction - mediated clustering of cell surface receptors in association with membrane lipid rafts has further been shown to deliver signals for either cell activation or cell death depending on the conditions [ 20 , 2430 ] .
recent observations on how environment - linked chemical or redox reactions control genetically prescribed signaling for cell activation or cell death are reviewed here , following several related review articles on this matter [ 9 , 10 , 28 , 3138 ] .
additionally , significance of these observations is briefly discussed from the viewpoint of crosstalk between environment - linked chemical or redox reaction- ( covalent bond)-mediated and genetically prescribed ( protein tertiary structure - linked noncovalent bond - oriented ) regulation of intracellular signaling for either cell activation or cell death .
initial activation of ptks is usually linked to recognition by cell surface receptors of specific ligands .
thus , receptor type ptks such as egfr first bind specific ligands such as egf to be dimerized on cell membranes for activation . on the other hand , src family ptks as nonreceptor type ptks with a hydrophobic structure at the n - terminus are activated following specific ligand - induced clustering of cell surface receptors in association with membrane lipid rafts .
lipid rafts are known to work as a station of molecules for signal transduction to which the nonreceptor type ptks are attached under the cell membrane [ 3943 ] .
earlier experiments showed that this receptor - ligand bond - mediated step for activation of either receptor type or src family nonreceptor type [ 17 , 2023 , 44 , 45 ] ptks in cells can be bypassed by environment - linked chemical or redox reactions .
these reactions induce clustering of receptors and membrane lipid rafts , which somewhat mimics ligand - mediated receptor clustering . environmental agents for such chemical or redox reactions include thiol - reactive hg ions [ 2022 , 4446 ] , arsenite [ 24 , 29 ] and methane sulfonate compounds ( 1,4-butanediyl - bismethanethiosulfonate ( bmts ) [ 45 , 47 ] , protein amino group - reactive carbonyl compounds ( glyoxal , methylglyoxal ) [ 23 , 48 , 49 ] , and protein sulfhydryl , amino , and imidazole groups - reactive 4-hydroxynonenal ( hne ) [ 26 , 27 , 50 ] .
this pathway of activation of ptks has been most extensively studied in cells carrying src family ptks such as c - src ( primary and tertiary structures of human c - src are illustrated in comparison with those of human c - ret in figure 1 ) and lck , which direct other ptks such as zap70 and syk and a large family of protein serine / threonine kinases downstream of the signal transduction cascade .
exposure of cells carrying c - src or lck to hg , arsenite , glyoxal , or 1,4-butanediyl - bismethanethiosulfonate ( bmts ) induced chemical or redox reaction - mediated clustering of membrane rafts .
this occurred in close association with clustering of glycosylphosphatidyl inositol- ( gpi- ) anchored proteins in the rafts as the potential direct targets of the redox reactions and src family ptks that are bound to lipid rafts with a myristoylated membrane - targeting structure at the n terminus ( 20 , 53 ) . this signaling event
was followed by activation of src family ptks as well as generation of ros [ 23 , 46 ] .
generation of ros possibly underlay the mechanism of activation of ptks and ultimately led to cell activation or cell death .
all of these signal transduction pathways were inhibited by methyl beta cyclodextrin , a compound that disrupts lipid rafts by removing cholesterol from membranes .
this suggested a crucial requirement of the integrity of membrane lipid rafts in the redox reaction - linked signal pathway that apparently mimics the genetically prescribed receptor - ligand bond - mediated signal transduction .
hne , which is able to pass through the plasma membrane , also triggered another signal transduction pathway for cell death , possibly through scavenging intracellular glutathione .
these results suggest that genetically prescribed signal transduction pathways for cell activation or cell death , including the step of ptk activation , can be bypassed or modified at different steps by environment - linked chemical or redox reaction - mediated pathways .
as briefly mentioned in the introduction section , the receptor - ligand bond - mediated signaling pathway for activation of src kinase is originally prescribed by genes .
this pathway includes phosphorylation of tyr527 in the tail of src protein by csk to be bound to the sh2 domain of src for stabilizing and inactivating the kinase molecule [ 24 , 7 ] .
binding between cell surface receptors attached to membrane lipid rafts , including gpi - anchored proteins , and their ligands may trigger the signal pathway for dephosphorylation of phosphorylated tyr527 of src kinase ( or its equivalent tyrosine residues of other src family ptks ) by specific ptps .
this should result in disruption of the bond between phosphorylated tyr527 and sh2 domain of src and induction of autophosphorylation of tyr416 for activation of the catalytic activities of src or other src family ptks [ 5 , 6 , 8 ] .
recently , the question of whether this is the only mechanism for triggering tyr416 autophosphorylation and activation of src or other src family ptks has been repetitiously discussed [ 10 , 3137 ] .
pu et al . investigated whether the environment - linked chemical or redox reaction - oriented signaling pathway that bypasses the genetically prescribed cell surface receptor - ligand interaction - mediated pathway includes the above - described tyr527-mediated regulatory mechanism of src kinase .
they revealed that cells carrying tyr527-missing v - src or cells missing csk for phosphorylation of tyr527 , which are thereby already active , were still reactive to hg for superactivation of the kinases .
it was concluded from these results that the environment - linked chemical or redox reaction - mediated pathway triggers a new mechanism for tyr416 autophosphorylation and activation of src kinase independent of dephosphorylation of previously phosphorylated tyr527 .
oxidative agents are known to inactivate ptps of which cysteine residues in the catalytic domain are sensitive to the agents .
it has long been suggested that this is the central or even the only mechanism of upregulation of ptks by oxidative agents , maintaining the autophosphorylation level of major tyrosine residues in the catalytic domain of ptks [ 11 , 12 , 52 ] .
ros produced in cells through the mechanism triggered by environment - linked chemical or redox reaction - mediated clustering of cell surface receptors / membrane rafts might actually inactivate ptps , which in turn upregulate the catalytic activity of ptks .
. exposed src kinase proteins that had been immunoprecipitated from cell lysates in a medium with detergents to sulfhydryl - reactive hg or nitric oxide- ( no- ) generating s - nitroso - n - acetyl penicillamine ( snap ) .
they then measured the catalytic activities of src proteins for tyr416 autophosphorylation and tyrosine phosphorylation of specific substrates .
pu et al . showed that the catalytic activity of immunoprecipitated c - src from nih3t3 cells was elevated severalfold by exposure to low to moderate concentrations ( 0.550 m ) of hg , whereas it was decreased by exposure to a high concentration ( 500 m ) of hg .
n - acetylcysteine neutralized this hg effect , suggesting the involvement of a redox reaction in the mechanism .
addition of the protein tyrosine phosphatase inhibitor na3vo4 to the reaction mixture did not inhibit the hg - mediated activation .
this result provisionally eliminates the possibility that hg - mediated inactivation of ptps was the central mechanism for upregulation of the c - src activity .
hg was further found to be capable of activating tyr527-defective v - src kinase and c - src kinase from mutant cells defective in tyr527-phosphorylating csk kinase .
cyanogen bromide cleavage maps revealed that hg selectively promoted the autophosphorylation at tyr416 and that the previously in vivo radiolabeled phosphorus on tyr527 was not deleted .
these observations suggested that hg triggered the redox reaction - linked mechanism for promotion of both autophosphorylation and general catalytic activity of src kinase in vitro independent of tyr527-linked or ptp - mediated regulation .
the exact target structure of the redox reaction for promotion of autophosphorylation of c - src was not , however , identified in these experiments .
exposure of src kinase to snap was also shown to promote tyr416 autophosphorylation independent of tyr527-linked or ptp - mediated regulation .
interestingly , promotion of tyr416 autophosphorylation , which accompanied increase in overall kinase activity of c - src , was found to be closely linked to s s bond - mediated aggregation of src molecules .
this observation supported a hypothetical view that the environment - linked oxidative agent - triggered signal transduction pathway for upregulation of c - src activity includes sulfhydryl - oxidation - mediated structural modification of src proteins .
further experiments demonstrated that exposure of cells carrying ret kinases to ultraviolet ( uv ) [ 17 , 54 ] or osmotic pressure intracellularly induced activation of these kinases .
either c - ret proto - oncogene as a receptor type ptk or its consistently active oncogene mutants , including an extracellular domain - deleted ( nonreceptor type ) mutant ret - ptc-1 ( primary and tertiary structures of c - ret in comparison with those of c - src are illustrated in figure 1 . ) , were activated .
interestingly , uv- or osmotic pressure - mediated activation of ret - ptc-1 accompanied an increase in the amount of s s bond - mediated dimers of the kinase proteins .
s bond - mediated dimerization from 1% as the background level to 4% of total ret - ptc-1 proteins , and the fraction of dimerized ret - ptc-1 selectively displayed a high level of autophosphorylation [ 17 , 18 ] .
this was demonstrated by an in vitro kinase assay under the condition with or without a reducing agent ( 2-mercaptoethanol ) for the kinase proteins that had been immunoprecipitated from the uv- or osmotic stress - exposed cells .
it was speculated that the uv - induced or osmotic - stress - generated ros in cells attacked intracellular domains of the ret kinase to oxidize the domains for s s bonded dimerization , ultimately resulting in promotion of autophosphorylation and activation of the kinase .
more recently , direct evidence has been provided by kemble and sun [ 19 , 38 ] for oxidative modification of purified src proteins accompanying changes in the levels of their catalytic activities .
they isolated src proteins from bacteria lysates containing recombinant src proteins by adding iminodiacetic acid - agarose beads charged with nicl2 to the lysates .
the isolated src proteins were active when reduced by adding dithiothreitol ( dtt ) to a medium containing src proteins and retained 825% of their full activity when oxidized by adding h2o2 to the medium .
this downregulation of the kinase activity was shown to be caused by oxidation of specific cysteine residues on src , resulting in an s s bridge for dimerization of src molecules .
their study results formally proved that some cysteine residues on src proteins are capable of being oxidized for dimer formation with downregulation of the kinase activity .
this effect of dimer formation is , however , apparently opposite to the above - described effect on immunoprecipitated ret - ptc-1 kinase , in which the kinase activity was upregulated in linkage to s s bond - mediated dimerization of kinase proteins [ 17 , 18 ] .
the reason why dimer formation of src or ret - ptc-1 due to s - s bonds between specific cysteine residues induced apparently opposite effects on their kinase activities in the experiments performed by two different study groups [ 1719 ] is not clear .
however , in the model of ret - ptc-1 , kinase molecules must have been exposed to possibly mildly oxidative environments in cells .
intracellular environments are normally maintained for reducing because of the presence of large amounts of reducing agents such as glutathione and thioredoxin , and the oxidizing / reducing balance in cells therefore does not change drastically even after production of ros in cells ( 57 ) .
the mildly oxidative environment possibly selectively affected sh groups of specific cysteines for fine structural modification of kinase proteins , which was needed for maintenance and upregulation of the kinase activity .
ret - ptc-1 molecules were possibly located in a row under the cell membrane , which should make two neighboring kinase molecules easily accessible to each other for s s bonded dimerization at specific cysteines .
s s bonded dimerization in close linkage to upregulation of the kinase activity [ 17 , 18 ] is direct evidence of the involvement of oxidative structural modification in redox - mediated upregulation of ptk activities .
s s bonded dimerization may not , however , be an absolute requirement for the oxidative effects for upregulation of ptks as discussed in the next section .
in contrast , isolated recombinant src proteins prepared by kemble and sun , which were probably arranged in a random manner in vitro , were affected by rather heavily oxidative agents such as oxygen in air and h2o2 , which possibly oxidized free sh groups on multiple cysteines on src but selectively induced dimerization through s
s bonds between specific cysteines , probably inducing a major structural change of src for downregulation of the kinase activity .
kemble and sun studied the target cysteine residues responsible for s s bonded dimer formation of src proteins by preparing mutant proteins in which each of totally ten cysteine residues of src was replaced with alanine .
they showed that dimer formation occurred only through oxidation of cys277 , which is located in the catalytic domain of src . on the other hand ,
kato et al . [ 17 , 54 ] and takeda et al . examined the sensitivity to ultraviolet light irradiation or osmotic stress of ret - ptc-1 ( a nonreceptor type pkc ) mutants .
each of two cysteine residues ( cys365 and cys376 ) on the alpha - helix h region downstream of the catalytic domain of ret - ptc-1 was replaced with alanine .
they found that the mutants in which cys376 of ret - ptc-1 is replaced with alanine almost totally lost their background ability for inducing autophosphorylation of tyr294 as the major autophosphorylation site .
the mutants at the same time lost their abilities to form s s bonded dimers of kinase proteins in cells to which a high level of autophosphorylation was associated ( in vivo ) , even after exposure to ultraviolet irradiation or osmotic stress .
these results are in contrast to another observation that replacement of cys365 of ret - ptc-1 , a cysteine located near cys376 on alpha - helix h , with alanine did not detectably alter either background ability for autophosphorylation of tyr294 or the ability to form s s bonded dimer formation in cells .
correspondingly , a single mutation in cys498 of v - src , an equivalent of cys376 of ret - ptc-1 , but not in other cysteines yielded clear suppression of kinase activity and temperature sensitivity in cell transformation .
takeda et al . further confirmed that all ret - ptc-1 mutants in which c376 of ret - ptc-1 was replaced with glycine , lysine , threonine or serine lost their background level of activity for autophosphorylation of tyr294 in cells , even though comparable amounts of ret proteins were obtained from each of these mutants , suggesting that possibly accelerated degradation of the mutant proteins is not the reason for the above - mentioned loss of abilities .
all of these results suggested that cys376 of ret - ptc-1 , an equivalent of cys498 of src or cys987 of c - ret , is crucially involved in the maintenance and upregulation of catalytic activities of these kinase proteins in cells .
moreover , because both s s bonded dimer formation and tyr294 autophosphorylation were simultaneously lost in the cys376-missing ret - ptc-1 mutant , it is likely that cys376 plays a crucial role in the redox regulation of the kinase activity of this ptk through oxidative structural modification .
cys277 of src is located in the sequence context of gqgcfg on the small n - terminal lobe of the catalytic domain and is conserved in 8 of more than 90 human ptks examined . on the other hand , cys376 of ret - ptc-1 or cys498 of src is located in the mxxcw motif on the c - terminus of alpha - helix h and is conserved in all but one ( fgr : threonine in place of cysteine ) of 82 human ptks examined in the database swiss - prot ( 18/11/2004 ) , suggesting a crucial role in regulation of the kinase activity of ptks in general [ 32 , 33 , 55 ] .
structural modification of cys376 of ret - ptc-1 , cys498 of src or its equivalent cysteine residues on almost all ptks in a mildly oxidative environment in the cell could thus work to basically maintain or to upregulate the kinase activity .
in contrast , modification of src proteins at cys277 or its equivalent cysteine residues on 8 out of more than 90 human ptks in possibly heavily oxidative environments evidently acted to downregulate it ( see figure 2 ) .
the reason of the different effects of s s linked dimerization on ret - ptc-1 and src might simply be that ret - ptc-1 is missing the cysteine equivalent to cys 277 of src .
have , however , shown that exposure of immunoprecipitated src proteins to low to moderate levels of sulfhydryl - reactive hgin vitro caused upregulation of the kinase activity , whereas exposure of these proteins to a high level of hg downregulated the activity .
it is possible that long - term exposure to oxygen in air ( in vitro ) during preparation of recombinant src proteins caused the proteins to be oxidized at a number of free sh groups of cysteines possibly including both cys277 and cys498 .
s bonded dimerization , however , occurred only between cys277 , of which the downregulatory effect mostly overcame the potential upregulatory effect by oxidization of cys498 .
taken together , the results indicate that oxidative structural modification of pkts might bidirectionally control their kinase activities in the following manner .
fine structural modification by oxidization of cys376 of ret - ptc-1 or its equivalent cysteines in mildly oxidative environments works for maintenance and upregulation of kinase activities of almost all ptks carrying equivalent cysteine residues .
in contrast , more extensive structural modification of ptks through oxidization of cys277 of src or its functionally equivalent cysteine residues on other ptks in heavily oxidative environments downregulates their kinase activities .
the fine structural modification through oxidization of cys376 of ret - ptc-1 , cys498 of src or its equivalent cysteine residues on other ptks for kinase activation may not necessarily require s s bonded dimerization of kinase proteins .
x seems still effective , because exposure of immunoprecipitated src proteins to sulfhydryl - reactive hg successfully induced upregulation of the kinase activity without s s bonded dimerization of the kinase proteins in vitro .
fgr , an exceptional ptk that does not carry cys376 of ret - ptc-1-equivalent , carries threonine in place of cysteine in the mxxcw motif .
takeda et al . therefore investigated the mechanism for this exceptional ptk to keep its kinase activity .
they prepared a number of ret - ptc-1 mutants in which cys376 was replaced with glycine , lysine , threonine , or serine and examined the kinase activities of imunoprecipitated mutant ret - ptc-1 from cell lysates in vitro .
as described in the previous section , all of these mutant proteins almost totally lost their basic kinase activities in vitro .
surprisingly , however , cells carrying cys376ser or cys376thr mutant ret - ptc-1 , but not those carrying cys376gly or cys376lys mutant ret - ptc-1 , displayed almost normal levels of autophosphorylation at tyr294 in vivo ( in cells ) .
correspondingly , cys376thr / ser , but not cys376gly / lys , was capable of inducing cell transformation almost as effectively as parental ret - ptc-1 as an active oncogene .
further study demonstrated that the essential role of the cysteine at the mxxcw motif in initiating the kinase activity of ret - ptc-1 could be partially replaced by the activity of protein kinase c as a serine / threonine kinase in vivo ( but not in vitro ) .
this result suggested that the ptk missing the cysteine at the mxxcw motif uses an alternate pkc - mediated pathway as a rescue system for maintaining the kinase activity .
pkc itself in the rescue pathway should , however , be activated by another ptk that possesses the cysteine at the mxxcw motif .
it is therefore speculated that there is a new pathway of crosstalk between genetically prescribed and environment - linked mechanisms for regulation of ptks .
crosstalk between src and ret kinases has also been reported as src - mediated repair of function - impaired ret - men2a ( another oncogenic ret mutant ) with substitutions of tyrosines in the cooh - terminal kinase domain for phenylalanine .
environment - linked oxidative signals have also been shown to induce apoptotic cell death , basically sharing a common initial step for cell activation , by inducing clustering of cell surface receptors in association with membrane lipid rafts . this signaling pathway was shown to induce generation of reactive oxygen species ( ros ) and activation of apoptosis signal - regulating kinase 1 ( ask-1 ) , c - jun amino - terminal kinase ( jnk ) , and p38 mapk and reciprocal regulation of bcl-2/bax [ 24 , 48 , 50 , 59 , 60 ] or downregulation of erk .
this pathway was further shown to cause mitochondria to release cytochrome c , leading to activation of caspases for dna fragmentation and apoptotic cell death , or to release of a high level of ros for necrotic cell death .
environment - linked oxidative agents might also more directly affect mitochondria or induce an intracellular reduced glutathione level , ultimately leading to apoptotic or necrotic cell death .
these observations suggest extensive crosstalk between microenvironment - linked and genetically prescribed signals for inducing either cell activation or cell death ( see figure 3 ) .
switching for either cell activation or cell death seems to depend on the nature and levels of environment - linked chemical or oxidative signals and on the time of involvement of environment - linked signals in relation to the stage of genetically prescribed development and functions of the cells .
a number of environment - linked chemical or oxidative signals seem to be changed to intracellular oxidative signals through generation of ros as potential second messengers in signal transduction in cells [ 34 , 35 ] .
ros may lead to either cell activation or cell death depending largely on the level of ros generated [ 20 , 25 ] .
thus , a low level of environment - linked oxidative stress probably induces a low grade of ros production for upregulation of ptks .
this causes downstream activation of map family kinases such as erk , serine / threonine kinases , and transcriptional factors for production of specific cytokines and cytokine receptors , ultimately leading to cell activation and proliferation .
in contrast , a slightly increased level of oxidative stress induces apoptotic cell death through a cascade of signaling such as a higher grade of ros production and downstream activation of the ask-1-mediated signal pathway including jnk / p35mapk , bax / bh3 , cytochrome c , and caspases .
higher levels of stress induce necrosis accompanying a high - grade ros production possibly through serious damage of mitochondria . a low level of ros is always produced by mitochondria and may be used for controlling the activation levels of ptks physiologically . a higher level of ros can be produced in cells after direct attack of mitochondria by environment - linked oxidative agents .
evidence has been provided for a mechanism to localize ros signal at a specific subcellular compartment , which is essential for promoting redox - linked signaling pathways after cell surface receptor activation .
ros production for inducing either cell activation or cell death has been suggested to be membrane raft linked [ 24 , 62 , 63 ] and might be localized through interactions of nadph oxidase with signaling platforms associated with lipid rafts . favoring in part this speculation , kawamoto et al .
recently obtained experimental results suggesting that cells exposed to sulfhydryl - reactive arsenite generate ros outside mitochondria ( unpublished observation ) .
both the development and functions of living organisms are basically self - regulated by information natively included in genes .
thus , genes code for tertiary structures of proteins , including receptors and their ligands , which provide noncovalent bond - oriented molecular switches in signal transduction for either cell activation or cell death .
however , the environment conditionally gives some covalent bond - mediated nongenetical information to living organisms , particularly through environment - linked oxidizing or reducing ( redox ) reactions .
the redox reactions are principally reversible and might therefore replace some of the genetically prescribed noncovalent bond - mediated molecular switches for signal transduction . recent evidence introduced here
suggests that the environment - linked covalent bond ( chemical or redox reaction- ) mediated information plays a crucial role in controlling the genetically prescribed noncovalent bond - oriented signaling systems for cell activation or cell death .
the key target molecules for the covalent bond - mediated information are membrane lipid raft - associating cell surface receptors and intracellular ptks .
ptks as crucial targets of redox regulation are capable of triggering subsequent cascade reactions of signaling elements such as protein serine / threonine kinases and transcription factors .
the idea for general importance of redox regulation of ptks in overall signal transduction in living organisms corresponds well to the fact that the specific cysteine residue as the target of redox regulation is highly conserved in a number of human ptks .
environment - linked chemical or redox reactions control the genetically prescribed signal delivery system for either cell activation or cell death in living organisms .
accumulated evidence indicates that this includes environment - mediated control of ptks as a master key of intracellular signal transduction for cell activation and of several elements of signal transduction for cell death .
the pathway for environment - linked control of ptks include ( 1 ) clustering of cell surface receptors in association with membrane lipid rafts , potentially bypassing receptor - ligand interaction , and ( 2 ) membrane lipid raft - linked production of ros beneath the membrane .
this leads to ( 3 ) ros - mediated inactivation of ptps , which in turn upregulate ptks , and to ( 4 ) ros - mediated oxidative structural modification of ptks for bi - directional direct control of ptks through oxidization of sh groups of specific cysteines on ptks to either s s or s x .
upregulated ptks promote the subsequent signal transduction , ultimately resulting in either cell activation or cell death .
it is concluded that environment - linked chemical or redox reactions , which are mediated by covalent bonds , bidirectionally control the genetically prescribed and thereby protein tertiary structure - linked noncovalent bond - oriented signaling systems for either cell activation or cell death in living organisms . | recent observations on environment - linked control of genetically prescribed signaling systems for either cell activation or cell death have been reviewed with a focus on the regulation of activities of protein tyrosine kinases ( ptks ) .
the environment - linked redox reactions seem to primarily affect cell surface receptors and cell membrane lipid rafts , and they induce generation of reactive oxygen species ( ros ) in cells .
ros thus generated might upregulate the catalytic activities of ptks through inactivating protein tyrosine phosphatases that dephosphorylate and inactivate autophosphorylated ptks .
recent evidence has , however , demonstrated that ros could also directly oxidize sh groups of genetically conserved specific cysteines on ptks , sometimes producing disulfide - bonded dimers of ptk proteins , either for upregulation or downregulation of their catalytic activities .
the basic role of the redox reaction / covalent bond - mediated modification of protein tertiary structure - linked noncovalent bond - oriented signaling systems in living organisms is discussed . |
metastatic or loco - regionally advanced non - small cell lung carcinoma ( nsclc ) is one of the leading causes of death attacked with cancer in india and though treatment outcomes in advanced disease remain modest , with the paradigm shift in the approach to treatments
customized treatment based on the tumor histology and genetic profiles have become the standard of care .
systemic chemotherapy with the platinum based doublets is the standard of care in metastatic or advanced nsclc .
however , patients with advanced or metastatic disease eventually present disease progression and needs second - line systemic therapy in a selected group of patients or other supportive measures . second - line chemotherapy with docetaxel had shown to improve overall survival ( os ) by means of 3 months and to delay the deterioration of quality - of - life in patients with good performance status .
pemetrexed is another anti - folate approved for use in non - squamous histology nsclc as second - line chemotherapy and has emerged as a preferred option in this sub - group .
the ncic trial br21 had shown that the benefit of erlotinib after of first - line chemotherapy in nsclc .
treatment with erlotinib improves progression free survival ( pfs ) ( hazard ratio : 0.61 , p=0.001 ) and then erlotinib approved for the treatment of advanced nsclc after the failure of upfront chemotherapy .
interest , phase iii high - powered randomized study and the patients randomized to receive gefitinib or docetaxel as second - line therapy .
the analysis showed that the objective response and survival were similar in the two groups and consequently approved as second - line therapy in nsclc . in spite of the clinical benefit of the second - line treatment ,
there are reliable predictors of objective response , those who received first - line chemotherapy , but very little knowledge available from literature about the prognostic variables in patients , who receive second - line therapy .
there is a modest survival advantage in second - line therapy , at the same time leaves toxic side - effect .
we need to find out robust clinical and demographic predictive and prognostic factors to guide the second - line therapy in nsclc after disease progression .
the objective of this study was to identify the clinical or demographic predictive factors for survival of advanced or metastatic nsclc who received second - line therapy .
we retrospectively reviewed 329 patients received second - line treatment from july 2007 to september 2011 in the department of radiation oncology , burdwan medical college and hospital .
they met the following inclusion criteria ;
histological or cytological proof of nsclcstage iii - iv ( brain metastasis excluded)received first - line platinum based doublet chemotherapythey had measurable disease as defined by who criteriathey were 18 years or older in age
.
histological or cytological proof of nsclc stage iii - iv ( brain metastasis excluded ) received first - line platinum based doublet chemotherapy they had measurable disease as defined by who criteria they were 18 years or older in age .
a total of 12 potential prognostic variables were chosen as the basis of previously published clinical trials .
the variables were categorized as
age ( 60 years vs. > 60 years)sex ( male / female)performance status ( ecog 0 - 1/2 - 3)smoking status ( never smoker / smoker or formerly smoker)serum lactate dehydrogenase ( normal / elevated)histology ( adenocarcinoma / non - adenocarcinoma)stage at diagnosis ( stage iiia and iiib or stage iv)first - line chemotherapy ( platinum based doublet , etoposide vs. paclitaxel or gemcitabine or pemetrexed)response to first - line chemotherapy ( clinical disease control or progressive disease)pfs after first - line chemotherapy ( 6 months/>6 months)site of failure ( intrathoracic vs. intra - thoracic and distant / distant / brain metastasis)second - line chemotherapy ( base supportive care vs. docetaxel / pemetrexed / erlotinib / gefitinib )
age ( 60 years vs. > 60 years ) performance status ( ecog 0 - 1/2 - 3 ) smoking status ( never smoker / smoker or formerly smoker ) serum lactate dehydrogenase ( normal / elevated ) histology ( adenocarcinoma / non - adenocarcinoma ) stage at diagnosis ( stage iiia and iiib or stage iv ) first - line chemotherapy ( platinum based doublet , etoposide vs. paclitaxel or gemcitabine or pemetrexed ) response to first - line chemotherapy ( clinical disease control or progressive disease ) pfs after first - line chemotherapy ( 6 months/>6 months ) site of failure ( intrathoracic vs. intra - thoracic and distant / distant / brain metastasis ) second - line chemotherapy ( base supportive care vs. docetaxel / pemetrexed
/ erlotinib / gefitinib ) all of the analysis performed using the spss statistical software program package ( spss version 16 , spss inc . , chicago ) .
the difference of the clinical characteristic between the groups was analyzed by chi - square test and student t - test .
os calculated from the first cycle of chemotherapy to the date of death from any cause or the date of last follow - up .
survival after second - line chemotherapy calculated from the 1 day of starting second - line therapy to the date of death from any cause or date of last follow - up .
pfs defined as the period from the first day of chemotherapy until the progression of disease or death .
we retrospectively reviewed 329 patients received second - line treatment from july 2007 to september 2011 in the department of radiation oncology , burdwan medical college and hospital .
they met the following inclusion criteria ;
histological or cytological proof of nsclcstage iii - iv ( brain metastasis excluded)received first - line platinum based doublet chemotherapythey had measurable disease as defined by who criteriathey were 18 years or older in age
.
histological or cytological proof of nsclc stage iii - iv ( brain metastasis excluded ) received first - line platinum based doublet chemotherapy they had measurable disease as defined by who criteria they were 18 years or older in age .
a total of 12 potential prognostic variables were chosen as the basis of previously published clinical trials .
the variables were categorized as
age ( 60 years vs. > 60 years)sex ( male / female)performance status ( ecog 0 - 1/2 - 3)smoking status ( never smoker / smoker or formerly smoker)serum lactate dehydrogenase ( normal / elevated)histology ( adenocarcinoma / non - adenocarcinoma)stage at diagnosis ( stage iiia and iiib or stage iv)first - line chemotherapy ( platinum based doublet , etoposide vs. paclitaxel or gemcitabine or pemetrexed)response to first - line chemotherapy ( clinical disease control or progressive disease)pfs after first - line chemotherapy ( 6 months/>6 months)site of failure ( intrathoracic vs. intra - thoracic and distant / distant / brain metastasis)second - line chemotherapy ( base supportive care vs. docetaxel / pemetrexed / erlotinib / gefitinib )
age ( 60 years vs. > 60 years ) performance status ( ecog 0 - 1/2 - 3 ) smoking status ( never smoker / smoker or formerly smoker ) serum lactate dehydrogenase ( normal / elevated ) histology ( adenocarcinoma / non - adenocarcinoma ) stage at diagnosis ( stage iiia and iiib or stage iv ) first - line chemotherapy ( platinum based doublet , etoposide vs. paclitaxel or gemcitabine or pemetrexed ) response to first - line chemotherapy ( clinical disease control or progressive disease ) pfs after first - line chemotherapy ( 6 months/>6 months ) site of failure ( intrathoracic vs. intra - thoracic and distant / distant / brain metastasis ) second - line chemotherapy ( base supportive care vs. docetaxel / pemetrexed / erlotinib / gefitinib )
all of the analysis performed using the spss statistical software program package ( spss version 16 , spss inc . , chicago ) .
the difference of the clinical characteristic between the groups was analyzed by chi - square test and student t - test .
os calculated from the first cycle of chemotherapy to the date of death from any cause or the date of last follow - up .
survival after second - line chemotherapy calculated from the 1 day of starting second - line therapy to the date of death from any cause or date of last follow - up .
pfs defined as the period from the first day of chemotherapy until the progression of disease or death .
between july 2007 and september 2011 , 329 patients received second - line systemic therapy or best supportive care ( bsc ) after failing platinum based doublet chemotherapy .
patients with good ( ecog 0 - 1 ) and poor performance ( ecog 2 - 3 ) status was 66.3% and 33.7% respectively .
the median survival after the starting of second - line treatment or bsc was 5 months .
baseline characteristic of patients the results of univariate analysis of survival after second - line treatment and os are summarized in table 2 . among the 12 variables included for binary logistic analysis ,
all the above identified to have significant prognostic factors for survival after second - line treatment except the stage at diagnosis , 1 line chemotherapy and site of failure after 1 line chemotherapy .
univariate analysis of categorical variables multivariate analysis by cox regression model included the prognostic significant variables from the univariate analysis .
multivariate analysis by cox regression model has shown that performance status ( ps ) and second - line treatments were independent prognostic factors for survival after second - line treatment . furthermore , performance status , age , smoking habits , pfs after 1 line therapy and second - line therapy were the variables considered an independent prognostic factor for os [ tables 3 and 4 , figures 14 ] .
multivariate analysis of categorical variables by cox regression model for survival after second line therapy and os prognostic variables , os and survival after second - line treatment kaplan - meier curve estimates of overall survival in patients with ecog 0 - 1 or ecog 2 - 3 performance status kaplan - meier curve estimates of survival after second - line therapy in patients with ecog 0 - 1 or ecog 2 - 3 performance status kaplan - meier curve estimates of overall survival in patients treated with docetaxel / pemetrexed / erlotinib / gefitinib or best supportive care kaplan - meier curve estimates of survival after second line therapy in patients treated with docetaxel / pemetrexed / erlotinib / gefitinib or best supportive care
between july 2007 and september 2011 , 329 patients received second - line systemic therapy or best supportive care ( bsc ) after failing platinum based doublet chemotherapy .
patients with good ( ecog 0 - 1 ) and poor performance ( ecog 2 - 3 ) status was 66.3% and 33.7% respectively .
the median survival after the starting of second - line treatment or bsc was 5 months .
the results of univariate analysis of survival after second - line treatment and os are summarized in table 2 . among the 12 variables included for binary logistic analysis , all the above identified to have significant prognostic factors for survival after second - line treatment except the stage at diagnosis , 1 line chemotherapy and site of failure after 1 line chemotherapy .
univariate analysis of categorical variables multivariate analysis by cox regression model included the prognostic significant variables from the univariate analysis .
multivariate analysis by cox regression model has shown that performance status ( ps ) and second - line treatments were independent prognostic factors for survival after second - line treatment . furthermore , performance status , age , smoking habits , pfs after 1 line therapy and second - line therapy were the variables considered an independent prognostic factor for os [ tables 3 and 4 , figures 14 ] .
multivariate analysis of categorical variables by cox regression model for survival after second line therapy and os prognostic variables , os and survival after second - line treatment kaplan - meier curve estimates of overall survival in patients with ecog 0 - 1 or ecog 2 - 3 performance status kaplan - meier curve estimates of survival after second - line therapy in patients with ecog 0 - 1 or ecog 2 - 3 performance status kaplan - meier curve estimates of overall survival in patients treated with docetaxel / pemetrexed / erlotinib / gefitinib or best supportive care kaplan - meier curve estimates of survival after second line therapy in patients treated with docetaxel / pemetrexed / erlotinib / gefitinib or best supportive care
docetaxel , pemetrexed and tkis ( erlotinib and gefitinib ) have significantly changed the treatment paradigm for advanced nsclc .
however , the optimal integration of first - line and second - line chemotherapy is yet to be defined . at this time
, the selection of second - line cytotoxic agent or only the bsc may depends numbers of factors including physician preferences , patients preferences , smoking history , smoking history and tumor histology .
give the incurable nature of the advanced nsclc and modest survival seen after second - line setting , patient 's preferences and convenience should considered when selecting the second - line agents .
few prior studies had shown that performance status , gender , histopathology , stage at diagnosis , type of initial chemotherapy , the best response to first - line chemotherapy , pfs after upfront chemotherapy have strong predictive value in os and os after second - line chemotherapy in univariate analysis . however , this trend did not persist in multivariate regression analysis ; the only variables have a strong correlation where the ps of patients and response to first - line chemotherapy .
this retrospective study has shown the potential influence of ps and the second - line cytotoxic therapy on the outcome of the second - line treatments and the ps as well as age , smoking habits , pfs and second - line therapy had strong predictive value in os .
the outcomes were similar regardless of the response to first - line cyto - toxic therapy and did not accord with the results of .
this might be due in inadequate treatment with first - line chemotherapy containing etoposide and retrospective review analysis .
the importance of ps also confirmed in our study and has shown poor ps had a negative effect on os and survival after second - line therapy or bsc only .
the current study demonstrated that the poor ps not only had a negative effect on os , also did not tolerate second - line cytotoxic treatment due to toxicity .
it remains ambiguous whether stage at the time of diagnosis ( iiia , iiib or iv ) in patients receiving second - line cytotoxic agent will ensure prognostic knowledge for os .
, di maio et al . , scartozzi et al . had shown significant predictive value of stage at diagnosis on the os while di maio et al . and zietemann and duell did not mention the prognostic value of the stage . in our study ,
stage at diagnosis did not identify as a prognostic variable for os or survival after second - line treatment .
this may be due to retrospective analysis , the difference of the treatment choice and etoposide included in the first - line chemotherapy regimen .
the main limitation of this study was retrospective study , we did not evaluate the molecular characteristic of tumor and first - line chemotherapy did not base upon the histopathology and small number of patients included for analysis .
the ps has shown consistent result as a prognostic factor in univariate and multivariate analysis for os and survival after second - line systemic treatment .
these findings may also facilitate pretreatment prediction of survival and be used for selecting patients for the correct choice of cyto - toxic therapy .
more prospective multicentric randomized studies are required to answer the treatment option in the second - line therapy in nsclc or beyond .
two additional areas that may be worth pursuing in the data collection would be quality - of - life improvement and toxicity experiences by the patients during or after second - line therapy . | background : platinum based doublets chemotherapy are the standard of care for metastatic or advanced non - small cell lung carcinoma .
this leads to modest survival advantage and improve quality - of - life .
however , patients with advanced or metastatic disease eventually present disease progression and needs second - line systemic therapy in a selected group of patients or other supportive measures .
there is very little knowledge available from the literature about the prognostic variables in patients , who receive second - line therapy.materials and methods : we retrospectively reviewed 329 patients received second - line treatment from july 2007 to september 2011 in the department of radiation oncology , burdwan medical college and hospital . for statistical analysis ,
12 potential prognostic variables included .
univariate and multivariate regression analysis carried out to identify the prognostic variables associated with survival.results:the results of univariate analysis for overall survival ( os ) and survival after second - line therapy identified to have prognostic significance : age , sex , performance status , smoking history , serum lactate dehydrogenase , histopathology , first - line chemotherapy and its response and second - line therapy except the stage at diagnosis and site of failure after first - line therapy .
the multivariate cox regression analysis has shown that only performance and second - line therapy were independent prognostic variables for survival after second - line treatment and above these prognostic factors ; age , smoking status and progression free survival also for os.conclusion:the performance status has shown consistent result as a prognostic factor in univariate and multivariate analysis for os and survival after second - line therapy .
these findings may also facilitate pretreatment prediction of survival and be used for selecting patients for the correct choice of cytotoxic therapy . |
one concept that unites all therapeutic modalities including the surgical , medical , pharmacological , physiotherapeutic ( as well as other health professions - related ) and the psychological , is change ; specifically the valid confirmation of an improvement in a patient s health status due to intervention .
this issue has been at the core of medicine and rehabilitation since their very inception as clinicians always aspired to treat successfully those in need of acute as well as chronic conditions .
primum non nocerefirst ( above all ) , do no harm-was attributed to thomas sydenham , a 17th century prominent physician ( 16241689 ) , ( smith , 2005 ) .
both attest to the fact that it is the moral / professional duty of the physician not to bring upon a negative change i.e. to worsen the health status of the patient .
indeed , what is change , in terms of either improvement or worsening ( aggravation ) , is a complicated issue and , as this paper will indicate , involves clinical as well as statistical considerations .
however change relates very intimately to the concept of difference i.e. change is judged based on a difference in some measurable entity and almost always it relates to a time span .
for example , if a due to exercise rehabilitation , a patient with an arthroscopically reconstructed anterior cruciate ligament ( acl ) increases his lysholm score ( lysholm and gillquist , 1982 ) from 61 to 72 within , say 2 weeks , this will be considered , prima faciae , a positive change . on the other and , if a patient is deemed to be moving his neck
better based on a visual inspection of his cervical movement , such a decision is qualitatively - based and hence the confirmation of a change is , at best , questionable .
that said , in some instances the difference may be so dramatic that no measurement is actually necessary but in this paper , such changes are not the issue .
rather , the objective is to find out why change is not simple as it may seem to be and how is it applied at the level of individual patient / subject . why is the confirmation of change such a critical issue in rehabilitation ? there are , in principle , two instances which mandate the estimation of change .
one is during the intervention phase when a decision has to be made whether the patient has improved sufficiently to justify progressing from one regimen to another .
for example , in many orthopedic cases , e.g. following shoulder ligamentous tears , the initial stage is the restitution of functional active range of motion .
once this is achieved progression ( change ) , in the form of increased muscular exertion may follow , consisting initially of isometric contractions and then concentric contractions . only after
judging at what point in the rehabilitation process , such changes may be implemented , is a challenge for all parties involved .
the other instance relates to termination of rehabilitation i.e. when the patient is judged to have entered the treatment plateau at which no fundamental , functional or physiological change can be expected within reasonable medical probability in spite of continuing medical or rehabilitative procedures , the so - called maximal medical improvement ( mmi ) . here
the treating clinician as well as the patient wish to have a well - based decision regarding the mmi .
moreover , rehabilitation , much like medicine , is a costly component in health economics .
the insuring bodies , whether the government , public or private companies , insist nowadays on informed expenditure . in other words , therapeutic means
therefore , the issue of efficiency depends at least partly in showing progress or proving mmi . in its simplest form ,
change derives from the difference between the values of a specific outcome score taken over a time interval : t1 to t2 namely :
change = outcome measure score t2-outcome measure score t1 straightforward examples may relate e.g. to blood pressure , body temperature , white blood cell count , ecg etc . , but often the rate at which these differences s take place : (om)/t ( where om
it should be borne in mind that as far as the typical medical tests are concerned , the tests are performed using very sensitive and accurate instruments , and almost always without the active cooperation of the patient . moreover ,
modern testing systems and highly reliable test parameters are based on minimal involvement of the tester and thus his skill in performing the test is not a significant factor . on the other hand ,
assessing rehabilitation outcomes depends strongly on measuring human performance , and as a result rehabilitation - related changes take on a drastically different dimension . in this case , the human factor , both of the patient / subject and that of the tester , is decisive . as far as the previous is concerned , the patient / subject is an integral part of the test and his full cooperation is a sine qua non condition for its validity .
sub - optimal cooperation in performing the test due to factors such as fatigue , motivation , intelligence , emotional status etc .
have a direct effect on the test / assessment outcomes . as for the latter ,
these background factors , which are largely absent from the abovementioned ordinary clinical tests introduce
noise into the measurement process namely the outcome scores include factors that are irrelevant to the measured entity and reflect temporary effects .
this situation has led to the acute need in accounting for these confounding factors which result in apparent but largely irrelevant effect on the scores of outcome measures .
the solution was to analyze the reproducibility of the findings using a test - retest paradigm , where the retest is undertaken under the same conditions of the
test. what such paradigm yields is a measure of the consistency of the results . as a rule , the closer the results in the retest are to those obtained in the test , the higher the reproducibility and vice versa .
moreover , a high reproducibility means that the effect of the confounding factors is limited . since with the lapse of time ( normally days or weeks )
some inevitable discrepancy between the test and retest scores occurs , no human performance - related parameter enjoys a perfect reproducibility .
however , assuming equal conditions and ideal reproduction of the test scores , the analysis allows one to state with statistical confidence that based on a previous reproducibility study , whether a change ( difference ) in the scores reflects normal fluctuations ( the confounding part ) or that it is the result of intervention .
thus in assessing change , irrespective of the measurement instrument ( the most sophisticated or a simple questionnaire ) , two critical factors must be addressed : the reproducibility ( consistency ) and the accuracy of the measurement . as mentioned above , the reproducibility is the degree to which repeated measurements under unchanged conditions show the same results while the accuracy of a measurement is the degree of closeness of the measurement of a quantity to that quantity s reference ( true ) value ( fig .
1 ) . these two elements are independent , namely measurements may be accurate but not reproducible or vice versa , neither accurate nor reproducible or both .
for example , if an experiment contains a systematic error , then increasing the sample size generally increases reproducibility but does not improve accuracy in the sense that the measurements fall away from the true value due to a flawed test or experiment . eliminating the systematic error improves the accuracy without changing the reproducibility .
in addition to accuracy and reproducibility , measurements are also characterized by their resolution ( responsiveness ) which is the smallest change in the underlying physical quantity that produces a response in the measurement .
measurement systems that successfully address all 3 characteristics at a high level are valid for clinical applications .
to demonstrate the application of these concepts to real life situation consider the following question : in terms of accuracy , reproducibility and resolution , how do manual muscle testing , isometric and isokinetic , differ from each other in testing muscle strength ? starting first with manual muscle testing ( mmt ) , it should be borne in mind that this is a semi - quantitative measurement tool which is also highly nonlinear e.g. the difference between grade 1 and 2 is not equal for the difference between 4 and 5 . if the strength of the subject or patient is above 3 , it must be 4 or 5 .
subdivisions into 4 , 4 , and 4 + have been made since the difference between grades 4 and 5 spans about 90% of the strength potential of the muscle ( dvir , 1997 ) .
however , this subdivision does not help in making the system more accurate and it even renders it less reproducible as the results of repeated tests do not zoom on the same value ( e.g. 4 + ) .
moreover , in terms of responsiveness mmt is specifically poor ; increasing or decreasing the contraction level of the muscles does not necessarily lead to a change in the mmt grades unless the increase / decrease is at least 35% of the total force ( sapega , 1990 ) .
this all leads to a clinical assessment tool that is very poor in confirming change .
using instrumented isometric testing in the form of a hand held dynamometer ( hhd ) is a decisive step forward .
the reproducibility of hhd has been extensively studied and found to be satisfactory ( bohannon , 2012 ) .
this means that the test - retest results of isometric strength performed over days are sufficiently close to be clinically acceptable .
these instruments are also quite accurate when correlated with other dynamometers but may suffer from bias , particularly when the tester himself is not strong enough . moreover , hhds are not suitable for e.g. trunk muscles testing , limiting their applicability .
however hhds are responsive namely , increase or decrease in the contraction output of the muscle can be traced with a resolution of 1n . finally ,
isokinetic assessment of muscle strength is accurate , reproducible and responsive ( dvir , 2004 ) as long as the muscle torque exceeds that of the gravitational torque ( when the plane of testing is anti - gravitational ) which satisfies the basic requirements of clinical validity of a measurement system .
thus isokinetic dynamometers can validly be used in subjects / patients that do not suffer from debilitating weakness ( typically mmt grades 1 , 2 , 3 ) and produce data that allow clinicians to judge even subtle changes .
finally , it should be emphasized that the cruder the resolution of the system , the chances of being more reproducible are higher .
thus if a tester is asked to rate the strength of muscles as either 4 or 5 ( based on bilateral testing ) , it is likely that the reproducibility will be high . in other words
if it is rated as 4 , on retest and under the same conditions , it will remain 4 .
however , the ability to make any clinical interpretation from such a result is absolutely limited as grade 4 is particularly wide .
if on the other hand the resolution is made higher e.g. one of the following : 3 + , 4 , 4 , 4 + , 5 then the clinical content is much higher but as the reproducibility is extremely low , there is little that can be inferred from such measurements .
the basic tenet is that measurements of the same clinical entity differ from one test session to the other , when spanned over days or weeks .
this is true for normal subjects and , especially , for patients . as mentioned above such a difference
this is the normal fluctuation taking place with respect to all biological parameters and in the relevant context .
suppose for example that stature ( height ) of an adult is measured by cm , standing upright and shod , and the result is 176 cm .
it is highly likely that on a second test , one week later , the result will be the same .
if however , the measurement is in mm , the first score of 1,763 mm may change to 1,765 mm a week later . has the subject increased in height ?
now , let us get back to the previous example where due to exercise rehabilitation , a patient with a reconstructed acl increased his lysholm score from 61 to 72 from week 6 to week 8 after the operation .
another patient who suffered from shoulder pain due to subacromial bursitis scored 78 mm on the visual analogue scale ( vas ) at the first visit to the therapist .
is this a clinically meaningful change ? before trying to answer these questions , the concepts of significant change vs clinically meaningful change have to be defined .
thus , if for instance a treatment method is assessed , and the design is based on an experimental , a control and a sham group , significant differences may be noted , for relevant outcome parameters on a within- and inter - group basis .
this can lead to a conclusion that the method changes ( improves ) significantly the health status of the patient group .
however it says nothing about the individual patient in the group which is best exemplified by fig .
in this example the mean value of a specific outcome measure of the group changes significantly to a higher level as indicated by the upward pointing solid arrow . on the other hand , 3 individuals who are included in this group , might not change ( =0 ) , improve with the group , although not as drastically ( >0 ) , or even deteriorate over the duration of the experiment ( <0 ) . therefore , whereas on the whole , the treatment is effective , at the level of the individual patient / subject this is not necessarily so .
significant change in the context of improvement or aggravation of a health status is misleading inasmuch as it relates to the individual .
this contrasts with the main objective of clinical practice , that is , to improve the condition of the individual or at least not to cause any harm .
this conflict furnished the basis for the relatively new research field relating to the concept of change at the individual level .
the research has initially focused on what is known as the error of measurement and its various descriptors , also known as clinimetric parameters . the term
smallest real difference ( srd ) , became increasingly applied and it related to some numerical value , the cutoff , that delineates what should be the clinically smallest meaningful change at the level of the individual from what is known as the error of measurement. it should be mentioned that these values may apply ( with the necessary variations ) to a group , but they are not used as often .
the srd is one of the mainstays of the distribution model which compares the change in a patient - reported outcome score to some measure of variability such as the sd , the effect size or the standard error of measurement ( sem ) .
the error of measurement which is the aggregate of factors that collectively blurs the true value of the measurement , serves as the main indicator for change .
included in the error of measurement are elements related to the measuring instruments , e.g. mechanical or electronic drift , the tester ( high vs low skill ) , the patient / subject , the test protocol and the test environmental conditions .
when weighted together , these factors result in the fact that the margins of uncertainty regarding an individual subject / patient are much wider than those acting for the group .
the srd derives directly from the sem ( lexell and downham , 2005 ) . in this context
, the sem is based on a test - retest study of a specific outcome measure .
the correlation coefficient - pearson s r or icc - is calculated along the common standard deviation ( sd ) and the sem then derives from the following equation : sem = sd(1-icc ) .
as can be seen , the sem takes into account both the dispersion around the mean and the degree of correlation between the two measurements .
thus , if the test and retest scores perfectly overlap , the icc is 1 and the sem is 0 . on the other hand if the icc is 0 , then the sem is equal to the pooled sd .
however , to derive the srd , which is applicable to the individual subject / patient ( heretofore srdi ) , at a level of confidence of 95% , the sem has to be multiplied by 1.962 .
thus , according to the distribution model approach , the smallest real difference indicating change at the level of the individual subject or patient , is 2.77 sem .
this fairly large margin accounts for the multitude of factors that can affect the real score . to indicate a clinically meaningful change at the group
lever - srdg - the cutoff is smaller and is equal to 1.96 sem . clearly , if a reproducibility study proves , that the test - retest correlation under strict experimental conditions is 1.0 , then the srd=0 ( individual or group ) which means that any difference in an outcome score is clinically meaningful .
for instance if a patient reports an improvement of one point up on the lysholm scale , say from 66 to 67 , this would be considered a clinically meaningful change .
obviously this is not the case , since perfect test - retest correlation based on a reasonable time interval ( e.g. 1 w ) , in patients , is a practical impossibility . moreover , even for the undiscerning eye , a difference of one point over a scale of 100 points , would be considered within the measurement error . on the other hand ,
how many points are needed to establish a clinically meaningful difference in terms of the srd is something that can not be estimated unless a proper reproducibility study is undertaken . to further illuminate the use of the srd for assessing individual change
consider the following example in which a group of patients suffering from chronic cervical pain were assessed before and after the administration of a new exercise - based treatment for cervical pain . in order to find out who among the patients really benefitted from this treatment ,
namely exceeded the srdi / improved meaningfully , and whether the group as a whole benefitted from the treatment , namely the group s mean score was greater than the srdg , a test - retest study was undertaken prior to the study in this group .
the patients were asked to rate their cervical pain twice over a period of 1 week .
the test - retest correlation was 0.72 while the pooled ( test+retest ) sd was 25 mm .
the srdi was therefore 2.7725(10.72 ) = 37 mm whereas the srdg was 26 mm .
this means that only those patients who have lowered their vas score by 37 mm or more underwent a clinically meaningful change .
for instance a patient who has rated his vas as 88 mm and 60 mm before and after treatment , respectively , did not undergo a clinically meaningful change as the difference |88 - 60| = 28 mm was less than the cutoff value .
this would have been the same decision if the patient started with a vas score of e.g. 62 mm and completed the protocol with a vas of 28 mm .
furthermore , while in the previous case the improvement was by about 33% , in the latter it was by more than 50% , yet for both cases the result is the same : no meaningful clinical improvement . on the other hand if the group s mean score before the exercise program was 88 mm and after it 60 mm -the difference was greater than 26 mm and therefore , the treatment was effective .
after - before difference in the vas scores was highly significant ( p<0.001 ) and still remained significant ( p<0.05 ) even if the improvement was reduced to 20 mm .
this underlines the profound difference between an analysis which is based on straightforward statistical tests of significance ( e.g. t - test for matched samples ) and the srdg approach which is more vigorous . clearly , neither apply at the individual level .
the surprising result vis - - vis the individual patient namely that even differences as high as 35 mm are not sufficient to judge a clinically meaningful change stems from the strict standards imposed by the modern understanding of the meaning of change .
there is little doubt that with reasonable sds , had a group of patients with neck pain improve by 50% or even 33% , such an improvement could qualify as significant , paving the way for the possible further application of the new protocol , subject to its relevancy .
however , when the individual patient is concerned , the margin of error is much wider as
errors do not even out in the same way as they do for a group .
thus , in this case , even a dramatic improvement in the vas may not qualify for a change .
on the other hand , setting strict criteria , and applying them to the individual patient , allows e.g. a more focused look at the possible advantages and pitfalls of a specific treatment approach while enabling the clinician to apply the approach with more confidence .
looking at the sem - based srd formula , it is clear that two factors have a direct effect of the magnitude of the srd : the sd and the icc ( r ) .
largely , increasing the sample size should reduce the sd and thus bring down the srd .
equally , increasing the icc will decrease the value of (1-icc ) with the same effect .
easier for the individual patient to exceed the srdi and increase the rate of clinically meaningful
under the same conditions , the more homogenous the group , the lesser is the icc ( r ) .
thus , extreme cases ( outliers ) will push the icc up but at the same time will make the group less homogenous , inevitably limiting the generalizations that may operate with respect to a certain intervention . reproducibility studies that furnish
the building stones for srd analysis tend to include relatively small number of participants with the inevitable consequence that the srd is relatively large leading to sometimes paradoxical results .
one solution for this weakness is to increase the sample size as a larger n plays a decisive role in reducing the sd .
in addition , sticking to well established protocols and using accurate and responsive instruments should further help in having a larger percentage of the participants cross the srdi cutoff .
it should be emphasized that the srd is not the only parameter used for the analysis of change but certainly one of the most common .
as mentioned above , the distribution model is heavily based on reproducibility studies that quantify the error of measurement , enabling the separation of the true change from the
this is an objective approach that does not consider either the patient s or the clinician s subjective feeling / impression / judgment regarding the success , no effect or even failure of the intervention .
this realization led to the application of an alternative approach to the issue of change , the so - called anchor - based approach .
the mainstay of this approach is the minimal clinically important difference ( mcid ) , a concept that differs quite sharply from the clinimetric srd .
the mcid has been defined in multiple ways but the most essential definition is probably the one by jaeschke et al .
( 1989 ) which stipulates that it is the smallest difference in a score , in a domain of interest , that patients perceive as beneficial and that would mandate , in the absence of side - effects , a change in the patient s management . another often quoted definition is by the american thoracic society ( 2007 ) the smallest difference that clinicians and patients would care about .
according to this model , the change in a patient - reported outcome score is compared to some other measure of change - the anchor or the external criterion .
thus the objective error of measurement is essentially eliminated and replaced by a coupling of subjective and an objective measures .
the mcid is also known by the terms smallest detectable difference ( sdd ) , minimal detectable change ( mdc ) or minimal detectable difference ( mdd ) .
to demonstrate the specific qualities of this approach consider the study by michener et al .
( 2011 ) where 136 patients presenting with shoulder pain were assessed twice , before and after 34 weeks of rehabilitation .
prior to treatment the patients filled the 11-point numeric pain rating scale ( nrs , fig .
they did so with respect to 3 conditions of pain : at rest , with normal daily activities , and with strenuous activities .
patients also filled the penn shoulder score ( pss ) which includes questions relating to pain ( based on an 11 p scale ) , satisfaction ( based on 7 categories scale ) , and function ( based on 5-category scale and 20 questions ) .
ten and sixty , respectively , bringing the total score to 100 points ( for a full view of the pss see , http://www.eliterehabsolutions.com/pdfs/penn%20shoulder%20score.pdf . )
previous research has indicated that for the function scale , 8.6 points ( about 15% ) is the mcid for the function section namely , patients can be classified as meaningfully improved if their score is 8.6 point , or not improved , otherwise .
the mcid for the average nprs for all patients was 2.17 , for both the surgical and nonsurgical subgroup .
this means that a change of slightly more than 2 points , around 20% of the full scale , is the smallest difference value associated with a clinically meaningful change .
obviously with the existence of another parameter - the anchor and an associated ( mcid ) begs the question why use the other one .
the answer is the efficiency of using the latter and the relatively straightforward findings it provides .
for example , although smaller mcids have been found , e.g. 1.3 on the 010 numeric pain rating scale ( cleland et al . , 2008 ) , the majority of studies looking at different disorders , have indicated that using similar scales for pain the typical mcid was 2 and above .
thus it could be argued that a meaningful improvement could roughly be traced when a decrease of about 20% in the rating of the pain was achieved .
another issue is the relationship between the mcid and the level of statistical significance when an intervention is applied .
it is possible an mcid might be larger than the difference associated with statistical significance , especially a clinical trial involving a large population . under such circumstances the
significant difference could be of little practical importance as the situation regarding the individual participant in such a study , or indeed a patient , is unclear .
an opposite situation may also arise when clinicians believe they know fairly well what the mcid might look like , e.g. an improvement of 20 in knee rom following some surgical intervention although the difference was not significant .
it should be understood that the mcid is instrument - dependent , where instrument means most often a questionnaire of some sort . moreover , although such questionnaires , that frequently refer to quality of life ( qol ) or function , look quite similar , they are objective - dependent . thus for example , a qol questionnaire relating to problems with lower limb , should dwell particularly on ambulation whereas for the upper limb , an important issue would be manipulation of objects . to sum up , judging the existence of change in rehabilitation is very complex .
this situation is a result of the special type of assessment instruments , the role of the patient and the clinician in performing the test , and the fluctuating nature of some of the main outcome measures e.g. pain and function . as a result
. moreover , even significant differences fail to tell whether a change occurs , unless the objective of the decision is the group .
thus , in order to judge the existence of change , in most cases due to intervention but occasionally as a natural resolution of the symptoms , specific approaches to the problem have to be used . the former model is based on reproducibility parameters such as the sem and normally mandates large and homogenous samples whereas the latter is particularly suitable when functional measures are employed .
the change parameters derived from these approaches are not identical in value and the decision which is more appropriate is context - dependent .
clearly , although much has yet to be done , our current understanding already permits a deeper insight into the fascinating problem of change .
determination of change in the context of rehabilitation is an involved issue but that requires critical attention .
modern statistical methods combined with profound clinical reasoning may give the clinical community appropriate tools for deciding whether a change observed as a mere difference score is also a clinically meaningful one . | the valid confirmation of a positive change ( improvement ) in a patient s health status due to intervention has been at the core of medicine and rehabilitation since their very inception as clinicians always aspired to ensure that treating their patients had led to successful outcomes both in acute and chronic conditions .
however what is change : either improvement or worsening ( aggravation ) , is a complicated issue which involves clinical as well as statistical considerations .
change invariably relates to a difference in some measurable entity and almost always it relates to a time span .
the confirmation of clinical change is important both for varying the treatment course ( if necessary ) and for the termination of treatment when the latter has reached wither its prescribed objective or a plateau . since in the context of rehabilitation , the outcome measures ( om ) are strongly linked to performance , determination of change in the latter is confounded by many factors , collectively known as the error of measurement , which render a decision regarding clinically meaningful change , highly involved .
this is further complicated by the stability of the observed om , the so - called reproducibility of the om , and the accuracy of the measurement instrument .
the higher the reproducibility the lower is the error .
moreover , in order to proclaim change , in most cases a positive one , it is necessary for the difference in outcome scores ( i.e. the change ) to surpass the error of measurement , in varying degree of rigor .
this paper describes selected methods associated with determination of change and focuses predominantly on the difference between a simple difference in scores ( simple change ) , a significant difference in scores and the so - called clinically meaningful change in scores which is considered today as the benchmark for confirmation of a real change . |
functional popliteal artery entrapment syndrome ( paes ) is an important and possibly underrecognized cause of exertional leg pain .
it shares many features with other causes of exertional leg pain , and more than one condition can be present at once , confusing the clinical picture .
an understanding of the typical presenting features of the common causes of exertional leg pain is essential , allowing the clinician to determine those suggestive of paes and requiring further investigation . investigating functional paes
is fraught with potential problems and , if performed incorrectly , can result in false negative and false positive findings .
the authors believe that currently accepted vascular investigations such as ankle - brachial indices and doppler ultrasound performed at rest are not accurate in investigating functional paes . rather , a review of the literature would suggest that investigations such as provocative doppler ultrasound and mri angiography are performed as soon as possible after reproducing symptoms to capture the occlusion while it is occurring .
once the diagnosis of functional paes is confirmed , there are a number of treatment strategies available . until recently , definitive intervention was only available in the form of vascular surgery with variable myotomies and releases .
we provide information on a pilot study suggesting a new less invasive intervention of guided botulinum toxin injection to the level of entrapment , as an alternative to surgical intervention .
paes shares many clinical features with other causes of exertional leg pain , most of which are thought to be more common .
adding to the complexity , more than one cause of exertional leg pain may be present in an individual patient at any one time .
chronic exertional compartment syndrome ( cecs ) in particular has many of the same features of paes and the two conditions can be confused [ 2 , 3 ] . also , it is not possible to distinguish between anatomical and functional paes on clinical symptoms alone .
a detailed history and examination provide very useful information and help determine whether further investigation is warranted .
an outline of the more common differential diagnoses of exertional leg pain and their features follows and a summary is provided in table 1 .
this condition is thought to represent the most common cause of exertional leg pain with an incidence between 13 and 42% .
patients describe typically bilateral leg pain along a strip of the distal third of the posteromedial tibial border that comes on early with exercise and may warm up but usually will ache for some time after stopping .
examination at rest will usually reveal palpable tenderness along the distal third of the posteromedial tibial border and pain with hopping .
some studies suggest a possible predisposition of mtss in females . while stress fractures are not common in the nonexercising population , they are thought to represent 0.720% of sports medicine clinic presentations .
patients typically complain of pain that is focal and palpable ( unless the site is covered by a significant muscle layer where symptoms can be vague and nondescript ) . with lower limb stress fractures
, pain often presents early with impact related exercise and is worse during the landing phase of running .
stress fractures are typically unilateral and can affect any bone in the lower limbs , although the tibia is most common [ 1 , 9 ] .
female athletes are thought to be at greater risk of developing stress fractures than males and bennell et al .
suggest that menstrual disturbances , caloric restriction , lower bone density , muscle weakness , and leg length differences are important risk factors for stress fracture .
patients by definition are pain - free at rest and develop a steadily worsening pain with exertion .
the pain will gradually build up over 1020 minutes and is not rapid in onset ( unlike some cases of paes ) .
the pain is typically isolated to one or more compartments , most commonly anterior and deep posterior , with the least most common being the superficial posterior compartment ( where the majority of paes pain is described ) [ 5 , 9 ] .
the pain will typically dissipate slowly over minutes to hours but will return quickly if the patient attempts to return to exercise again .
the leg pain is overwhelmingly bilateral ( although unilateral cases can present ) and there is no difference in predisposition according to the sex of the individual [ 7 , 11 ] .
paes can be further divided into two groups , anatomical and functional . in the case of anatomical paes , there is a clearly defined anatomical lesion that directly leads to entrapment and subsequent occlusion of the popliteal artery .
the second and larger subgroup of anatomically normal or functional paes is particularly poorly understood . in functional paes
, there is evidence of popliteal artery occlusion and subsequent claudicant symptoms , but no defined lesion can be found that directly causes the occlusion .
patients with anatomical paes are thought to be older and more sedentary while functional paes patients are thought to be younger , more commonly female , and more active .
baltopoulos et al . suggest that functional paes may be bilateral in 2576% of cases .
the true incidence of functional paes is unknown , and it is described as being rare [ 8 , 13 , 14 ] . however , it is possible that this condition is underreported and the incidence may be greater than previously recognized .
it occurs most commonly in the back of the calf but it can be anterior and lateral especially if the anterior tibial artery is involved [ 2 , 16 ] .
patients can have pain with minimal exertion or provocative leg positioning and occasionally at rest .
symptoms such as coldness and paraesthesia have also been reported . running or walking on an incline
the pain follows a claudicant pattern similar to cecs but unlike cecs will resolve more quickly on cessation of exercise .
provocative manoeuvres may result in the development of a reduced peripheral pulses and/or a popliteal bruit on auscultation of the popliteal fossa although this may also be possible in the asymptomatic occluder group as outlined later .
when examining patients with suspected functional paes , it is important to diagnose and/or exclude other causes of exertional leg pain .
examination includes palpation and percussion over the lower limb to look for signs of bone stress , be it focal at the site of a stress fracture or along a strip of the distal posteromedial tibial border in the case of mtss .
some authors suggest that paes must have a combination of pain with hopping , in conjunction with the development of ischaemic signs like pallor , coldness , and reactive hyperaemia . in our experience ,
pain with hopping more likely indicates some form of bone stress , and the development of physical signs of ischaemia in younger individuals with suspected functional paes is extremely uncommon .
some authors describe physical examination as unreliable and rely on clinical suspicion as justification for progressing to vascular investigations .
some do not describe their clinical assessment , while others attempt to provoke symptoms by encouraging the patient to hop or climb stairs , followed by a cursory physical examination .
most will assess resting peripheral pulses [ 12 , 18 ] which should be examined at true rest .
the popliteal fossa should be auscultated to determine the presence of a resting bruit . in general , bruits are not audible until an artery is approximately 50% occluded .
the sound increases in pitch as the lumen becomes more narrowed to a critical size .
thereafter , the sound may no longer be detectable as the volume of blood flow becomes greatly reduced . if pulses are reduced or a bruit is present at rest , than this would indicate an underlying vascular malformation or significant luminal narrowing ( such as in advanced atherosclerosis ) and further radiological imaging is indicated .
we have developed a simple clinical test that can be performed in the consulting rooms . after assessing for a popliteal fossa bruit and examining peripheral pulses at rest , the patient is required to perform 1520 single leg eccentric heel drops off the edge of a step , while asking about any developing or worsening leg pain .
immediately after the test is performed , the popliteal fossa is again auscultated for a bruit , and peripheral pulses are examined for any reduction . in our experience , it is worth auscultating for a number of minutes after exertion , as more significant cases ( with complete occlusion ) may initially have no bruit , or at least delayed onset of a bruit as flow reestablishes .
we believe that if patients do not develop pain or discomfort with this test , or if a bruit and/or pulse reduction is not evident , then it is unlikely that the patient is suffering from paes .
unfortunately , the development of pain / bruit / pulse reduction does not mean the patient has paes , as it is possible that they may fall into the asymptomatic occluder group .
provided that the patient has clinical features suggestive of paes , a positive result warrants further vascular investigation .
despite paes being a well - defined condition , no clear - cut consensus regarding the diagnostic work - up of these patients exists .
the general population can be divided into four groups , including asymptomatic nonoccluders , who presumably will not present for assessment or investigation .
therefore , the three symptomatic groups that investigation must distinguish areanatomical paes , asymptomatic occluder ( i.e. , exertional leg pain due to another cause , but in whom the artery can incidentally occlude),symptomatic occluder , that is , functional paes .
asymptomatic occluder ( i.e. , exertional leg pain due to another cause , but in whom the artery can incidentally occlude ) , symptomatic occluder , that is , functional paes .
the abi is calculated by dividing the systolic blood pressure at the ankle by that at the arm .
some authors recommend the use of ankle brachial indices in standard work - up for functional paes .
however , as occlusion in functional paes occurs during exertion , testing after exercise is likely to result in a false negative result .
there are also difficulties in obtaining abi measurements during exercise and pillai suggests that abis during forceful plantarflexion are difficult to interpret and not as helpful when assessing graded compression of a patent artery .
some authors will routinely perform compartment pressure studies in addition to vascular studies in the work - up of suspected paes .
this seems to be excessive and our review of the literature could find no compelling reason as to why this should be mandatory .
for this reason we do not recommend routine compartment pressure studies , unless symptoms and history are strongly suggestive of possible concomitant cecs .
doppler ultrasound provides a relatively cheap , noninvasive , and accessible procedure to assess flow through the popliteal artery , and it is generally recommended that this be the first line investigation for paes [ 20 , 21 ] . despite this , a review of the literature shows no definite consensus on how to perform doppler ultrasound in investigating paes .
this assesses loss of the posterior tibial artery distal pulse during sustained passive dorsiflexion and plantarflexion of the foot .
also , there is a risk of overcalling entrapment with movement of the artery , muscles , or probe during exercise giving the illusion of occlusion , but technique variations can usually overcome this .
many authors researching paes suggest that the most important feature in its diagnosis is the reproduction of symptoms with the help of provocative manoeuvres and verification by duplex ultrasonography [ 17 , 18 , 21 ] .
typically , investigations at rest will show a patent popliteal artery with normal distal pulses .
in functional paes , occlusion will often only occur during exertion and can resolve almost immediately .
even the 3060 seconds taken from getting off a treadmill to an examination bed and applying the ultrasound probe can be enough for arterial occlusion to cease . because occlusion can resolve very quickly , immediate assessment after reproduction of symptoms is essential .
the position of most occlusions occurs when the patient has their knee extended , and they hold active plantarflexion .
it is not possible to hold this position for sustained periods during a mri due to discomfort and fatigue .
ultrasound is a useful modality , as it allows real time assessment whilst some form of provocation to reproduce symptoms is performed . despite the agreement that provocation is necessary , a clear and detailed description of the protocols to do this is generally not provided .
we perform a resting anatomical study first , with the patient in the supine position .
we record waveforms and velocities of the peripheral arteries as well as assess for evidence of intimal thickening or fixed arterial disease .
the anatomy of the popliteal fossa is assessed , looking particularly for any popliteal artery or soft tissue structure variations or whether the anterior tibial artery has a high bifurcation .
hoffman et al . found that the force of plantarflexion required to occlude the popliteal artery during provocative positioning is important , with the majority of asymptomatic occluders occluding the popliteal artery with sustained holding of 70% of maximal plantarflexion force .
for this reason , we use a graded ultrasound protocol progressing throughprone patient holding a position of provocation ( knee extension and plantarflexion ) against no load , patient is prone and pushing against approximately 25% of maximal plantarflexion force ( figure 1(a)),dynamic loading by pushing against 50100% of maximal plantarflexion force ( figure 1(b ) ) .
prone patient holding a position of provocation ( knee extension and plantarflexion ) against no load , patient is prone and pushing against approximately 25% of maximal plantarflexion force ( figure 1(a ) ) , dynamic loading by pushing against 50100% of maximal plantarflexion force ( figure 1(b ) ) .
symptomatic patients who occlude in the first 2 categories are presumed to represent more severe cases of functional paes .
if the initial 2 assessments are negative , the patient is assessed in an erect weight - bearing position while cycling through range of motion .
this is thought to represent somewhere between 50 and 100% maximal plantarflexion force and may more reliably represent what is occurring functionally during activity . if the erect assessment is negative , the patient may attempt similar exertion that elucidates symptoms in a normal training experience , until they are symptomatic . at this point
we will use cine loops to demonstrate the popliteal artery from resting patency through compression to occlusion and mark the site of compression on skin for mri correlation .
we feel that doppler ultrasound does not adequately demonstrate the anatomy to rule out anatomical paes and confirmation of occlusion in a symptomatic patient means the patient may be in either the anatomical or functional paes groups .
mri is a valuable noninvasive modality that allows optimal visualization of the popliteal artery as well as the surrounding structures [ 23 , 24 ] .
mri can demonstrate a variety of findings including abnormal lateralized insertion of the medial head of gastrocnemius , medial displacement , and occlusion of the popliteal artery in the popliteal fossa and fat tissue filling the normal location of the medial head of gastrocnemius [ 23 , 25 ] . in this way
, anatomical paes can be distinguished from functional paes and the muscles and anatomical boundaries contributing can be accurately delineated .
this in turn can direct injection therapy ( as discussed in section 5 ) or the site of surgical intervention .
mri angiography can demonstrate the level of occlusion , but limitations include the development of movement artifact with forceful plantarflexion positions and inability to hold such a position for prolonged periods .
this is a significant limitation as for reasons outlined earlier ; investigating for occlusion in functional paes should occur during provocation .
most papers reviewed do not describe provocative manoeuvres with mri angiography [ 2325 ] while one suggests holding sustained forceful plantarflexion continuously for 2029 seconds whilst the angiogram is performed .
after positive ultrasound studies , a fiducial marker is placed on the patient 's skin where the popliteal artery is being occluded to help correlate between the ultrasound and mri site of occlusion . t1
weighted axial and coronal images are acquired to demonstrate the medial head and lateral heads of gastrocnemius , popliteus , and plantaris muscles and their alignment and associations with the femoral condyles and popliteal arteries and nerves .
the patient is instructed to dorsiflex and plantarflex their feet whilst acquiring t2 weighted 2d steady state images axially across the popliteal region . before the final contrast mri angiogram is performed ,
the patient is instructed to alternate a neutral ankle position with maximal plantarflexion force until they stimulate the pain that they usually experience ( rather than a single sustained forceful contraction ) .
once they experience this pain , they keep their ankles in plantarflexion whilst we inject the contrast and perform the angiogram ( figure 2 ) .
one of the disadvantages of this technique is that there is an approximate 30-second delay before the contrast arrives at the popliteal artery and the angiogram can be performed and the artery may re - establish flow during this time .
patients quite often are in pain and or exhausted during this last series and may shake because of this .
based on our review of the literature , figure 3 represents a decision - making flowchart in the assessment , work - up , and treatment of suspected paes .
given the described rapid progression of arterial injury , it is recommended that anatomical paes patients undergo surgery to remove the site of entrapment .
this typically involves exploration , limited fasciotomy , myotomy to varying degrees , and possible excision of occlusive fibrous bands [ 2 , 17 ] .
results of popliteal fossa exploration , bypass , or muscle detachment , or a combination of these , and fossa decompression are generally good .
most series report a small number of patients , but > 90% appear to return to activities in sports 3 months with resolution of all previous symptoms .
the pathogenesis and progression of functionalpaes are uncertain , but it may be that these patients develop arterial injury more gradually with onset of more significant symptoms later in life .
turnipseed recommends resection of plantaris muscle and the crural band of soleus fascia that forms the outlet of the popliteal fossa as he feels that this fascial band is the fulcrum against which the neurovascular bundle is compressed in functional paes .
however the site and amount of muscle necessary to be removed to prevent further occlusion is not always obvious .
the popliteal artery will need exploration and there are increased risks of postoperative complications such as seroma ( 4.6% ) and infection ( 2% ) .
also surgery for functional paes does not seem to be as successful as that for anatomical paes , with reports suggesting only 77% of patients after surgery reporting complete resolution of symptoms .
the use of botulinum injections for paralysis of muscles to manage medical conditions is well established .
there are several descriptions of the use of botulinum in the treatment of muscle spasticity , particularly in cerebral palsy patients [ 2729 ] , and piriformis injection of btx - a has been used successfully to treat sacral plexus and proximal sciatic nerve compression .
described the use of btx - a injection into a crus of the hemidiaphragm to treat renal artery stenosis .
the proposed mechanisms of action for intramuscular periarterial botulinum therapy for paes areparalysis of the muscular slip of muscle responsible for the dynamic arterial occlusion , localised muscle atrophy caused by the botulinum which may increase space for the vessel and would explain the prolonged effect of botulinum on this condition beyond the expected therapeutic effect of the medication , possible arterial smooth muscle relaxation of the popliteal artery resulting in vasodilatation .
paralysis of the muscular slip of muscle responsible for the dynamic arterial occlusion , localised muscle atrophy caused by the botulinum which may increase space for the vessel and would explain the prolonged effect of botulinum on this condition beyond the expected therapeutic effect of the medication , possible arterial smooth muscle relaxation of the popliteal artery resulting in vasodilatation .
unfortunately , to date , there is no published data on the efficacy of botulinum injection in the management of functional paes .
we have commenced a pilot study using intramuscular periarterial injection of btx - a to treat functional paes with promising initial results .
we hope to publish the outcomes as our cohort size increases , but at present this remains an unproven intervention .
functional paes is a condition that is possibly underrecognized and , if left untreated , can result in progressive arterial damage and the risk of developing lower limb ischaemia .
it shares many features with other causes of exertional leg pain ( especially chronic exertional compartment syndrome ) and may coexist with one or more of these .
a suggestive clinical history includes features of pain aggravated by exercise , but also possibly at rest with positions of knee extension and plantarflexion .
the pain will typically resolve quickly once provocative manoeuvres are ceased , although an ache may persist for hours .
anatomical and functional paes can not be distinguished on clinical features alone , and possibly over half of the normal population can demonstrate some arterial occlusion with provocative manoeuvres .
for this reason , specialized vascular investigations are indicated , particularly a doppler ultrasound protocol performed at rest and during provocation and immediately after , which can demonstrate real time arterial occlusion and the level it is occurring at .
once occlusion is demonstrated , mri can demonstrate the definitive anatomy of the popliteal fossa , whether anatomical paes exists , and the site and extent of functional entrapment .
from here , the best treatment can be provided , with consideration of guided botox injection for functional paes as a potential new intervention , or progression to surgical intervention . | functional popliteal artery entrapment syndrome ( paes ) is an important and possibly underrecognized cause of exertional leg pain ( elp ) .
as it is poorly understood , it is at risk of misdiagnosis and mismanagement .
the features indicative of paes are outlined , as it can share features with other causes of elp .
investigating functional paes is also fraught with potential problems and if it is performed incorrectly , it can result in false negative and false positive findings .
a review of the current vascular investigations is provided , highlighting some of the limitations standard tests have in determining functional paes .
once a clinical suspicion for paes is satisfied , it is necessary to further distinguish the subcategories of anatomical and functional entrapment and the group of asymptomatic occluders . when definitive entrapment is confirmed , it is important to identify the level of entrapment so that precise intervention can be performed .
treatment strategies for functional paes are discussed , including the possibility of a new , less invasive intervention of guided botulinum toxin injection at the level of entrapment as an alternative to vascular surgery . |
the cardiovascular health study ( chs ) is a national institutes of health national heart , lung , and blood institute funded , population - based longitudinal study of adults aged 65 years ( 7 ) .
( a full list of principal chs investigators and institutions can be found at http://www.chs-nhlbi.org/pi.htm . )
the main objective of the study was to study the onset and course of coronary heart disease and stroke .
communities : forsyth county , north carolina ; sacramento county , california ; washington county , maryland ; and pittsburgh , pennsylvania .
subsequent biannual in - person or telephone contacts were used to ascertain and verify the incidence of outcome events . for our analysis ,
we excluded 78 women with missing information on parity , 59 with missing information on baseline diabetes status , and 45 with missing information on other covariates , leaving a final eligible sample of 3,211 women .
our main exposure of interest was parity , assessed in women at the baseline interview , with the question , how many live births have you had ?
the response was modeled as a categorical variable : nulliparous ( 0 births ) , 12 livebirths , 34 livebirths , and grand multiparity ( 5 livebirths ) .
information on medication use was ascertained annually with detailed medication inventories ( 8) . fasting serum blood glucose and insulin were measured at the central laboratory at the university of vermont in 19891990 , 19921993 , and 19961997 ( 9 ) . in this cohort of older women , we studied both the cross - sectional relationship between parity and prevalent diabetes at baseline and the association of parity with incident diabetes only among those who were nondiabetic at baseline ( n = 2,761 ) .
baseline diabetes was assessed using data from the baseline clinic examination and defined with american diabetes association criteria as the use of hypoglycemic medications or a fasting blood glucose of 126 mg / dl .
incident diabetes was defined as the first use of diabetes medications or by a criterion of fasting blood glucose of 126 mg / dl during the follow - up years for which fasting blood glucose was available .
insulin resistance was estimated at baseline using homa - ir , calculated by the following formula : homa - ir = fasting serum insulin ( microunits per milliliter ) fasting plasma glucose ( millimoles per liter)/22.5 .
we used descriptive statistics to characterize participant 's demographic characteristics at baseline . to examine the relationship between parity and prevalent diabetes , we estimated the prevalence ratios using generalized linear models with a log - link and poisson distribution ( 10 ) .
models using a binomial link yielded similar point estimates and confidence intervals but did not converge in all cases . to examine the association between parity and incident diabetes in those without diabetes at baseline , we used cox proportional hazard models to estimate hazard risk ratios related to parity . for both the incident and prevalent diabetes outcomes , we examined the relationship between parity and outcomes in multivariable sequentially adjusted models .
the next models controlled for several possible confounders including age , race , income ( three categories ) , education ( more than high school , high school , or less than high school ) , marital status ( married , single , widowed , or divorced ) , height ( centimeters ) smoking ( current , never , or former ) , study site , and alcohol intake ( user / abstainer ) .
our final models contained all of the previously listed potential confounders and in addition controlled for body anthropometrics as potential mediators ( baseline bmi [ calculated from measured weight and height ] , height and measured waist circumference , and self - report of bmi at age 50 years ; because of missing data , age 50 years bmi was imputed from age , race , and baseline bmi in 128 women ) . in these final models
, we examined the degree of mediation using the sas mediate macro ( 11 ) .
we also conducted multivariable linear regression analyses to examine whether parity was associated with baseline fasting serum glucose , insulin levels , and homa - ir only among those without diabetes at baseline .
because insulin and homa - ir were , as expected , right - skewed , both were log - transformed to improve normality .
our main exposure of interest was parity , assessed in women at the baseline interview , with the question , how many live births have you had ?
the response was modeled as a categorical variable : nulliparous ( 0 births ) , 12 livebirths , 34 livebirths , and grand multiparity ( 5 livebirths ) .
information on medication use was ascertained annually with detailed medication inventories ( 8) . fasting serum blood glucose and insulin were measured at the central laboratory at the university of vermont in 19891990 , 19921993 , and 19961997 ( 9 ) . in this cohort of older women , we studied both the cross - sectional relationship between parity and prevalent diabetes at baseline and the association of parity with incident diabetes only among those who were nondiabetic at baseline ( n = 2,761 ) .
baseline diabetes was assessed using data from the baseline clinic examination and defined with american diabetes association criteria as the use of hypoglycemic medications or a fasting blood glucose of 126 mg / dl .
incident diabetes was defined as the first use of diabetes medications or by a criterion of fasting blood glucose of 126 mg / dl during the follow - up years for which fasting blood glucose was available .
insulin resistance was estimated at baseline using homa - ir , calculated by the following formula : homa - ir = fasting serum insulin ( microunits per milliliter ) fasting plasma glucose ( millimoles per liter)/22.5 .
we used descriptive statistics to characterize participant 's demographic characteristics at baseline . to examine the relationship between parity and prevalent diabetes , we estimated the prevalence ratios using generalized linear models with a log - link and poisson distribution ( 10 ) .
models using a binomial link yielded similar point estimates and confidence intervals but did not converge in all cases . to examine the association between parity and incident diabetes in those without diabetes at baseline , we used cox proportional hazard models to estimate hazard risk ratios related to parity . for both the incident and prevalent diabetes outcomes , we examined the relationship between parity and outcomes in multivariable sequentially adjusted models .
the next models controlled for several possible confounders including age , race , income ( three categories ) , education ( more than high school , high school , or less than high school ) , marital status ( married , single , widowed , or divorced ) , height ( centimeters ) smoking ( current , never , or former ) , study site , and alcohol intake ( user / abstainer ) .
our final models contained all of the previously listed potential confounders and in addition controlled for body anthropometrics as potential mediators ( baseline bmi [ calculated from measured weight and height ] , height and measured waist circumference , and self - report of bmi at age 50 years ; because of missing data , age 50 years bmi was imputed from age , race , and baseline bmi in 128 women ) . in these final models , we examined the degree of mediation using the sas mediate macro ( 11 ) .
we also conducted multivariable linear regression analyses to examine whether parity was associated with baseline fasting serum glucose , insulin levels , and homa - ir only among those without diabetes at baseline .
because insulin and homa - ir were , as expected , right - skewed , both were log - transformed to improve normality .
those with grand multiparity ( 5 livebirths ) were more likely to be black , have larger bmi and waist circumferences at baseline , more likely to abstain from alcohol , and less likely to have graduated from high school .
in unadjusted analyses , diabetes was considerably more prevalent in women with grand multiparity ( 25% ) compared with those with fewer livebirths ( 12% ) and with those who were nulliparous ( 15% ; p < 0.001 ) .
baseline characteristics of women enrolled in chs data are means sd or n ( % ) . in regression models controlling for age and race
, we observed a nearly 60% increase in prevalent diabetes associated with grand multiparity compared with women who were nulliparous ( table 2 ) .
after addition of demographic and clinical factors ( including measures of socioeconomic status ) to the model , the observed increase in prevalent diabetes associated with grand multiparity was reduced to 33% but remained statistically significant . in a final model that controlled for body anthropometrics ,
the prevalence of diabetes associated with grand multiparity was 21% higher than that for the referent nulliparous group and no longer significant .
the degree of attenuation was somewhat less if we adjusted only for bmi recalled from age 50 years rather than measured bmi also ( prevalence ratio 1.27 [ 95% ci , 0.961.68 ] ) , consistent with a greater degree of measurement error in recalled bmi . comparing generalized estimating equation models with and without adjustment for anthropometrics , these factors explained 44% ( [ 286% ] ; p = 0.04 ) of the adjusted association of grand multiparity with prevalent diabetes .
multivariable models of parity and prevalent diabetes data are prevalence ratio ( 95% ci ) .
* model 2 : age , race , marital status ( 4 ) , income ( 3 ) , education ( 3 ) , height ( in centimeters ) , alcohol yes / no , clinic ( 4 ) , and smoking ( 3 ) .
model 3 : also for bmi , bmi at age 50 years , and waist circumference .
we observed similar associations between parity and prevalent diabetes in blacks and whites ( table 2 ) . although the association of grand multiparity with prevalent diabetes was numerically stronger in blacks , interaction terms of race with grand multiparity were not significant , even in models that included anthropometrics ( p > 0.16 ) .
we next examined the relationship of parity with incident diabetes . despite the strong association of grand multiparity with diabetes at baseline ,
it was not associated with the incident development of diabetes in older women ( table 3 ) .
multivariable models of parity and incident diabetes data are n or hrs ( 95% ci ) .
* model 2 : age , race , marital status ( 4 ) , income ( 3 ) , education ( 3 ) , height ( in centimeters ) , alcohol yes / no , clinic ( 4 ) , and smoking ( 3 ) . unadjusted analysis suggested a modest trend toward increased fasting insulin and homa - ir associated with grandmultiparity , whereas fasting glucose was not significantly associated with parity ( table 4 ) . in multivariable models , controlling for age , race , marital status , income , education ,
height , alcohol use , clinic site , and smoking , we again observed no association with fasting glucose but a trend toward increased insulin and homa - ir with higher categories of parity ( ptrend = 0.01 for insulin and 0.02 for homa - ir ) .
parity and metabolic markers among those without diabetes data are means sd or coefficient ( sem ) .
* multivariable linear regression : models include age , race , marital status ( 4 ) , income ( 3 ) , education ( 3 ) , height ( in centimeters ) , alcohol yes / no , clinic ( 4 ) , and smoking ( 3 ) . in analyses stratified by race , there was no interaction between parity and race and fasting glucose ( p = 0.3 ) .
intermediate degrees of parity appeared to have a greater impact on insulin and homa - ir in black women than in white women , whereas the reverse was true for grandmultiparity ( table 4 ) .
interaction terms of parity ( defined categorically ) and race were statistically significant in the adjusted models with log(insulin ) and log(homa - ir ) as the outcome ( p = 0.02 for both ) .
when we tested the interaction specifically between race and grandmultiparity , the p values for effect modification by race were 0.90 , 0.06 , and 0.09 for glucose , log(insulin ) , and log(homa - ir ) , respectively .
the apparent differences by race in the relationship of parity with insulin resistance were not markedly different in analyses that further adjusted for anthropometrics ( data not shown ) .
in regression models controlling for age and race , we observed a nearly 60% increase in prevalent diabetes associated with grand multiparity compared with women who were nulliparous ( table 2 ) .
after addition of demographic and clinical factors ( including measures of socioeconomic status ) to the model , the observed increase in prevalent diabetes associated with grand multiparity was reduced to 33% but remained statistically significant . in a final model that controlled for body anthropometrics ,
the prevalence of diabetes associated with grand multiparity was 21% higher than that for the referent nulliparous group and no longer significant .
the degree of attenuation was somewhat less if we adjusted only for bmi recalled from age 50 years rather than measured bmi also ( prevalence ratio 1.27 [ 95% ci , 0.961.68 ] ) , consistent with a greater degree of measurement error in recalled bmi . comparing generalized estimating equation models with and without adjustment for anthropometrics , these factors explained 44% ( [ 286% ] ; p = 0.04 ) of the adjusted association of grand multiparity with prevalent diabetes .
multivariable models of parity and prevalent diabetes data are prevalence ratio ( 95% ci ) .
* model 2 : age , race , marital status ( 4 ) , income ( 3 ) , education ( 3 ) , height ( in centimeters ) , alcohol yes / no , clinic ( 4 ) , and smoking ( 3 ) . model 3 : also for bmi , bmi at age 50 years , and waist circumference .
we observed similar associations between parity and prevalent diabetes in blacks and whites ( table 2 ) .
although the association of grand multiparity with prevalent diabetes was numerically stronger in blacks , interaction terms of race with grand multiparity were not significant , even in models that included anthropometrics ( p > 0.16 ) .
we next examined the relationship of parity with incident diabetes . despite the strong association of grand multiparity with diabetes at baseline ,
it was not associated with the incident development of diabetes in older women ( table 3 ) .
multivariable models of parity and incident diabetes data are n or hrs ( 95% ci ) .
* model 2 : age , race , marital status ( 4 ) , income ( 3 ) , education ( 3 ) , height ( in centimeters ) , alcohol yes / no , clinic ( 4 ) , and smoking ( 3 ) .
unadjusted analysis suggested a modest trend toward increased fasting insulin and homa - ir associated with grandmultiparity , whereas fasting glucose was not significantly associated with parity ( table 4 ) .
in multivariable models , controlling for age , race , marital status , income , education , height , alcohol use , clinic site , and smoking , we again observed no association with fasting glucose but a trend toward increased insulin and homa - ir with higher categories of parity ( ptrend = 0.01 for insulin and 0.02 for homa - ir ) .
parity and metabolic markers among those without diabetes data are means sd or coefficient ( sem ) .
* multivariable linear regression : models include age , race , marital status ( 4 ) , income ( 3 ) , education ( 3 ) , height ( in centimeters ) , alcohol yes / no , clinic ( 4 ) , and smoking ( 3 ) . in analyses stratified by race , there was no interaction between parity and race and fasting glucose ( p = 0.3 ) .
intermediate degrees of parity appeared to have a greater impact on insulin and homa - ir in black women than in white women , whereas the reverse was true for grandmultiparity ( table 4 ) .
interaction terms of parity ( defined categorically ) and race were statistically significant in the adjusted models with log(insulin ) and log(homa - ir ) as the outcome ( p = 0.02 for both ) .
when we tested the interaction specifically between race and grandmultiparity , the p values for effect modification by race were 0.90 , 0.06 , and 0.09 for glucose , log(insulin ) , and log(homa - ir ) , respectively .
the apparent differences by race in the relationship of parity with insulin resistance were not markedly different in analyses that further adjusted for anthropometrics ( data not shown ) .
our study , among the first in a population - based cohort of older women , helps to elucidate the relationship of parity with diabetes among older women .
we found that grand multiparity ( 5 live births ) was associated with higher risk of prevalent diabetes in analyses controlling for age and race .
this association was attenuated but continued to be statistically significant after adjustment for demographic and clinical factors . when anthropometric measures were added to the models , the magnitude of association was further attenuated and became statistically insignificant .
thus , the association of grand multiparity with increased prevalence of diabetes seems to be confounded or mediated , in large part , by variation in sociodemographic factors and higher body weight associated with grand multiparity .
further , we did not observe any significant variation in the association of parity with diabetes between blacks and whites . on the other hand , among women who remained free of diabetes into older age , parity
was not associated with incident diabetes nor with fasting glucose among those free of diabetes .
however , there was a statistically significant , albeit weak in magnitude , association of parity and fasting insulin and homa - ir at baseline in women free of diabetes .
our data are generally consistent with the hypothesis that parity appears to play an ongoing role in the development of diabetes through middle age , but its effect wanes among older women who have remained nondiabetic . previous studies examining the relationship between parity and diabetes have come to discordant conclusions .
some have found that parity , particularly higher levels of parity , is associated with increased risk of diabetes .
for example , nicholson et al . ( 5 ) analyzed a cohort of middle - aged black and white women and found that those who had borne 5 children had twice the risk of incident diabetes in unadjusted analyses ( hazard ratio [ hr ] 2.10 [ 95% ci 1.732.53 ] ) . in their study , the increased risk of diabetes was attenuated but persisted after adjustment for factors including sociodemographic factors and anthropometrics ( adjusted hr 1.27 [ 1.021.57 ] ) .
consistent with our findings , an earlier study examining the relationship between parity and glucose homeostasis in middle - aged to older women found that after adjustment for covariates , each pregnancy was associated with increased fasting insulin and decreased insulin sensitivity that was not explained by obesity and body composition measures ( 12 ) .
this study suggested that changes in insulin sensitivity related to parity persist many years after child - bearing .
( 6 ) examined a cohort of > 120,000 registered nurses , aged 3055 years at baseline .
similar to our findings , their unadjusted analyses suggested that women with high parity ( defined in their study as 6 live births ) had a 50% higher risk of incident diabetes over 12 years of follow - up .
however , after adjustment for age and bmi , they observed no significant relationship between parity and incident diabetes .
their results are concordant with ours in connecting high levels of parity with diabetes in middle age and in the finding that the effects of high parity are largely related to the impact on midlife weight .
together , ours and the previous study emphasize the importance of weight management targeted to multiparous women .
we also had standardized measures of several potential confounding and mediating variables , including directly measured weight and waist circumference , allowing us to examine the mechanism of association of our relationship of interest .
first , some information that may have been helpful in the interpretation of the results , such as history of gestational diabetes mellitus , was not available .
next , our results may not be generalizable to other races and ethnicities , such as native americans and asians ( 13,14 ) , and we may have had limited power to detect differences between blacks and whites .
in addition , we had a limited number of cases of incident diabetes , particularly among grand multiparous women , 25% of whom already had diabetes at baseline , limiting our power to detect a small effect of grand multiparity on incident diabetes .
finally , because our analysis examines the relationship between a relatively early life exposure with outcomes much later in life , our results may be influenced by survivor bias .
accordingly , selective survival of women into older age may result in an attenuated measured association of parity with later diabetes outcomes . in summary , grand multiparity , but not lesser degrees of parity ,
much of the higher prevalence of diabetes associated with past child - bearing seems to be mediated ( or confounded ) by the heavier bmi associated with grand multiparity .
this finding presents an opportunity for education and intervention related to weight control among grand multiparous women to reduce diabetes prevalence . in nondiabetic women who have reached older age , higher parity
is not associated with increased risk of developing diabetes but tended to be associated with small increases in fasting insulin and homa - ir .
additional longitudinal studies designed specifically to study the long - term medical effects of child - bearing should be conducted .
future studies should include multiple longitudinal measures of diabetes related factors over time to better understand the interplay of risk factors over time . | objectiveto examine the relationship of parity with diabetes and markers of glucose homeostasis in older women.research design and methodswe used data from the female participants in the cardiovascular health study , a longitudinal cohort of adults aged 65 years .
these data included an assessment of parity ( baseline ) and fasting serum levels of glucose , insulin , and medication use ( baseline and follow - up ) .
we estimated both the cross - sectional relationship of parity with baseline diabetes and the relationship of parity with incident diabetes.resultsin unadjusted analyses , women with grand multiparity ( 5 live births ) had a higher prevalence of diabetes at baseline compared with those with fewer births and with nulliparous women ( 25 vs. 12 vs. 15% ; p < 0.001 ) . in regression models controlling for age and race , grand multiparity was associated with increased prevalence of diabetes ( prevalence ratio 1.57 [ 95% ci 1.202.06 ] ) ; with addition of demographic and clinical factors to the model , the association was attenuated ( 1.33 [ 1.001.77 ] ) . in final models that included body anthropometrics , the association was no longer significant ( 1.21 [ 0.861.49 ] ) . in those without diabetes at baseline , parity was not associated with incident diabetes or with fasting glucose ; however , there was a modest association of parity with fasting insulin and homeostasis assessment model of insulin resistance.conclusionsgrand multiparity is associated with diabetes in elderly women in cross - sectional analyses .
this relationship seems to be confounded and/or mediated by variation in body weight and sociodemographic factors by parity status . in older nondiabetic women , higher parity
does not pose an ongoing risk of developing diabetes . |
the use of nickel - titanium ( niti ) rotary instruments for root canal system preparation has increased due to their undeniably favorable qualities ; however , unexpected fracture is an important disadvantage of these instruments [ 13 ] .
controversy remains regarding the contribution of torsional fracture , fatigue fracture and the combination of both to the separation of niti rotary instruments .
some have implicated fatigue fracture to be a main reason for the separation of endodontic files in the clinical setting [ 2,5 , 6 ] .
fatigue fracture occurs due to repeated compressive and tensile stresses accumulated at the point of maximum flexure of an instrument rotating in a curved canal without the instrument being bind to the root canal .
it should also be noted that controversy exists regarding the effect of un - retrieved separated files in root canals on the outcome of endodontic treatments .
considering that the retrieval of separated instruments from the root canal system is very difficult and in some cases impossible and that un - retrieved fractured files may have an adverse effect on the outcome of endodontic treatments , overcoming or reducing the risk of this problem is of high clinical significance .
neoniti ( neolix , chtres - la - fort , france ) is a newly introduced niti rotary system with a non - homogeneous rectangular cross section and multiple taper in a single instrument ; it consists of one c1 and three a1 ( with # 20 , # 25 and # 40 tip sizes ) files .
the taper in the a1 # 25 file is 0.08 from d0 up to d5 ; whereas from d5 to d16 the taper is 0.04 . it is manufactured using a newly developed wirecut electrical discharge machining ( wedm ) process .
the manufacturer claims that this manufacturing process is highly precise down to the micron , oil - free and clean and stress is limited to the metal surface during this process .
furthermore , it produces a rough surface , resulting in abrasive properties that enhance the speed of root canal preparation .
moreover , the manufacturer claims that these files undergo appropriate heat treatment that results in high flexibility and shape memory of this system .
protaper next ( dentsply , tulsa dental specialties , tulsa , usa ) is another newly marketed rotary file system made of m - wire niti with a rectangular cross - sectional design .
twisted file ( sybronendo , orange , ca , usa ) is manufactured by twisting a niti wire with a triangular cross - section and r - phase crystalline structures created by heating and cooling .
after completion of twisting , the file is heated and cooled again to maintain its new shape while converting back into its austenite crystalline structure .
mtwo ( vdw gmbh , munich , germany ) and race ( fkg dentaire , la - chaux - de - fonds , switzerland ) rotary instruments are two routinely used systems for root canal cleaning and shaping .
s cross - section with two cutting edges resembling that of the s - file .
race instruments have a triangular or square - shaped cross - section and alternating cutting edges . considering that no research has been done regarding the mechanical properties of neoniti rotary files ,
the aim of this study was to compare the cyclic fatigue resistance of neoniti a1 # 25 , race 25/.06 , mtwo 25/.06 , twisted file 25/.06 and protaper next x2 file .
artificial grooves simulating three canals measuring 1.5 mm in width , 20 mm in length , and 2.5 in depth with a u - shaped cross - section were machined into 316l stainless steel blocks by computer - assisted milling and hardened with polished chrome plating .
three types of trajectories were designed according to the following parameters : group a : a straight cervical segment measuring 5.29 mm with an arc length of 9.42 mm , a curvature radius of 6 mm , a straight apical segment measuring 5.29 mm , and the arc in the middle portion of the canal ; group b : a straight segment measuring 7.44 mm with an arc length of 12.56 mm , a radius of 6 mm , and the arc in the apical portion of the canal ; group c : a straight cervical segment measuring 10.58 mm with an arc length of 9.42 mm , a curvature radius of 6 mm , and the arc located in the apical portion of the canal .
a 4-mm - thick glass was screwed in front of the simulated canals to prevent the instrument from slipping out ( fig .
1 ) . comparison between trajectories a and c was done to evaluate the influence of the arc location ( i.e. in the apical vs. the middle portion of the canal ) on the ncf of rotary instruments .
comparison between trajectories b and c was done to evaluate the influence of the arc length ( i.e. 9.42 mm or 12.56 mm ) on the ncf of rotary instruments .
five types of rotary files with a similar tip size were used and evaluated in this study :
neoniti a1 # 25race 25.06mtwo 25.06twisted file 25.06protaper next x2 file protaper next x2 file three types of trajectories designed for this study .
b. trajectory with curvature located in the apical third . c. trajectory with curvature located in the apical third with smaller arc length .
the hand - piece of an endodontic electromotor ( x - smart ; dentsply maillefer , ballaigues , switzerland ) with a reduction ratio of 1:20 was fixed above the block so cyclic fatigue could be assessed in a static mode ( fig .
2 ) . after putting a glass slab on the block and fixing the hand - piece to the block with the file in it , the instruments ( n=10 from each subgroup ) freely rotated in a
the rotational speed and torque suggested for all evaluated rotary instruments by their manufacturers were similar except for that of twisted file . to standardize the study ,
twisted file placed in the trajectory of group a ( right image ) liquid paraffin ( kimiagar toos , mashhad , iran ) was used as lubricant during the file rotation and it was applied on the canal walls by a micro - brush .
the instruments were used until fracture occurred and the time to fracture was recorded in seconds by two methods : ( a ) direct visualization with a 2.5 loupe ( heine hr binocular loupes , heine optotechnik , herrsching , germany ) and listening ( b ) playing captured videos by corel video studio prox2 software ( corel corp . , ottawa , canada ) .
the ncf was calculated using the following formula : ncf = time ( seconds ) to failure rotational speed/60 .
the exact angle and radius of the curvature in which each file was constrained in , were calculated from the snap shots taken from the files while rotating in the trajectories as measured by pruett et al . .
fractured tips were evaluated using a stereomicroscope ( model la - szm45-b1 , nanjing sunny optical instrument co. , ltd . ,
nanjing , china ) at 7 magnification and am423x dinoeye digital eyepiece and rll64 led light source ( led model ) .
images were saved in jpeg format by dinocapture 2.0 software and the length of the fractured file tips was measured .
one - way anova and tukey s hsd test were used for inter- and intra - group pairwise comparisons of ncf , separated fragment length , angle and radius of curvature ; the significance was determined at the 95% confidence level .
the overall mean time of fracture calculated visually and on the captured film was 82.0291.09 seconds and 80.5289.53 seconds , respectively .
the difference seen between the two different methods of calculation was not statistically significant ( p=0.059 ) .
the mean , minimum and maximum values of ncf for each group are shown in table 1 . according to the results of the current study in all curve types neoniti showed the highest and race showed the lowest ncf values ( p<0.05 ) , indicating the highest and lowest fatigue resistance , respectively . in group
a , a significant difference was seen in the ncf among the evaluated files ( p<0.001 ) .
3 ) : the mean standard deviation of ( a ) number of cycles to fracture ( b ) fragment length ( c ) angle of curvature and ( d ) radius of curvature of experimental subgroups . the mean standard deviation ( sd ) , minimum and maximum ncf values of each subgroup .
groups with statistically significant differences are shown with similar symbols ( alphabets or numbers ) .
neoniti and twisted file showed a significant difference when compared with each other ( p<0.001 ) .
furthermore , both neoniti and twisted file showed significant differences when compared to other subgroups ( p<0.001 ) . in groups
b and c , the ncf values from the lowest to the highest were as follows ( fig .
3 ) : race < protaper < twisted file < mtwo < neoniti . in groups
b and c , a significant difference was seen in the ncf among the evaluated files ( p<0.001 ) .
race , protaper and twisted file showed no statistically significant differences ( p>0.3 ) ; twisted file and mtwo had no statistically significant difference either ( p>0.4 ) ; whereas , neoniti had a statistically significant difference with all other groups ( p<0.001 ) .
the mean length of separated fragment of instruments in each subgroup is shown in table 2 .
overall , the fragment length of separated instrument in group a was significantly longer than that in group b ; furthermore , these values in group b were significantly longer than those in group c. in group a , no statistically significant difference was seen ( p>0.1 ) . the twisted file separated fragments were the longest while the race separated fragments were the shortest . in groups b and c ,
significant differences were seen in the separated fragment length of different groups ( p<0.001 ) .
b , the longest separated fragment length was seen in race ; whereas , in group c , the longest separated fragment length was seen in protaper . in group b , protaper , mtwo and twisted file
had no significant difference with all other file types ( p>0.2 ) ; whereas , race had a statistically significant difference with neoniti ( p=0.003 ) . in group c
, neoniti had a statistically significant difference when compared to all other file types ( p<0.001 ) ; whereas , twisted file and mtwo had no statistically significant difference ( p=0.4 ) .
the mean angle and radius of curvature of each subgroup are shown in table 2 .
overall , the highest and the lowest curvature angles were seen in race and neoniti , respectively . regarding the radius of curvature ,
neoniti and protaper showed the lowest and mtwo showed the highest angle of curvature . in groups
b and c , the angle and radius of curvature also showed significant differences ( p<0.001 ) . the highest and the lowest angle of curvature in group b belonged to protaper and neoniti , respectively ; whereas , in group c the highest and the lowest values were seen in race and neoniti , respectively ( fig .
3 , table 2 ) . in group a , neoniti showed the lowest radius of curvature ; whereas in groups b and c it showed the highest values ( fig .
the overall mean time of fracture calculated visually and on the captured film was 82.0291.09 seconds and 80.5289.53 seconds , respectively .
the difference seen between the two different methods of calculation was not statistically significant ( p=0.059 ) .
the mean , minimum and maximum values of ncf for each group are shown in table 1 . according to the results of the current study in all curve types neoniti showed the highest and race showed the lowest ncf values ( p<0.05 ) , indicating the highest and lowest fatigue resistance , respectively . in group
a , a significant difference was seen in the ncf among the evaluated files ( p<0.001 ) .
3 ) : the mean standard deviation of ( a ) number of cycles to fracture ( b ) fragment length ( c ) angle of curvature and ( d ) radius of curvature of experimental subgroups . the mean standard deviation ( sd ) , minimum and maximum ncf values of each subgroup .
groups with statistically significant differences are shown with similar symbols ( alphabets or numbers ) .
neoniti and twisted file showed a significant difference when compared with each other ( p<0.001 ) .
furthermore , both neoniti and twisted file showed significant differences when compared to other subgroups ( p<0.001 ) . in groups
b and c , the ncf values from the lowest to the highest were as follows ( fig .
3 ) : race < protaper < twisted file < mtwo < neoniti . in groups
b and c , a significant difference was seen in the ncf among the evaluated files ( p<0.001 ) .
race , protaper and twisted file showed no statistically significant differences ( p>0.3 ) ; twisted file and mtwo had no statistically significant difference either ( p>0.4 ) ; whereas , neoniti had a statistically significant difference with all other groups ( p<0.001 ) .
the mean length of separated fragment of instruments in each subgroup is shown in table 2 .
overall , the fragment length of separated instrument in group a was significantly longer than that in group b ; furthermore , these values in group b were significantly longer than those in group c. in group a , no statistically significant difference was seen ( p>0.1 ) .
the twisted file separated fragments were the longest while the race separated fragments were the shortest . in groups b and c , significant differences were seen in the separated fragment length of different groups ( p<0.001 ) .
in group b , the longest separated fragment length was seen in race ; whereas , in group c , the longest separated fragment length was seen in protaper . in group b , protaper , mtwo and twisted file
had no significant difference with all other file types ( p>0.2 ) ; whereas , race had a statistically significant difference with neoniti ( p=0.003 ) . in group c , neoniti had a statistically significant difference when compared to all other file types ( p<0.001 ) ; whereas , twisted file and mtwo had no statistically significant difference ( p=0.4 ) .
the mean angle and radius of curvature of each subgroup are shown in table 2 .
overall , the highest and the lowest curvature angles were seen in race and neoniti , respectively . regarding the radius of curvature ,
neoniti and protaper showed the lowest and mtwo showed the highest angle of curvature . in groups
b and c , the angle and radius of curvature also showed significant differences ( p<0.001 ) .
the highest and the lowest angle of curvature in group b belonged to protaper and neoniti , respectively ; whereas , in group c the highest and the lowest values were seen in race and neoniti , respectively ( fig .
3 , table 2 ) . in group a , neoniti showed the lowest radius of curvature ; whereas in groups b and c it showed the highest values ( fig .
different types of rotary files exhibit differences in resistance to fatigue failure due to differences in various determinants such as their manufacturing process , structural characteristics and geometric designs , surface texture , the type of curve they are introduced into and the method used for fatigue failure calculation .
according to the results of this study , no significant difference was seen between the fracture time estimated by direct visualization and watching a captured video clip . when reviewing the captured video clips ,
the time of fracture can be determined more precisely and easily due to lack of eye strain .
fatigue failure can be evaluated in two different modes of static and dynamic . in the dynamic mode ,
the hand - piece is promoted in a back - and - forth axial motion while the instrument is driven in a full rotational movement inside the trajectory .
this model is more similar to clinical conditions in which rotary files are used ; but to standardize the amplitude and speed of the axial movement of the hand - piece , a device must be designed .
it should be noted that the fatigue resistance of rotary files may be higher when used in the dynamic mode due to the axial movement , which prevents stress concentration in the same area allowing stress distribution along a larger portion of the instrument .
conducting another research that compares the fatigue failure of these rotary instruments in the dynamic mode is suggested .
three ways for improving the longevity of endodontic files have been suggested which include the following : ( i ) thermal treatments before machining ; ( ii ) choosing machining conditions adapted to the niti alloy and ( iii ) electro - polishing .
this study compared the cyclic fatigue resistance of different types of niti rotary files with different manufacturing techniques and different improvement strategies .
twisted files are formed by twisting a triangular blank in combination with heat treatment and special surface conditioning , which conserves the natural grain structure .
race files are developed by a grinding process , which is followed by electro - polishing .
the mean ncf of all rotary files was lower in group a compared to groups b and c implying that the resistance to fatigue failure is lower in files used in root canals exhibiting a more coronal curvature than apically placed curvatures . in more coronally located curvatures ,
the portion of the instrument situated in the critical stress concentration point has a greater diameter and undergoes higher levels of metal fatigue opposed to when the arc is located in the apical portion .
also these values were higher in group b in comparison to group c indicating that the shorter the length of the arc of curvature the higher the resistance of the file used in that trajectory to fatigue failure .
controversy remains regarding the effect of electro - polishing on the fatigue failure of rotary files .
some studies have shown that electro - polishing improves the instrument s working properties such as resistance to failure by producing a smooth and homogeneous protective surface oxide layer with less defects and residual surface stress ; whereas , herold et al .
inferred that electro - polishing did not prevent the development of microfractures and barbosa et al . concluded that electrochemical polishing had no influence on the resistance to fracture of the rotary instruments tested . according to the findings of our study race had the lowest ncf values in comparison to all other experimental rotary files in all canal types evaluated .
mtwo files are expected to show lower fatigue resistance than files undergone electro - polishing , such as race , due to the crack - like surface features created subsequent to their machining manufacturing process . on the contrary , the mtwo group exhibited higher ncf values than race , although the difference was not statistically significant .
in addition , while m - wire alloys are considered to be more flexible and more resistant to fatigue than conventional niti alloys , mtwo showed no statistically significant difference when compared to protaper next .
gambarini el al , also concluded that instruments made of m - wire alloy did not show higher resistance to fatigue when compared to instruments produced by the traditional niti .
twisted files showed significantly higher ncf values than race , protaper next and mtwo in curvatures located in the middle third of the trajectory ; whereas , in more apically located curvatures no significant difference was seen between the aforementioned files .
this may be due to the unique manufacturing process and heat treatment of this file .
further research regarding the physical and mechanical properties of this newly marketed file is required .
it should be noted that there is a slight inconsistency between the shape of the files and that of the trajectory they are rotating in .
this was seen when evaluating the angle and radius of curvature that each rotary file formed in the trajectories .
it is unclear whether the slight differences seen in the angle and radius of curvature between rotary file types have a clinically significant effect on the resistance to fatigue failure of the evaluated files .
for instance , while neoniti files used in the group a trajectory had a similar angle of curvature and shorter radius of curvature compared to other files in group a , this file had the highest ncf values .
it should be noted that lower fatigue resistance is expected in shorter radius of curvatures . in group c , although race files showed the highest angle of curvature , these files exhibited the lowest ncf values .
furthermore , neoniti files in this group exhibited the highest resistance to fatigue even though they had the lowest angle of curvature .
further research is required regarding the clinical impact of slight differences in the angle and radius of curvature on the resistance to fatigue failure of endodontic rotary files .
finally it should be noted that in clinical conditions , rotary files are used in a dynamic mode , endure torsional stresses and cyclic fatigue at the same time and are bind to the root canal walls ; therefore , their fatigue resistance may be different from the results of this study .
the fatigue resistance of the evaluated rotary files was lower in more coronally located curvatures .
neoniti exhibited the highest and race exhibited the lowest fatigue resistance compared to other evaluated files . | objectives : the purpose of this study was to evaluate the resistance to fatigue failure of five different nickel - titanium rotary files in three different curved trajectories.materials and methods : a total of 150 neoniti a1 , race 25.06 , mtwo 25.06 , twisted file 25.06 and protaper next x2 files with the tip size of 25 were tested ( n=30 for each group ) .
three groove types simulating curved canals were used differing in radius , arc length and position of the arc , each measuring 1.5 mm in width , 20 mm in total length and 2.5 in depth .
resistance to cyclic fatigue was determined by counting the numbers of cycles to failure .
furthermore , the fragment length of the fractured tips and angle and radius of curvature formed by each file in each trajectory were evaluated .
the data were analyzed by t - test , one way anova and tukey s hsd test.results:neoniti showed the highest and race showed the lowest number of cycles to fracture ( ncf ) values ( p<0.05 ) , indicating the highest and lowest fatigue resistance , respectively .
the highest and lowest curvature angles were seen in race and neoniti , respectively . regarding the radius of curvature ,
twisted file had the lowest and neoniti had the highest values .
the mean ncf of all rotary files was lower in the more coronally curved trajectory.conclusion:the fatigue resistance of the evaluated rotary files was lower in more coronally located curvatures .
neoniti exhibited the highest and race exhibited the lowest fatigue resistance compared to other evaluated files . |
congenital hereditary endothelial dystrophy ( ched ) is a corneal dystrophy characterized by bilateral diffuse clouding of the corneas , causing impaired vision . depending on the mode of inheritance , it manifests either in infancy or young childhood with a cloudy cornea , light sensitivity , tearing , and sometimes nystagmus.1
ched is primarily a disease of the corneal endothelium , but it has traditionally been treated with penetrating keratoplasty.2 targeted corneal endothelium transplantation techniques , such as descemet s stripping automated endothelial keratoplasty ( dsaek ) , have become a standard of care for the treatment of corneal endothelial dysfunction in adults,3 but few reports exist describing its use in the pediatric age group.47 in this report , the authors describe a case of dsaek being performed in a child with ched .
a 10-year - old boy presented to the corneal and refractive surgery unit as a referral from a local ophthalmologist due to poor vision and hazy corneas since infancy .
the father and both of the child s siblings were affected by the same condition .
a diagnosis of ched was made on the basis of clinical findings and the positive family history .
the corneas of both eyes were diffusely edematous ( figure 1 ) , with central corneal thicknesses of 921 m and 891 m in the right and left eyes respectively , measured with ultrasonic pachymetry .
intraocular pressures were 15 mmhg in the right eye and 18 mmhg in the left eye .
axial lengths of the eyes were slightly shorter than normal , being 22.53 mm and 22.49 mm , as confirmed on a - scan ultrasound biometry .
the patient was scheduled for dsaek on the right eye , to be performed under general anesthesia . to assist in graft sizing
, the eye was measured with a pair of calipers at the beginning of the procedure and was found to be 10.5 mm .
the endothelial graft was prepared using a moria cb microkeratome head ( 350 m blade depth ) and a moria altk artificial ( ac ) anterior chamber ( moria surgical , doylestown , pa ) .
the thickness of the donor cornea was 673 m , as measured by ultrasonic pachymetry .
after the microkeratome pass , the posterior lamella tissue was punched from the endothelial side using a disposable trephine , 8 mm in diameter .
the donor tissue was then covered with optisol solution ( chiron ophthalmics , irvine , ca ) until use . with the surgeon sitting temporally ,
the central corneal epithelium was debrided to improve visualization of the ac ( figure 2a ) .
a 1-mm side port was created at the 6 oclock position , through which an ac maintainer was inserted and the irrigating fluid turned on .
a 2.75-mm temporal clear corneal incision was created , through which descemet s membrane ( dm ) scouring was performed at the required diameter using a reverse sinskey hook ( figure 2b ) .
the ac maintainer was then turned off , and trypan blue dye was injected into the ac .
the ac maintainer was then turned back on to wash the dye out of the ac .
a 90 descemet s stripper was then used to strip dm from the overlying corneal stroma ( figure 2c ) .
a peripheral iridectomy was performed at the 11 oclock position using a vitrector ( figure 2d ) .
a 4.5-mm sized nasal clear corneal incision was created to prepare for insertion of the tip of the busin glide ( moria surgical).8,9 the donor disk was transferred to the busin glide with the endothelial surface oriented upwards .
the central area of the endothelium was coated with viscoelastic , taking care not to let viscoelastic reach the stromal side of the graft . with the surgeon sitting superiorly ,
the loaded glide was inverted and positioned at the entrance of the nasal clear corneal incision .
busin forceps were then passed through the temporal incision across the ac to grasp the donor disk at the glide opening .
the donor disk was pulled into the ac ( figure 2e ) and allowed to unfold spontaneously under continuous irrigation from the ac maintainer ( figure 2f ) .
an air bubble was injected under the graft and the ac maintainer was turned off ( figure 2 g ) .
the epithelial surface was then gently massaged using a blunt , bent , irrigating cannula centered on the disk ( figure 2h ) .
after centration was achieved , a large air bubble was injected into the ac to keep the disc adherent to the overlying stroma for around 15 minutes ( figure 3 ) . at the conclusion of the procedure
, some air was exchanged with balanced salt saline ( bss ) , reducing the bubble size to approximately 2/3rd of the ac volume .
postoperatively , the patient was positioned in a face - up position for the first 3 to 4 days .
alcon , fort worth , tx ) and 1% prednisolone acetate suspension ( pred forte , allergan , irvine , ca ) administered hourly for the first week .
best spectacle corrected visual acuity ( bscva ) at 3 months was 20/400 . at 1 year
, the patient had bscva of 20/100 with a refraction of + 4 0.5 180 , and was able to play soccer at school with this vision .
the corneal stroma , although it became clearer , did not become totally transparent , with moderate residual stromal haze being present even at 1 year while the central corneal thickness was 658 m ( figure 4a postoperative appearance at 2 weeks ; figure 4b appearance at 1 month postoperatively ; figure 4c postoperative appearance at 3 months ; figure 4d postoperative appearance at 1 year ) .
conventional penetrating keratoplasty has a higher risk of graft failure and poorer visual prognosis than that of adult keratoplasty .
children are harder to examine , their eyes have low scleral rigidity , and produce more of a fibrin reaction . during surgery , positive vitreous pressure may be experienced .
there is an increased risk of infection and rejection due to sutures loosening frequently , necessitating their early replacement or removal .
pkp wounds are more prone to traumatic rupture in children than in adults.10 targeted endothelial keratoplasty now forms an accepted standard of care for corneal endothelial dysfunction in adults.3 the benefits that endothelial transplantation procedures such as dsaek have over conventional pkp include faster visual rehabilitation , fewer or no sutures , and a structurally stronger globe .
these benefits are especially desirable in the pediatric age group . to date , however , reports of dsaek being performed in children are few in number , with varying degrees of success , with some being successful and others being converted to pkp . while performing dsaek in both cases , we found that the ac view was poor due to the corneal edema . debriding the corneal epithelium did little to improve visibility during the procedure .
movement inside the eye was made difficult by the shallow ac and phakic state of the eye ( figure 1 ) .
pineda et al , attempted to perform dsaek on an eye with ched , but the procedure was converted to a pkp due to difficulty in detaching descemet s membrane and poor clarity of the cornea.4 in contrast , the case the authors performed was successfully completed in spite of an inability to remove dm .
recipient interface can predispose to graft failure in dsaek.11 as the photos show , the time taken for the corneal stroma to clear after surgery was prolonged ( figure 4a d ) .
the corneal stroma retained some anterior haze for up to 1 year after surgery ( figure 5a appearance of corneal stroma at 3 months ; figure 5b appearance of corneal stroma at 6 months ; figure 5c appearance of corneal stroma at 1 year ) .
this suboptimal outcome may have been due to the persistent stromal haze or the coexistent amblyopia due to hyperopia .
had surgery been performed in a younger patient , there might have been less corneal haze and more potential for treating the amblyopia .
other recent reports describe dsaek being performed for ched in babies as young as 6 months12 and neonates.13 these cases could potentially reach better levels of vision as there is sufficient time to treat any associated amblyopia .
no examinations under anesthesia were required in this case , as the child was cooperative enough to have his sutures removed at the slit lamp .
this is in contrast to pediatric pkp , where multiple hospital admissions are usually needed during the postoperative course .
moreover , as the final refraction showed in this child , the procedure was refractively neutral , due to the absence of sutures .
in conclusion , dsaek can be used successfully in the treatment of ched in children , as an alternative to pkp .
dsaek allows for a more rapid visual rehabilitation of patients and fewer subsequent postoperative hospital admissions for treatment . | congenital hereditary endothelial dystrophy ( ched ) , presents in infancy or early childhood with bilateral clouding of corneas .
this condition has previously been managed surgically with penetrating keratoplasty ( pkp ) .
performing pkp in pediatric patients has its own set of difficulties .
more recently , there has been growing interest in treating this condition with descemet s stripping automated endothelial keratoplasty ( dsaek ) .
the purpose of this study is to report our experience of successfully performing dsaek in a child with ched . |
adrenohepatic fusion is a relatively common condition that has been reported in the literature and explained as a result of normal aging . however , the presence of a neoplastic process in the adrenohepatic fusion is relatively uncommon , especially when it is a malignant neoplasm . here ,
we present a patient with a malignant mass consisting of an adrenocortical carcinoma and hepatocellular carcinoma arising from an adrenohepatic union .
a 48-year - old male presented with his ruq abdominal pain that is relieved by lying on the right side and sleeping .
the patient had no past history of any disease , nor diagnosed of any chronic illnesses .
the computed tomography ( ct ) scan for abdomen was done and showed large retroperitoneal mass of heterogeneous density , with heterogeneous contrast enhancement posterior to the liver ( fig .
the mass measured 11 11 12 cm displacing the right kidney , and the right lobe of the liver and ivc anteriorly .
there was focal invasion of the liver measuring 3.6 6.4 cm at segment 7 .
the patient underwent an us - guided core biopsy of the mass that showed malignant cells consistent with both adrenocortical carcinoma and hepatocellular carcinoma ; however , the former diagnosis was favored based on the clinical picture .
the patient went for a right hepatectomy with right adrenalectomy , cholecystectomy and excision of associated lymph nodes .
the lesion and the excised structures were sent for histopathology that showed a right liver lobe mass attached to it measuring 17 10 7 cm , and the cut section of the mass showed nodular surface with multiple areas of necrosis .
slicing of the liver showed a gray white mass measuring 5 5 3 cm , which is adherent to the mass and the remaining liver parenchyma appear unremarkable .
the microscopic examination of the mass revealed a moderately differentiated hcc measuring 5 cm in diameter , confined to the liver , < 5 cm from resection margins and no lymph - vascular invasion ( fig .
2 ) , and a separate adrenocortical neoplasm measuring 17 cm in diameter , multiple foci of necrosis , no capsular or lymph - vascular invasion and resection margins were not involved by the tumor ( fig .
3 ) . excised lymph nodes included a single right portal vein lymph node , a gallbladder lymph node and seven para aortic lymph nodes .
figure 1:sections of the ct showing a large retroperitoneal mass , focal absence of fat gland and an invasion into the liver.figure 2:section from the hepatic mass showing positive staining of the tumor cells with the hepar ihc stain confirming the hepatic nature of the liver mass.figure 3:section from the adrenocortical mass showing strong positive cytoplasmic staining with low molecular weight cytokeratin - cam 5.2 ihc stain .
sections of the ct showing a large retroperitoneal mass , focal absence of fat gland and an invasion into the liver .
section from the hepatic mass showing positive staining of the tumor cells with the hepar ihc stain confirming the hepatic nature of the liver mass .
section from the adrenocortical mass showing strong positive cytoplasmic staining with low molecular weight cytokeratin - cam 5.2 ihc stain .
adrenohepatic fusion is a relatively common observation during autopsies ; however , neoplasms arising from the union are very poorly reported . in our case , an adrenohepatic fusion was detected using a ct scan of the abdomen that showed focal areas of loss of fat gland between the retroperitoneal mass and the liver .
dolan defined adrenohepatic union as the adhesion of the liver and the right adrenal cortex with the partial or complete absence of a fibrous capsule dividing the two organs .
adrenocortical adenoma arising from an adrenohepatic union was reported in several cases ; however , we could not find a report that describes an adrenocortical carcinoma combined with a hcc from an adrenohepatic union in the literature as the case we present here .
a differential diagnosis of hcc must be considered in such a case as the histopathology of the resected lesion shows mixed malignancies of adrenocortical carcinoma in the adrenal part of the lesion and a hcc in the hepatic part and several cases were reported in the literature of a primary hcc extending into the right adrenal directly , or to both adrenals as the first presentation of hcc metastasis , other reports mentioned cases where an hcc has developed in an ectopic liver tissue . on the other hand , an adrenocortical tumor that developed from an adrenal rest inside the liver with radiological findings of hcc
okuda introduced a few cases of hcc presenting as pedunculated masses outside the liver most of which had dual blood supply of a hepatic and a suprarenal artery that might support the fusion phenomenon , furthermore , none of the masses were discovered in the left adrenal , which , again , supports fusion and invasion rather than hematogenous spread .
core biopsy was reported in different similar cases to be of diagnostic value that changed the presumed diagnosis based solely on imaging modalities . in our case , after using a ct scan to diagnose the patient , a trucut biopsy was done to confirm the diagnosis of an adrenocortical carcinoma invading the liver ; however , an incidental histopathologic finding of co - occurrence of a hcc in the same lesion raised our suspicions , so we went to do a positron emission tomography scan cap to look for any focus of metastasis , which revealed negative results .
the patient was followed up for 2 years after the operation without evidence of metastasis or recurrence .
| abstractadrenohepatic fusion is a relatively common condition that has been reported in the literature and explained as a result of normal aging . however , the presence of a neoplastic process in the adrenohepatic fusion is relatively uncommon . a 48-year - old male presented with ruq abdominal pain with findings of a huge liver mass with adrenohepatic fusion in the computed tomography scan .
histopathological studies revealed a mixed , although distinctly separated , adrenocortical carcinoma and a hepatocellular carcinoma within an adrenoheaptic fusion . |
d - amino acid oxidase ( ec 1.4.3.3 , daao ) is a flavoprotein that catalyzes the enantioselective oxidative deamination of d - amino acids to yield the corresponding -imino acids , which are spontaneously hydrolyzed to -oxoacids and ammonia .
reoxidation of the reduced fad by molecular oxygen is accompanied by the release of hydrogen peroxide [ 1 , 2 ] .
daao is almost ubiquitous in eukaryotic organisms and fulfills different physiological functions : from a catabolic role in yeasts , which allows the use of d - amino acids as carbon and energy sources , to a regulatory role in the human brain , where it controls the levels of the neuromodulator d - serine [ 3 , 4 ] . in recent years
, a major biotechnological application of daaos has emerged for the industrial production of 7-aminocephalosporanic acid ( 7-aca ) , a key starting material for the preparation of semisynthetic cephalosporin antibiotics .
d - amino acid oxidases can catalyze the conversion of cephalosporin c to glutaryl-7-aminocephalosporanic acid ( glutaryl-7-aca ) , the first intermediate in the two step route that leads to 7-aca . in the second step of the process
, glutaryl-7-aca is hydrolyzed to 7-aca by a glutaryl-7-aca acylase , an enzyme from the group of penicillin amidohydrolases ( ec 3.5.1.14 ) .
daaos from several microbial sources have been demonstrated to work efficiently in the oxidative deamination of cephalosporin c [ 710 ] whereas daao from pig kidney ( pkdaao ) has been considered a poor catalyst in the same reaction .
in fact , immobilized trigonopsis variabilis cells with d - amino acid oxidase activity are actually the most employed biocatalyst for 7-aca production in industry .
cloning of the daao gene of t. variabilis in different hosts , such as escherichia coli [ 1216 ] , saccharomyces cerevisiae , kluyveromyces lactis , schizosaccharomyces pombe , and pichia pastoris [ 19 , 20 ] , has allowed obtaining the recombinant enzyme for biotechnological applications . in many cases ,
overproduction of hexahistidine - tagged tvdaao has been successfully achieved in order to obtain an engineered form of the protein that could facilitate its downstream processing in e. coli [ 1216 ] and p. pastoris . as reported previously , a chimeric tvdaao containing a hexahistidine tag at the n - terminal end
was largely expressed as apoenzyme by a recombinant e. coli strain , and the pure apoprotein could be purified in a single step by using metal - chelate affinity chromatography .
the holoenzyme could be reconstituted from the recombinant apoenzyme by addition of exogenous fad to yield a fully active enzyme .
the dissociation constant ( kd ) for fad of this tvdaao , which was quite similar of that reported for pkdaao , is 10-fold larger than that for rhodotorula gracilis daao .
thus , the weak noncovalent binding of fad to the recombinant enzyme might explain the observation that the flavin cofactor is lost upon dilution or prolonged dialysis .
in the present paper , we report experiments demonstrating that recombinant tvdaao activity was inhibited by different salts and that the direct inhibition by nacl is due to fad dissociation as confirmed by measuring the fluorescence of tryptophanyl residues of the holoenzyme .
d - alanine , 2,4-dinitrophenyl - hydrazine ( dnph ) , edta , fad , imidazolem , and 2-mercaptoethanol were supplied by sigma .
potassium phosphate , potassium hydroxide , and glycerol were purchased from scharlab ( barcelona , spain ) .
his - tagged d - amino acid oxidase from trigonopsis variabilis ( tvdaao ) was produced and purified as previously described in .
recombinant tvdaao was largely expressed as apoenzyme ( about 90% ) , and further enzyme purification and dyalisis against the appropriate buffer lead to a 100% pure apoenzyme preparation .
this apoenzyme solution was further dialyzed against 20 mm potassium phosphate buffer ph 8.0 , 20% glycerol , 5 mm edta , and 5 mm -mercaptoethanol at 4c and , then , centrifuged at 11,600 g for 5 minutes .
an excess of exogenous cofactor fad ( 5-fold enzyme concentration ) was added to apoenzyme solution to obtain the holoenzyme solution .
apoenzyme concentration was determined spectrophotometrically using a molar absorption coefficient at 276 nm of 276 = 70.5 mmcm .
the standard method measured the release of -ketoacid from d - alanine during the reaction by the formation of the corresponding hydrazone from dnph , which could be monitored at 450 nm .
the reaction mixture contained 80 l of 100 m fad dissolved in distilled water and 100 l of 100 mm d - alanine dissolved in 100 mm potassium phosphate buffer ph 8.0 .
the mixture was preincubated for 5 minutes at 30c , and the reaction was started by adding 20 l of enzyme solution .
after 10 minutes , the reaction was stopped with 20 l of a saturated solution ( approximately 10 mm ) of dnph in 1n hcl .
after 15 minutes of incubation with dnph , the colour was developed by the addition of 180 l of 2n naoh .
the activity was calculated by spectrophotometric absorption measurement at 450 nm , using a pyruvic acid calibration curve .
one activity unit ( u ) was defined as the amount of enzyme producing 1 mol of pyruvic acid per minute under the conditions mentioned .
the activity assay was performed under standard enzyme assay conditions as mentioned above , at a buffer concentration of 50 mm .
intrinsic fluorescence measurements were performed in an slm aminco 8000c spectrofluorimeter ( spectronic instruments , usa ) equipped with a thermostated cell holder using a 2 ml cell at 25c .
emission spectra were recorded using an excitation wavelength of 295 nm ( tryptophan emission ) .
excitation and emission bandwidths were set at 5 nm and 6 nm , respectively .
as shown in figure 1 , we observed a significant decrease of enzyme activity when nacl concentration was increased in the reaction medium . at a concentration of 300 mm nacl ,
the influence of buffer concentration on enzyme inhibition was ignored as no differences in activity were observed .
the phenomenon of flavoenzyme inhibition by salts has been described in the literature [ 2426 ] . as reported
, enzyme inhibition by high salt concentrations often occurs due to the abundance of negatively charged ions , which lead to conformational perturbations preventing holoprotein formation .
in fact , flavoproteins that bind their cofactor rather weakly can be deflavinated using bromide ions at high concentration [ 2732 ] .
chloride has been reported to be less chaotropic and therefore less effective in removal of flavin . in our case ,
as mentioned above , chloride negative charges could interfere with the weak interactions between cofactor and enzyme , favouring fad dissociation . the same deactivation was observed when kcl concentration was increased in the reaction medium ( figure 2 ) .
apart from halide ions , also other negative charged groups were described to induce fad dissociation , such as cyanate or cyanide .
an extensive study of the inhibition effect of recombinant tvdaao activity by nacl was carried out in order to know the inhibition type and the inhibition constant .
the enzyme activity was measured with different concentrations of d - alanine in the absence and presence of several concentrations of sodium chloride under the same standard assay conditions , at 50 mm potassium phosphate buffer ph 8.0 . as shown in figure 3 ,
the inhibition exerted by nacl was competitive , indicating that the chloride anion diminishes the affinity of the enzyme for the substrate , probably due to conformational perturbation of the active site , which could be related to the binding of the negative ion to some residues .
nevertheless , such hypothesis should be checked when the tvdaao three - dimensional structure will be available .
the replot of the slope versus inhibitor concentration fitted to a straight line whose intercept in the x - axis indicated an inhibition constant value of ki = 85 mm . in order to check possible fad dissociation from the holoenzyme due to nacl
, fluorescence emission spectra of reconstituted tvdaao in the presence of different nacl concentrations were measured with excitation at 295 nm ( figure 4 ) .
excitation at this wavelength selectively allows measurements of the contribution of tryptophan residues to the emission spectra of the protein .
fluorescence intensity was increased up to threefold when increasing concentrations of nacl were added to the enzyme solution .
in addition , a small red shift from 338 nm to 340 nm was observed , indicating that the tryptophan residues were in a slightly more hydrophilic environment .
such an effect may be related to the loss of cofactor fad and the corresponding exposure of tryptophan residues , which were quenched by the cofactor prior to nacl addition . as a matter of fact
, tvdaao apoenzyme , which contains no fad , shows an emission maximum at 340 nm with intensity 3-fold higher than that of the holoenzyme , whose maximum is at 338 nm .
the decreased fluorescence intensity of holoenzyme can be explained by a conformational transition upon fad binding or subunit association that would affect the local environment surrounding the indole ring of tryptophan residues .
fad dissociation from the holoenzyme in the presence of halide anions such as iodide , bromide , or chloride has also been reported for pkdaao , in which tryptophanyl fluorescence intensity was stronger than that of apoenzyme . in our case ,
a significant increase of the intrinsic fluorescence intensity can be observed at salt concentrations higher than 77 mm . above this concentration
this result is quite similar to the inhibition constant obtained in the kinetic experiments , strongly indicating that competitive inhibition by nacl could be related to fad dissociation .
as flavin cofactor binding is essential for catalytic activity , fad dissociation induced by chloride anions would lead to enzyme inactivation .
recombinant tvdaao activity is inhibited by the presence of high salt concentrations in the reaction media .
sodium chloride behaved as a competitive inhibitor , leading to conformational perturbations in the active site that might induce fad dissociation . | inhibition of recombinant d - amino acid oxidase from trigonopsis variabilis ( tvdaao ) activity in the presence of different sodium salts and potassium chloride is reported .
a competitive inhibition pattern by sodium chloride was observed , and an inhibition constant value of ki = 85 mm was calculated .
direct connection of nacl inhibition with fad cofactor dissociation was confirmed by measuring the fluorescence of tryptophanyl residues of the holoenzyme . |
the long - appreciated
beneficial properties of the trifluoromethyl
group in medicinal chemistry and the imperatives of green chemistry provide the impetus for the
current resurgence of interest in the development of trifluoromethylation
methods .
the ability to produce ever more complex trifluoromethylated substances
gives rise to the need for efficient methods to unambiguously assign
their constitution , configuration , and conformation .
current projects
in our laboratory necessitated the synthesis and configurational assignment
of pairs of diastereomeric -trifluoromethylcyclohexanols and
related compounds .
prakash reagent or other systems delivering a nucleophilic cf3 moiety with cyclohexanones , it became apparent that assigning the configuration
relative to an existing stereogenic center in such compounds is not
straightforward in the absence of crystals suitable for x - ray analysis .
thus , in previous work , the relative configuration of diastereomeric
pairs of -trifluoromethyl tertiary alcohols , if assigned at
all , was based on considerations of relative polarities , differences
in ir stretching frequencies of the oh group , nmr chemical shift differences
of the tertiary alcohols or of their derivatives , noe measurements
of derivatives , and considerations of inherent face selectivity in
the precursors . to address this problem , we considered two potential solutions :
( i ) the application of heteronuclear noe - type experiments ( hoesy )
between the cf3 group and proximal substituents and ( ii )
the karplus - type correlation of the dihedral angle ( ) subtended by the
cf3 group and axial or equatorial hydrogen atoms at the
vicinal position ( h - c - c - cf3 )
with the jch heteronuclear
coupling constants ( figure 1 ) .
heteronuclear jch coupling constants are widely
applied in carbohydrate chemistry for the determination of glycosidic
bond and hydroxymethyl group torsion angles and also enable the determination of torsion angles about cc
however , with the exception of
their use for the configurational assignment of sialic acid glycosides , heteronuclear jch coupling
constants do not find wide application in the conformational analysis
of cyclic systems .
we report here on the successful development of a straightforward jch heteronuclear coupling method
for the determination of configuration in trifluoromethylated tertiary
alcohols and related compounds that we believe will also be of use
in determining the relative configuration of a broad range of cf3-bearing quaternary centers .
we also report related vicinal c h coupling constants in olefinic systems ,
which should apply in the assignment of configuration of trifluoromethyl
alkenes .
reaction
of the ruppert prakash reagent
with a series of representative cyclohexanones 16 in the presence of either tetrabutylammonium fluoride or
cesium fluoride gave rise to the -trifluoromethylated cyclohexanols
or the corresponding trimethylsilyl ethers 914 with the yields and selectivities listed in table 1 , entries 16 .
similarly , reaction of the
ruppert prakash reagent with gluconolactone 7 gave
ketose 15 ( table 1 , entry 7 ) .
prakash reagent with imine 8 , formed in situ from 4-tert - butylcyclohexanone
and benzylamine followed by hydrogenolysis , gave
diastereoisomeric -trifluoromethylamines 16 ( table 1 , entry 8) .
reaction of pentafluoroethyltrimethylsilane with 4-tert - butylcyclohexanone
catalyzed by tetrabutylammonium fluoride gave -pentafluoroethyl
4-tert - butylcyclohexanols 17 ( table 1 , entry 9 ) .
all reactions , with the exception
of entry 9 , were conducted with 2.0 equiv of tmscf3 in
thf in the presence of 0.1 equiv of either tbaf or csf . promoted with tbaf and worked up
with 6 n hcl . promoted with csf
and worked up
with 2 equiv of tbaf
. promoted
with 0.8 equiv of khf2 and 1.5 equiv of tmscf3 in acetonitrile , followed
by hydrogenolysis over pd / c in meoh .
3.0 equiv of tmscf2cf3 in thf in
the presence of 0.3 equiv tbaf was employed .
in addition , a diastereomeric mixture of two steroids 19 containing a quaternary carbon , in which one of the four
ligands
is a trifluoromethyl group , was prepared as described by blazejewski
and co - workers by radical reaction of
allyltrimethylstannane with s - methyl xanthate ester 18 derived from tertiary trimethylsilyl
ether 11 ( scheme 1 ) .
although it is not the
primary focus of this article , it is noteworthy and bears comment
that kinetically controlled reaction of the ruppert
prakash
reagent with five of the six cyclohexanones studied is selective for
the formation of the axial trifluoromethyl derivatives .
the use of
both 4-tert - butyl and 4-phenylcyclohexanone as substrate
gave approximate 4:1 mixtures of adducts favoring introduction of
the cf3 group syn to the remote substituent , with the products
retaining essentially undistorted chair conformations in the solution
phase , as determined by analysis of the h nmr spectra ,
despite the axial location of the bulky cf3 group .
previously ,
the major product from the reaction with 4-phenylcyclohexanone was
assigned the opposite configuration ( cf3 trans to phenyl )
on the basis of chemical shift differences in the derived xanthate
esters of the two isomers . in view of
this discrepancy and in support of the fdcs nmr method discussed below
,
we obtained an x - ray crystal structure of the major isomer from reaction
with 4-phenylcyclohexane ( supporting information and ccdc 1032684 ) , which confirms its chair conformation and the
axial location of the cf3 group .
this assignment corrects the earlier literature , focuses attention on the ambiguities arising from the assignment
of configuration in such -trifluoromethyl tertiary alcohols
on the basis of chemical shift arguments alone , and underlines the
need for unambiguous methods for assignment of configuration that
are preferably based on the analysis of coupling constants . in their original report on the reaction of trifluoromethyltrimethysilane
with aldehydes and ketones , prakash and
co - workers noted the formation
of a single , but unassigned , diastereomeric product in the reaction
with cholestanone 3 .
subsequent
workers reported a 96:4 ratio of isomers favoring the 3-cf3 epimer , but they did not provide any basis for the attribution
of configuration . in our hands , a single
diastereomer 11 was formed ( table 1 , entry 3 ) , to which we assign the 3-cf3 configuration
on the basis of the fdcs method discussed below and which is further
confirmed by hoesy .
similarly , triterpenoid methyl ester 4 gives a single isomer of adduct 12 with an axial cf3 group ( table 1 , entry 4 ) , as determined
by fdcs and confirmed by the hoesy relationship of the cf3 group to a single of the two vicinal methyl groups .
the glucopyranose-4-one
derivative 5 reacts in a highly selective manner with
the ruppert
prakash reagent , but it affords galacto - configured
trimethylsilyl ether 13 with the equatorial cf3 group ( table 1 , entry 5 ) .
the configuration
of this derivative was assigned by the fdcs method below and is confirmed
by noesy correlations between the trimethylsilyl methyl groups and
the axial hydrogen at position 2 as well as by hoesy correlations
between the cf3 group and the axial hydrogens at positions
3 and 5 . with apramycinone derivative 6 , as reported
previously , a return to axial selectivity
is observed ( table 1 , entry 6 ) , as confirmed
by hoesy measurements .
the axial selectivity observed for the
introduction of the cf3 group into cyclohexanones 14 and 6 is interesting
in view of the steric bulk of
the cf3 group itself ( steric a value 2.37 ) and , more pertinently , of the presumed pentacoordinate
species ( r2co - sime3-cf3 or f - sime3-cf3 ) that transfers the cf3 group to the ketone .
presumably ,
this selectivity arises from the coordination / activation of the ketone
to the trimethylsilyl group of the ruppert
prakash reagent ,
as suggested by prakash and yudin ; in
much the same way , the facial selectivity of alkyllithium attack on
cyclohexanones can be reversed from the equatorial face to the axial
face by complexation of the ketone with sterically bulky lewis acids .
the equatorial selectivity observed in the formation
of 13 is consistent with that observed for the addition
of the bulky trichloromethide anion to 13 and it is -anomer and for the reduction of the permethyl analogue
of 13 by sodium borodeuteride .
less hindered acetylide nucleophiles , on the other hand , are axial
selective in their reactions with 13 .
the single diastereomer observed in the formation of the
six - membered cyclic -trifluoromethyl hemiacetal 15 presumably is due to mutarotation subsequent to the initial attack
and so reflects both the steric bulk of the cf3 group and the influence of the anomeric effects .
the reduced selectivity observed in the
trifluoromethylation of
the n - benzylimine 8 , as compared to
reaction with the corresponding ketone 1 ( table 1 , entries 1 and 8) , presumably arises from the weaker
coordination of the imine nitrogen than the ketone oxygen to the reagent ,
resulting in a smaller effective bulk and more facile accommodation
of the c
consistent with the
greater steric bulk of the pentafluoroethyl group ( steric a value
2.67 ) , reaction of 4-tert - butylcyclohexanone 1 with pentafluoroethyltrimethylsilane ( table 1 , entry 9 ) was
less selective than that with trifluoromethyltrimethylsilane ( table 1 , entry 1 ) , resulting in a greater proportion of
adduct 17 with the equatorial fluoroalkyl group . in the sialic acids , the measurement of jch coupling constants between
the anomeric carboxyl carbon and the axial h3 is a rapid and reliable
method for the determination of anomeric configuration ( figure 2 )
. such experiments , which we dub single frequency off - resonance decoupling
( sford ) and which are a variation on standard , off - resonance decoupling methods ,
are usually conducted on the methyl esters and are carried out with
selective low - power decoupling of the methoxycarbonyl protons to facilitate
identification of the desired residual jch coupling constant in the c nmr spectrum ,
as illustrated in figure 3a .
an axial co2me group , with its antiperiplanar relation to the axial h3 ,
typically displays a vicinal coupling constant of 57 hz , whereas
its equatorial counterpart , flanked by two gauche hydrogens , is usually
devoid of coupling ( figure 2 ) .
inspired by this method , we developed an analogous protocol
for
the determination of jch coupling
constants between the carbon of a cf3 group and its vicinal
hydrogen atoms , which we dub the c h fluorine - decoupled spectroscopy ( fdcs ) method . in this experiment ,
a fully h - coupled , c - observed spectrum was
acquired with selective f irradiation , as illustrated
in figure 3b . in this manner ,
the cf3 resonance is observed to be free of jcf coupling , thereby revealing
the diagnostic jch couplings
in an unobstructed manner .
pulse sequences employed in the sford and fdcs
determinations and
in the confirmatory hoesy experiments .
( a ) pulse sequence for single
frequency off - resonance decoupling ( sford ) experiment .
c was the observed nucleus , and the desired h was irradiated
with low - power single frequency continuous wave ( cw ) pulse .
c spectrum was the observed nucleus , and f signal
was selectively decoupled with a waltz-16 composite decoupling scheme .
the standard pulse sequence from the vnmrj 3.2 software
library was used , with 0.3 s mixing time and 4.0 s relaxation delay .
the acquisition time was 0.17 s , with 16 or 32 scans for each of 128
increments .
( d ) pulse sequence for the h{f } 1d hoesy experiment . the standard pulse sequence from
the vnmrj 3.2 software library was used .
f noe difference
experiment , two fids were recorded : one with f selective
low - power irradiation and the second fid without f irradiation .
the resulting two spectra were subtracted digitally to observe the
h f noe spectrum .
application of the fdcs method to the various -trifluoromethyl
cycloalkanols or their trimethylsilyl ethers displayed in table 2 supports the initial premise that the c h heteronuclear coupling constant is a function
of the torsion angle .
thus , an axial cf3 group exhibits
a coupling constant of 7.39.8 hz with vicinal axial hydrogen
atoms ( 180 ) ( table 2 , entries
1 , 3 , 5 , 6 , and 8) .
the vicinal coupling constant between an axial
cf3 group and an equatorial hydrogen atom ( 60 )
is typically 1 hz , but it ranges as high as 4.2 hz in the
steroidal and triterpenoid examples ( table 2 , entries 1 , 3 , 5 , 6 , and 8) .
an equatorial cf3 group
exhibits a vicinal coupling constant of 1 hz with vicinal
axial and equatorial hydrogen atoms ( 60 ) in the systems
studied ( table 2 , entries 2 , 4 , 7 , 9 , and 10 ) .
in the case of both the 180 and 60 dihedral angles ,
the
larger coupling constants in the observed ranges are found in the
conformationally more rigid systems ( table 2 , entries 5 and 6 ) .
this suggests that the smaller coupling constants
observed in the simple cyclohexyl systems are the result of an appreciable
population of nonchair conformers at room temperature , consistent
with the most recent estimates of the steric a value for the cf3 group , which suggest that it is more bulky than an isopropyl
group but less so than a tert - butyl group .
although the present data set is not sufficiently
extensive for careful calibration , we note that , as is well - appreciated
in homonuclear jh , h coupling and as has been demonstrated in other heteronuclear jch systems , the general karplus - type relationship correlating the magnitude
of f3c - c - c - h coupling constant
with torsion angle will be modulated by the nature and orientation
of substituents .
the fdcs method is readily extended from trifluoromethylcyclohexanols
to trifluoromethylcyclohexylamines and allows the assignment of axial
and equatorial cf3 groups in the -trifluoromethyl
amines 16 ( table 2 , entries 11
and 12 ) . by way of example ,
figure 4 shows
the c resonances of the two cf3 groups in
a mixture of the diastereomeric amines 16 before ( figure 4a ) and after ( figure 4b )
decoupling of the f atoms .
cf3 resonances in a diasteromeric mixture
of trifluoromethylamines 16 before ( a ) and after ( b ) f decoupling with a partial expansion ( c ) .
the analysis of the fdcs spectra of the inseparable
diastereomeric
trifluoromethylated steroid derivatives 19 , in which
the trifluoromethyl group is appended to a quaternary carbon , were
complicated by the additional jch coupling of the cf3 resonances to the allylic
methylene protons in addition to the diagnostic couplings to the vicinal
hydrogens in the steroidal a ring ( table 2 ,
entries 15 and 16 ; figure 5 ) . nevertheless ,
application of the standard fdcs sequence to the standard c spectrum ( figure 5a ) gave a simplified spectrum
( figure 5b ) displaying a downfield cf3 resonance as a broad multiplet for one isomer and a narrower more
upfield multiplet for the second isomer .
the broader multiplet , expanded
in figure 5c , clearly represents the axial
cf3 group with its diaxial couplings to the axial hydrogens
at the vicinal 2- and 4-positions , whereas the narrower multiplet
lacks such large couplings to the vicinal hydrogens in the steroidal
a ring .
although both multiplets are convoluted with additional jch couplings to the two allylic
hydrogens , which renders actual measurement of the diagnostic jch couplings constants difficult ,
the clear difference in the width at half height of the two multiplets
( w1/2 = 17.2 and 26.3 hz ) allows relative
configuration to be assigned . to confirm this assignment ,
a sford
experiment was conducted in which the allylic hydrogens were selectively
decoupled , giving rise to the partial spectrum in figure 5d .
the signals in this sford experiment retain the
quartet due to coupling to the cf3 resonances and also display coupling to the vicinal hydrogens
at positions 2 and 4 in the a ring .
the lines that make up the more
downfield quartet are broader than those in the upfield quartet , as
they display the larger jch couplings to the axial hydrogens at the 2- and 4-positions , which
is observable on the expansion ( figure 5e ) .
further confirmation of these assignments was achieved by noesy experiments
showing the spatial proximity of the vinylic hydrogens with the axial
hydrogen at the 1-position in the a ring of the major isomer .
cf3 resonances in a diasteromeric mixture
of trifluoromethylsteroids 19 : ( a ) before f decoupling , ( b ) after f decoupling , ( c ) expansion
of panel b. ( d ) c sford experiment , and ( e ) expansion
of panel d. the fdcs method is not
limited to cf3 groups ,
as demonstrated by its application to the diastereomeric -pentafluoroethyl
cyclohexanols 17 ( table 2 , entries
13 and 14 ) involving observation of the cf2 resonance .
the fdcs spectra of the two isomers of 17 contain an additional jcf quartet coupling to the cf3 group , but as the extra coupling
constant is significantly larger , it is of no consequence and does
not complicate interpretation .
the fdcs method was also applied to
the known -mono- and di- fluoromethylcyclohexanols 20 and 21 ( table 2 , entries 1720 ) ,
which were donated by drs .
the spectra of 20 and 21 are complicated by convolution of the vicinal coupling
constants with the jch couplings ,
but spectral interpretation is not difficult , as the vicinal coupling
constants are more than an order of magnitude smaller .
commercially
available -trifluoromethyl ethanol 22 allowed
the determination of the c h vicinal
coupling constant between a hydrogen atom and a cf3 group
in a freely rotating acyclic system ( table 2 , entry 21 ) .
unless otherwise
stated , spectra
were recorded in cdcl3 . the descriptors ax and
eq refer
to the axial or equatorial location of the fluoroalkyl groups and
of the vicinal hydrogens to which they are coupled , respectively .
owing to complications arising from
convolution with additional cf coupling to the allylic
hydrogens , multiplicity and coupling constants are difficult to assign
for 19ax and 19eq ( see text for clarification ) .
as discussed above ( table 2 ) ,
the configuration of a number of the samples
employed in this study was confirmed by heteronuclear overhauser effect
( hoesy ) measurements between cf3 groups and spatially proximal
hydrogen atoms .
hoesy spectra were acquired using an autotriple
resonance broadband probe ( atb ) , which is simultaneously tuned to h and f on the high - band rf coil .
2d hoesy experiments
used the manufacturer supplied fh - hoesy pulse sequence implemented
in vnmrj 3.2 software ( figure 3c ) .
h - observed , f - irradiated 1d hoesy experiments used the
fh decoupling pulse sequence from the vnmrj 3.2 software ( figure 3d ) .
the main limitation of the hoesy method , as
implemented in these experiments , is the requirement for the three
channel or atb type probe and of a spectrometer with three channel
capabilities .
when such hardware is on hand , the hoesy method , 1d
or 2d , provides a rapid means of assessing the spatial proximity of
the cf3 and adjacent protons and therefore of inferring
configuration and/or conformation .
because the 1d hoesy sequence is
observed by the proton channel and the 2d hoesy sequence by the f channel , sensitivity is correspondingly high and data acquisition
times are relatively short .
the fdcs sequence , on the other hand ,
employs a standard two - channel probe and can be implemented on any
modern spectrometer .
it gives direct information on the dihedral angle
subtended by the coupled h and cf3 spins and therefore on the conformation and/or configuration
of the substance under investigation .
the fdcs spectrum is acquired
through the c channel , and data acquisition is correspondingly
slow .
overall , fdcs and hoesy provide complementary information , and
the combination of the two is a powerful tool for studying the configuration
and conformation of cf3 and other fluoroalkyl - containing
molecules . although
the primary focus of this investigation is the development of the
fdcs method for the assignment of configuration in saturated systems
carrying cf3 groups , using commercially available compounds ,
we also briefly investigated its application to unsaturated molecules .
thus , as illustrated in figure 6 , the fdcs
method allows distinction of regioisomers in trifluoromethylated arenes ,
as the cf3 group exhibits a measurable c h coupling only to an ortho - hydrogen .
likewise ,
the fdcs method may be applied to the determination of configuration
of trifluoromethyl - substituted alkenes , as the trans - jch coupling constant is
more than double that of the corresponding cis coupling
constant ; jch couplings are
even smaller and should not complicate assignment of configuration
( figure 6 ) .
the
fluorine - decoupled carbon spectroscopy method is readily implemented
on standard two - channel nmr spectrometers and provides a facile method
for the determination of the configuration and/or conformation of
cf3- and other fluoroalkyl - substituted molecules .
based
on the karplus - type relation of the h - c - c - cf3 torsion angle to the coupling constant , the method
is an alternative to h , f hoesy .
earlier methods
for the assignment of configuration of cf3-substituted
tertiary alcohols based on chemical shift differences in derived xanthate
esters are unreliable and should be succeeded by the fdcs and/or hoesy
methods .
all reactions were performed using
oven - dried glassware under an atmosphere of argon .
all reagents and
solvents were purchased from commercial suppliers and were used without
further purification unless otherwise specified .
reactions were monitored by analytical thin - layer chromatography on
precoated glass - backed plates ( w / uv 254 ) and visualized by uv irradiation
( 254 nm ) or by staining with 25% h2so4 in etoh
or ceric ammonium molybdate solution .
high - resolution
mass spectra were recorded with an electrospray source coupled to
a time - of - flight mass analyzer .
h , c , f , hoesy , and fdcs spectra were recorded on a 400 or 600
mhz spectrometer using vnmrj 3.2 as processing software .
chemical shifts
are given in ppm ( ) , and coupling constants j are given in hz .
compounds 9ax and 9eq were prepared according to a literature
protocol using 4-tert - butylcyclohexanone
( 400 mg , 2.6 mmol ) and ( trifluoromethyl)trimethylsilane ( 740 mg , 5.2
mmol ) in tetrahydrofuran ( 3.0 ml ) at room temperature with a catalytic
amount of tetrabutylammonium fluoride ( 0.25 ml , 1.0 m in tetrahydrofuran ) .
chromatographic purification ( gradient elution of diethyl ether / pentane :
2 to 10% ) gave 9eq ( 70 mg , 12% , mp 48.049.5 c )
as an off - white solid and 9ax ( 290 mg , 50% , mp 94.595.5
c ) as a white solid .
9eq : h nmr
( 600 mhz , cdcl3 ) : 1.82 ( dd , j =
14.3 , 2.2 hz , 2h ) , 1.73 ( br s , 1h ) , 1.67 ( m , 2h ) , 1.59 ( dt , j = 13.5 , 4.0 hz , 2h ) , 1.36 ( m , 2h ) , 1.00 ( tt , j = 12.1 , 2.9 hz , 1h ) , 0.88 ( s , 9h ) ; c nmr ( 150 mhz ,
cdcl3 ) : 126.0 ( q , jcf = 284.4 hz , cf3 ) , 72.2 ( q , jcf = 28.1 hz , c1 ) , 47.0 , 32.3 , 30.2 ,
27.3 , 21.1 ; f nmr ( 564 mhz , cdcl3 ) :
87.4 ( s , cf3 ) ; fdcs ( 150 ) mhz , cdcl3 ) : 131.5 ( br s ) .
9ax : h nmr
( 600 mhz , cdcl3 ) :
2.20 ( m , 3h ) , 1.70 ( d , j = 13.5 hz , 2h ) ,
1.48 ( dt , j = 13.9 , 2.2 hz , 2h ) , 1.30 ( q , j = 12.4 hz , 2h ) , 1.08 ( tt , j = 12.1 , 2.9
hz , 1h ) , 0.84 ( s , 9h ) ; c nmr ( 150 mhz , cdcl3 ) : 126.8 ( q , jcf =
286.1 hz , cf3 ) , 71.8 ( q , jcf = 27.5 hz , c1 ) , 46.3 , 33.3 , 32.2 , 27.4 ,
23.0 ; f nmr ( 564 mhz , cdcl3 ) :
80.4
( s , cf3 ) ; fdcs ( 150 mhz , cdcl3 ) : 131.5
( tt , j = 8.9 , 3.6 hz ) .
compounds 10ax and 10eq were prepared according to a literature
protocol using 4-phenylcyclohexanone
( 1.0 g , 5.7 mmol ) and ( trifluoromethyl)trimethylsilane ( 1.6 g , 11.2
mmol ) in tetrahydrofuran ( 5.0 ml ) at room temperature with a catalytic
amount of tetrabutylammonium fluoride ( 0.6 ml , 1.0 m in tetrahydrofuran ) .
the residue was purified by column chromatography ( eluting with dichloromethane )
followed by recrystallization from 2-propanol / water ( 7:1 ) to give 10eq ( 90 mg , 8% , mp 75.076.5 c ) as an off - white
solid , 10ax ( 356 mg , 24% , mp 70.571.3 c )
as a white solid , and a mixture of both isomers ( 750 mg , 52% ) .
10eq : h nmr ( 600 mhz , cdcl3 ) :
7.33 ( t , j = 7.7 hz , 2h ) , 7.25 ( m , 3h ) , 2.55
( tt , j = 11.7 , 4.0 hz , 1h ) , 1.93 ( m , 3h ) , 1.85 ( m ,
6h ) ; c nmr ( 150 mhz , cdcl3 ) : 146.1 ,
125.5 , 126.7 , 126.6 ( q , jcf = 284.4 hz , cf3 ) , 126.3 , 72.0 ( q , jcf = 28.1 hz , c1 ) , 43.2 , 30.1 , 27.7 ; f nmr ( 564 mhz , cdcl3 ) : 87.3 ( s ,
cf3 ) ; fdcs ( 150 mhz , cdcl3 ) : 131.5 ( br
s ) .
10ax : h nmr ( 600 mhz , cdcl3 ) : 7.33 ( m , 2h ) , 7.26 ( m , 3h ) , 2.77 ( br s , 1h ) , 2.18
( m ,
3h ) , 1.96 ( m , 4h ) , 1.69 ( m , 2h ) ; c nmr ( 150 mhz , cdcl3 ) : 144.7 , 128.5 , 126.8 , 126.7 ( q , jcf = 285.5 hz , cf3 ) , 126.1 , 72.0
( q , jcf = 25.8 hz , c1 ) , 40.4 , 31.0 , 28.0 ; f nmr ( 564 mhz , cdcl3 ) : 82.2 ( s , cf3 ) ; fdcs ( 150 mhz , cdcl3 ) : 124.2 ( t , j = 7.3 hz ) . to a stirred solution of cholestan-3-one
( 400 mg , 1.0 mmol ) in tetrahydrofuran ( 3.0 ml ) were added ( trifluoromethyl)trimethylsilane
( 300 mg , 2.1 mmol ) and a catalytic amount of tetrabutylammonium fluoride
( 27 mg , 0.1 mmol , 1.0 m in tetrahydrofuran ) at room temperature .
the
resulting reaction mixture was stirred at room temperature for 2 h.
the solvent was evaporated under reduced pressure , and the residue
was dissolved in dichloromethane ( 5.0 ml ) , washed with water followed
by brine , dried over na2so4 , and concentrated
under reduced pressure .
the residue was purified by column chromatography
on silica gel eluting with etoac / hexanes ( 2 to 10% ) to afford 11ax(38 ) ( 482 mg , 90% , mp 103104
c ) as a white solid .
[ ]d20 = + 20.4 ( c = 3.7 , dichloromethane ) ; h nmr ( 600 mhz , cdcl3 ) : 2.08 ( td , j = 14.6 , 1.8 hz , 1h ) , 1.95 ( td , j = 12.8 ,
2.9 hz , 1h ) , 1.851.77 ( m , 2h ) , 1.65 ( m , 2h ) , 1.611.42
( m , 6h ) , 1.391.29 ( m , 6h ) , 1.281.20 ( m , 3h ) , 1.200.94
( m , 11h ) ,
0.89 ( d , j = 6.6 hz , 3h ) , 0.86 ( d , j = 2.6 hz , 3h ) , 0.83 ( s , 3h ) , 0.67 ( dd , j = 12.4 , 4.0 hz , 1h ) , 0.64 ( s , 3h ) , 0.14 ( s , 9h ) ; c
nmr ( 150 mhz , cdcl3 ) : 126.6 ( q , jcf = 287.2 hz , cf3 ) , 75.2 ( q , jcf = 26.9 hz , c3 ) ,
56.3 , 56.2 , 53.9 , 42.6 , 41.8 , 39.9 , 39.5 , 36.8 , 36.1 , 35.8 , 35.4 ,
35.2 , 34.8 , 31.7 , 30.1 , 28.6 , 28.2 , 27.9 , 24.1 , 23.8 , 22.8 , 22.6 ,
21.1 , 18.6 , 12.0 , 11.7 , 2.2 ; f nmr ( 564 mhz , cdcl3 ) : 80.3 ( s , cf3 ) ; fdcs ( 150 mhz ,
cdcl3 ) : 131.5 ( tt , j = 9.6 , 4.1
hz ) . a solution of methyl 3-ketoolean-12-en-28-oate ( 40 mg , 0.08 mmol ) and ( trifluoromethyl)trimethylsilane
( 66.2 mg , 0.46 mmol ) in tetrahydrofuran ( 1.5 ml ) was treated with
a catalytic amount of tetrabutylammonium fluoride ( 4.4 mg , 0.02 mmol ,
1.0 m in tetrahydrofuran ) at room temperature . the resulting reaction
mixture was stirred at room temperature for 1 h. the solvent was evaporated
under reduced pressure , and the residue was dissolved in dichloromethane
( 2.0 ml ) , washed with water followed by brine , dried over na2so4 , and concentrated under reduced pressure .
the residue
was subjected to flash column chromatography ( gradient elution of
etoac / hexanes : 2 to 20% ) to afford 12ax ( 42 mg , 82% ,
mp 178179 c ) as a white solid .
[ ]d20 = + 62.1 ( c = 1.4 , dichloromethane ) ; h nmr ( 600 mhz , cdcl3 ) : 5.26 ( t , j = 3.6 hz , 1h ) , 3.60
( s , 3h ) , 2.84 ( dd , j = 13.9 , 4.4 hz , 1h ) , 1.991.78
( m , 5h ) , 1.701.54 ( m , 5h ) , 1.51 ( m , 2h ) , 1.44 ( m , 2h ) , 1.31
( m , 3h ) , 1.24 ( m , 2h ) , 1.18 ( t , j = 12.2 hz , 2h ) ,
1.12 ( m , 1h ) , 1.11 ( s , 3h ) , 0.96 ( br s , 3h ) , 0.94 ( s , 3h ) , 0.91 ( s ,
3h ) , 0.88 ( s , 3h ) , 0.82 ( s , 3h ) , 0.71 ( s , 3h ) , 0.11 ( s , 9h ) ; c nmr ( 150 mhz , cdcl3 ) : 178.2 , 143.8 ,
127.2 ( q , jcf = 291.7 hz ,
cf3 ) , 122.1 , 81.0 ( q , jcf = 24.6 hz , c3 ) , 51.4 , 51.2 , 47.5 , 46.7 , 45.8 ,
41.7 , 41.6 , 41.3 , 39.2 , 36.3 , 35.4 , 33.8 , 33.0 , 32.8 , 32.3 , 30.6 ,
27.7 , 24.1 , 23.5 , 23.3 , 23.0 , 21.1 , 19.1 , 16.9 , 15.5 , 2.1 ; f nmr ( 564 mhz , cdcl3 ) : 69.5 ( s , cf3 ) ; fdcs ( 150 mhz , cdcl3 ) : 124.6 ( dd , j = 9.8 , 3.1 hz ) ; esihrms calcd for c35h57o3f3sina ( [ m + na ] ) , 633.3916 ;
found , 633.3927 . to a solution of methyl 2,3,6-tri - o - benzyl--d - glucopyranoside ( 296 mg , 0.54 mmol ) in anhydrous dichloromethane ( 2.5 ml )
was added
dess - martin periodinane ( 296 mg , 0.69 mmol ) at room temperature .
the
resulting reaction mixture was stirred at room temperature for 3 h ,
quenched with saturated aqueous nahco3 ( 3.0 ml ) , and washed
with water ( 3.0 ml ) and brine ( 3.0 ml ) .
the solvent was evaporated
under reduced pressure to give the 4-ketone as yellow oil , which was
taken forward to the next step without further characterization . to
a solution of this ketone ( 220 mg , 0.47 mmol ) in anhydrous tetrahydrofuran
( 4.0 ml )
was added a catalytic amount of cesium fluoride ( 7.0 mg ,
0.04 mmol ) followed by ( trifluoromethyl)trimethylsilane ( 700 mg , 4.90
mmol ) at room temperature .
the resulting reaction mixture was stirred
at room temperature for 1 h. the solvent was evaporated under reduced
pressure , and the residue was dissolved in dichloromethane ( 5.0 ml ) ,
washed with water ( 5.0 ml ) followed by brine ( 5.0 ml ) , dried over
na2so4 , and concentrated under reduced pressure .
the residue was purified by silica gel column chromatography eluting
with 10% etoac in hexanes to give 13eq ( 198 mg , 69% )
as an oil .
[ ]d25 = + 36.5 ( c= 4.9 , dichloromethane ) ; h nmr ( 600 mhz , cdcl3 ) : 7.387.25
( m , 15h ) , 4.97 ( dd , j = 9.9 , 5.1 hz , 2h ) , 4.74 ( d , j = 10.6 hz , 1h ) , 4.64 ( d , j = 11.7 hz ,
1h ) , 4.60 ( d , j = 9.9 hz , 1h ) , 4.54 ( d , j = 11.7 hz , 1h ) , 4.33 ( d , j = 7.7 hz , 1h ) , 3.96
( dd , j = 11.4 , 2.2 hz , 1h ) , 3.81 ( dd , j = 7.3 , 2.2 hz , 1h ) , 3.74 ( d , j = 9.5 hz , 1h ) , 3.69
( dd , j = 11.3 , 7.7 hz , 1h ) , 3.65 ( d , j = 9.2 hz , 1h ) , 3.64 ( s , 3h ) , 0.04 ( s , 9h ) ; c nmr ( 150
mhz , cdcl3 ) : 138.3 , 138.1 , 137.5 , 128.7 , 128.4 ,
128.3 , 128.2 , 128.1 , 127.7 , 127.7 , 127.6 , 127.6 , 124.6 ( q , jcf = 291.1 hz , cf3 ) , 104.5 ,
80.3 , 79.5 , 78.5 ( q , jcf =
25.2 hz , c4 ) , 76.4 , 75.6 , 74.8 , 73.7 , 69.3 , 57.2 , 1.8 ; f nmr ( 564 mhz , cdcl3 ) : 64.7 ( s ,
cf3 ) ; fdcs ( 150 mhz , cdcl3 ) : 122.1 ( d , j = 1.1 hz ) ; esihrms calcd for c32h39o6f3sina ( [ m + na ] ) , 627.2366 ;
found , 627.2341 . to a solution of
2,3,4,6-tetra - o - benzyl - d - glucono-1,5-lactone ( 200 mg , 0.47 mmol ) in anhydrous tetrahydrofuran
( 4.0 ml ) , was added a catalytic amount of cesium fluoride ( 6.0 mg ,
0.04 mmol ) followed by ( trifluoromethyl)trimethylsilane ( 1.0 g , 7.40
mmol ) at room temperature .
the resulting reaction mixture was stirred
at room temperature for 1 h. the solvent was evaporated under reduced
pressure , and the residue was dissolved in dichloromethane ( 4.0 ml ) ,
washed with water ( 4.0 ml ) followed by brine ( 4.0 ml ) , dried over
na2so4 , and concentrated under reduced pressure .
the residue was dissolved in tetrahydrofuran ( 3.0 ml ) and was treated
with tetrabutylammonium fluoride ( 170 mg , 0.65 mmol , 1.0 m in thf )
at room temperature .
the solvent was evaporated under reduced
pressure , and the residue was purified by silica gel column chromatography
eluting with 10% etoac in hexanes to give 15eq ( 142 mg ,
65% ) as an oil .
[ ]d25 = + 50.3 ( c= 8.0 , dichloromethane ) ; h nmr ( 600 mhz , cdcl3 ) : 7.407.27
( m , 17h ) , 7.267.22 ( m , 3h ) , 4.97 ( d , j =
11.0 hz , 1h ) , 4.90 ( d , j = 2.9 hz , 1h ) , 4.87 ( br
s , 1h ) , 4.85 ( d , j = 10.6 hz , 1h ) , 4.75 ( d , j = 9.9 hz , 1h ) , 4.69 ( dd , j = 10.6 , 7.7
hz , 1h ) , 4.59 ( d , j = 12.5 hz , 1h ) , 4.05 ( d , j = 9.9 hz , 1h ) , 3.94 ( br s , 1h ) , 3.93 ( d , j = 5.1 hz , 1h ) , 3.92 ( br s , 1h ) , 3.85 ( dd , j = 11.4 ,
3.6 hz , 1h ) , 3.81 ( m , 1h ) , 3.76 ( dd , j = 11.7 , 1.8
hz , 1h ) ; c nmr ( 150 mhz , cdcl3 ) : 138.2 ,
138.1 , 137.9 , 136.9 , 128.5 , 128.5 , 128.4 , 128.3 , 128.3 , 128.2 , 127.9 ,
127.8 , 127.7 , 127.7 , 127.6 , 127.5 , 122.4 ( q , jcf = 287.2 hz , cf3 ) , 94.3 ( q , jcf = 31.9 hz , c1 ) , 83.4 , 78.3 , 76.8 ,
75.9 , 75.7 , 75.1 , 73.4 , 72.9 , 67.8 ; f nmr ( 564 mhz , cdcl3 ) : 85.8 ( s , cf3 ) ; fdcs ( 150 mhz ,
cdcl3 ) : 122.1 ( br s ) ; esihrms calcd for c35h35o6f3na ( [ m + na ] ) ,
631.2283 ; found , 631.2264 .
a suspension
of 4-tert - butylcyclohexanone ( 1.0 g , 6.49 mmol ) ,
benzylamine
( 1.0 g , 9.71 mmol ) , and dry powdered magnesium sulfate ( 4.0 g , 33.3
mmol ) in dichloromethane ( 10 ml ) was stirred at room temperature for
48 h. the reaction mixture was filtered through celite and washed
with water , the solvent was removed under reduced pressure , and the
residue was directly subjected to the next reaction without further
purification . to a stirred solution of the crude intermediate imine
in acetonitrile ( 10 ml )
were added trifluoroacetic acid ( 0.8 ml , 10.0
mmol ) , potassium bifluoride ( 405.0 mg , 5.19 mmol ) , and n , n - dimethylformamide ( 2.0 ml ) at 0 c . the
reaction mixture was stirred for 10 min and was then treated with
( trifluoromethyl)trimethylsilane ( 1.4 g , 9.8 mmol ) at 0 c , and
the mixture was stirred at room temperature for 48 h. the reaction
mixture was quenched with saturated aqueous na2co3 solution ( 3 ml ) , and diluted with water ( 25.0 ml ) , and extracted
with ethyl acetate ( 3 100 ml ) .
the combined organic layers
were washed with water and brine , dried over na2so4 , and evaporated under reduced pressure .
the residue was dissolved
in meoh ( 10.0 ml ) , and the solution was stirred for 36 h with pd / c
( 10 mg , 10% mol ) under 1 atm of hydrogen ( balloon ) .
after completion ,
the reaction mixture was filtered through celite , and the filtrate
was evaporated under reduced pressure .
the residue was dissolved in
2 m hcl / meoh and then concentrated to dryness .
the resulting hydrochloride
salt was crystallized from acetonitrile ( 3.0 ml ) , filtered , and washed
with diethyl ether to afford the hcl salts of 16ax and 16eq ( 154 mg , 68% , mp 240242 c ) in a 1:1 ratio
as a white solid .
h nmr ( 600 mhz , cd3od ) :
2.37 ( d , j = 14.3 hz , 2h ) , 2.06 ( d , j = 12.8 hz , 2h ) , 1.951.83 ( m , 6h ) , 1.75 ( t , j = 13.5 hz , 2h ) , 1.401.27 ( m , 4h ) , 1.16 ( m , 2h ) , 0.92 ( s ,
9h ) , 0.88 ( s , 8h ) ; c nmr ( 150 mhz , cd3od ) :
125.5 ( q , jcf = 284.9
hz , cf3 ) , 125.2 ( q , jcf = 282.2 hz , cf3 ) , 58.4 ( q , jcf = 25.8 hz , c1 ) , 57.2 ( q , jcf = 28.0 hz , c1 ) ,
46.5 , 45.5 , 31.7 , 31.6 , 29.6 , 27.6 , 26.3 , 26.2 , 22.0 , 20.3 ; f nmr ( 564 mhz , cd3od ) : 73.4 ( s , cf3 ) , 80.8 ( s , cf3 ) ; fdcs ( 150 mhz , cd3od ) : 125.3 ( tt , j = 10.4 , 3.4 hz ) ,
124.9 ( br s ) ; esihrms calcd for c11h21nf3 ( [ m + h ] ) , 224.1626 ; found , 224.1629 . to a stirred solution
of ( pentafluoroethyl)trimethylsilane ( 507 mg , 2.6 mmol ) in tetrahydrofuran
( 1.5 ml ) were added 4-tert - butylcyclohexan-1-one
( 136 mg , 0.88 mmol ) and tetrabutylammonium fluoride ( 0.26 ml , 0.26
mmol , 1.0 m tetrahydrofuran ) at room temperature .
after 2 h of stirring ,
6 m hcl ( 1 ml ) was added , and the reaction mixture was stirred for
1 h at room temperature .
the aqueous layer was separated and extracted
with diethyl ether ( 3 3.0 ml ) .
the combined organic layers
were washed with saturated aqueous nahco3 ( 10.0 ml ) and
brine ( 10.0 ml ) and dried over mgso4 .
the crude products
were purified by flash column chromatography ( gradient elution with n - pentane / diethyl ether = 98:2 , 96:4 , 94:6 , 92:8 ) to give
a white solid 17ax ( 116 mg , 54% ; mp 74 c ) as the
major isomer and a colorless oil 17eq ( 54 mg , 25% ) as
the minor isomer .
neither 17ax nor 17eq was
amenable to ionization by either electrospray or electron impact mass
spectrometry .
17ax : h nmr ( 600 mhz ,
cdcl3 ) : 2.372.30 ( m , 2h ) , 2.26 ( br s , 1h ,
oh ) , 1.731.66 ( m , 2h ) , 1.48 ( td , j = 13.8 , 4.2 hz , 2h ) , 1.35 ( tdd , j = 14.3 ,
8.3 , 2.8 hz , 2h ) , 1.171.09 ( m , 1h ) , 0.84 ( s , 9h ) ; c nmr ( 150 mhz , cdcl3 ) : 119.4 ( qt , jcf = 280 hz , jcf = 31.9 hz , cf3cf2 ) , 116.3 ( tq , jcf = 260 hz , jcf = 28.4 hz , cf3cf2 ) , 72.2 ( t , jcf = 22.7 hz , c1 ) , 46.0 , 34.0 , 32.3 , 27.4 ,
22.8 ; f nmr ( 564 mhz ) : 81.4 ( s , 3f , cf3cf2 ) , 121.9 ( s , 2f , cf3cf2 ) ; fdcs ( 150 mhz , cdcl3 ) : 116.3 [ qtt , j = 28.4 hz ( from
fluorine ) , 7.3 hz ( from axial hydrogen ) , 3.3 hz ( from equatorial hydrogen ) ] .
17eq : h nmr ( 600 mhz , cdcl3 ) : 1.911.85 ( m , 2h ) , 1.76 ( s , 1h , oh ) , 1.711.59 ( m , 4h ) , 1.38 ( m , 2h ) , 1.00 ( tt , j = 12.4 , 3.1 hz , 1h ) , 0.86 ( s , 9h ) ; c nmr ( 150 mhz ,
cdcl3 ) : 119.5 ( qt , jcf = 288.0 hz , jcf = 31.9 hz , cf3cf2 ) , 114.8
( tq , jcf = 258.0 hz , jcf = 33.4 hz , cf3cf2 ) , 73.2 ( t , jcf = 22.7 hz , c1 ) , 47.0 , 32.3 , 30.3 , 27.3 ,
21.1 ; f nmr ( 564 mhz ) : 80.9 ( s , 3f , cf3cf2 ) , 129.5 ( s , 2f , cf3cf2 ) ; fdcs ( 150 mhz , cdcl3 ) : 119.5 broad quartet [ j = 33.4
hz ( from fluorine ) ] . a solution of 11ax ( 350 mg , 0.66 mmol ) in tetrahydrofuran ( 3.0 ml ) was treated with
2 n hcl ( 0.6 ml ) and stirred for 5 h at room temperature . the solvent
was evaporated under reduced pressure , and the residue was dissolved
in ethyl acetate ( 5.0 ml ) , washed with saturated aqueous nahco3 solution ( 5.0 ml ) and water ( 5.0 ml ) followed by brine ( 5.0
ml ) , dried over na2so4 , and concentrated under
reduced pressure afford the 3-(trifluoromethyl)-3-cholestanol , which was taken forward to the next reaction
without further purification . a suspension of 3-(trifluoromethyl)-3-cholestanol
(
135 mg , 0.29 mmol ) and potassium hydride ( 24 mg , 0.60 mmol ) in tetrahydrofuran
( 6.0 ml ) was stirred for 10 min at room temperature followed by addition
of carbon disulfide ( 115 mg , 1.47 mmol ) and then methyl iodide ( 420
mg , 2.95 mmol ) at 65 c .
the resulting reaction mixture was stirred
for 1 h at 65 c , cooled to 0 c , and quenched with water
( 5.0 ml ) .
after extraction into dichloromethane ( 2 8 ml ) , the
combined organic layers were washed with water ( 10.0 ml ) followed
by brine ( 10.0 ml ) and dried over na2so4 .
the
solvent was evaporated under reduced pressure , and the residue was
purified by silica gel column chromatography eluting with hexanes
to give 18 ( 119 mg , 74% , mp 95.596.0 c )
as a white solid .
[ ]d25 = + 25.4 ( c= 0.7 , dichloromethane ) ; h nmr ( 600 mhz , cdcl3 ) : 3.34 ( dt , j = 14.3 , 5.5 hz , 1h ) , 3.14 ( t , j = 14.3
hz , 1h ) , 2.45 ( s , 3h ) , 1.99 ( td , j = 14.3 , 2.2 hz ,
1h ) , 1.95 ( td , j = 12.8 , 3.3 hz , 1h ) , 1.831.75
( m , 2h ) , 1.71 ( dd , j = 13.5 , 4.4 hz , 1h ) , 1.66 ( m ,
1h ) , 1.571.40 ( m , 5h ) , 1.381.18 ( m , 9h ) , 1.161.00
( m , 6h ) , 1.000.93 ( m , 5h ) , 0.88 ( d , j = 6.6
hz , 3h ) , 0.85 ( d , j = 2.9 hz , 3h ) , 0.84 ( d , j = 2.9 hz , 3h ) , 0.69 ( dt , j = 12.5 , 4.0
hz , 1h ) , 0.64 ( s , 3h ) ; c nmr ( 150 mhz , cdcl3 ) : 211.4 , 125.7 ( q , jcf = 286.7 hz , cf3 ) , 91.6 ( q , jcf = 27.5 hz , c3 ) , 56.3 , 56.2 , 53.5 , 42.5 ,
42.5 , 39.8 , 39.4 , 36.1 , 35.7 , 35.4 , 35.3 , 35.1 , 31.7 , 28.8 , 28.5 ,
28.2 , 27.9 , 24.1 , 23.8 , 22.8 , 22.5 , 22.4 , 21.2 , 19.0 , 18.6 , 12.6 ,
12.1 ; f nmr ( 564 mhz , cdcl3 ) : 76.7
( s , cf3 ) . to a stirred
solution of xanthate 18 ( 102 mg , 0.18 mmol ) in allyltributyltin
( 433.0 mg , 1.30 mmol ) was added triethylborane ( 20.0 mg , 0.20 mmol )
at 30 c .
the resulting reaction mixture was stirred
for 1 h at 30 c , quenched with diisopropyl azodicarboxylate
( diad ) ( 452.0 mg , 2.23 mmol ) , and stirred for 3 h at room temperature .
column chromatography of the reaction mixture ( silica gel , pentane )
afforded a mixture of 19ax and 19eq along
with allyltributyltin .
the residue was dissolved in anhydrous dichloromethane
( 2.0 ml ) , and propionaldehyde ( 84 mg , 1.44 mmol ) was added .
the reaction
mixture was cooled to 0 c , and boron trifluoride diethyl etherate
( 205 mg , 1.44 mmol ) was added ; the resulting reaction mixture was
stirred at 0 c for 1 h , quenched with saturated aqueous nahco3 solution ( 2.0 ml ) , and washed with water ( 2.0 ml ) and brine
( 2.0 ml ) .
the solvent was evaporated under reduced pressure , and the
residue was purified by silica gel column chromatography eluting with
hexanes to obtain a mixture of 19ax and 19eq ( 49.2 mg , 55% , mp 5152 c ) in a 1:2.5 ratio as a white
solid .
[ ]d25 = + 16.4 ( c= 2.2 , dichloromethane ) ; h nmr ( 600 mhz , cdcl3 ) : 5.71 ( m , 2h ) , 5.11 ( dd , j = 10.3 , 2.2 hz , 1h ) , 5.07 ( dd , j = 13.9 ,
1.8 hz , 1h ) , 2.37 ( d , j = 7.3 hz , 2h ) , 2.16 ( m , 1h ) ,
1.96 ( m , 2h ) , 1.81 ( m , 2h ) , 1.72 ( dd , j = 13.9 , 4.4
hz , 1h ) , 1.67 ( m , 2h ) , 1.621.41 ( m , 11h ) , 1.401.17
( m , 17h ) , 1.171.03 ( m , 9h ) , 1.030.94 ( m , 4h ) , 0.910.88
( m , 7h ) , 0.880.85 ( m , 8h ) , 0.79 ( s , 3h ) , 0.660.63
( m , 4h ) ; c nmr ( 150 mhz , cdcl3 ) : 133.7 ,
132.8 , 129.8 ( q , jcf = 286.1
hz , cf3 ) , 129.3 ( q , jcf = 283.3 hz , cf3 ) , 111.8 , 117.7 , 56.5 , 56.4 , 56.3 ,
56.2 , 54.3 , 54.0 , 42.5 , 41.0 , 40.1 , 39.9 , 39.5 , 36.1 , 35.8 , 35.5 ,
35.5 , 35.4 , 34.9 , 34.6 , 32.9 , 31.9 , 31.7 , 30.0 , 29.2 , 28.5 , 28.2 ,
28.0 , 27.4 , 25.2 , 24.1 , 23.8 , 23.4 , 22.8 , 22.5 , 20.9 , 18.6 , 13.7 ,
12.0 , 11.7 , 8.7 ; f nmr ( 564 mhz , cdcl3 ) :
73.1 ( s , cf3 ) , 79.3 ( s , cf3 ) ;
fdcs ( 150 mhz , cdcl3 ) : 19eq , 128.5
( narrow multiplet ) , 19ax , 129.0 ( broad multiplet ) . | the
synthesis of a series of -trifluoromethylcyclohexanols
and analogous trimethylsilyl ethers by addition of the ruppert
prakash
reagent to substituted cyclohexanones is presented . a method for the
assignment of configuration of such compounds , of related -trifluoromethylcyclohexylamines
and of quaternary trifluoromethyl - substituted carbons
is described
based on the determination of the 3jch coupling constant between the fluorine - decoupled 13cf3 resonance and the vicinal hydrogens .
this method is dubbed fluorine - decoupled carbon spectroscopy and abbreviated
fdcs .
the method is also applied to the configurational assignment
of substances bearing mono- , di- , and perfluoroalkyl rather than trifluoromethyl
groups .
the configuration of all substances was verified by either 19f1h heteronuclear overhauser spectroscopy
( hoesy ) or x - ray crystallography .
the relative merits of fdcs and
hoesy are compared and contrasted .
2jch , 3jch , and 4jch coupling constants to 19f decoupled cf3 groups in alkenes and arenes have also
been determined and should prove to be useful in the structural assignment
of trifluoromethylated alkenes and arenes . |
moringa oleifera lam . found in most parts of ghana belongs to the monogenetic family moringaceae ( order brassicales ) .
its nonmedical uses include use of the seeds in wastewater treatment due to their coagulant properties [ 1 , 2 ] .
its medicinal uses stem from the fact that the entire plant has high protein , vitamins , mineral , and carbohydrate content .
moringa leaves are rich in minerals such as iron , potassium , and calcium as well as vitamins , essential amino acids , and a number of glycosides [ 3 , 4 ] .
the seeds have high content ( 42% ) of edible oil that also has medicinal uses .
moringa is used for the management of various ailments , as a galactogogue in mothers of preterm infants [ 5 , 6 ] .
it is also used to manage heart diseases and eye problems as well as inflammations and dyspepsia [ 7 , 8 ] .
pharmacological studies have shown that the extracts of the plant have antioxidant [ 911 ] , anticarcinogenic , anti - inflammatory , antispasmodic and antidiuretic properties .
recent studies indicate that it also has antinociceptive as well as wound healing ability .
studies on the root bark have shown it to have analgesic , alexeteric , and antihelminthic properties .
toxicity studies have shown that aqueous extract of moringa leaf extract has no significant adverse effects in rats , rabbits [ 1518 ] or poultry . however , there are significant differences in the safety and composition of various moringa species from different locations . in ghana and most parts of west africa , powdered leaves of m. oleifera
are marketed under different brand names and consumed daily as a nutraceutical , prophylaxis , or cure for various conditions .
the prevailing socio - cultural / economic conditions and consequent difficulty in accessing healthcare services , especially by rural populations as well as the general perception that plant medicines are efficacious and free from side effects [ 19 , 20 ] will increase the frequent and widespread spread use of moringa leaf powder . in view of the reported differences in moringa leaf products , which may impact adversely on its safety , the micro- and macroelemental content of dried leaves of moringa oleifera were measured .
in addition , the leaf extract was investigated for its toxicological effects in sprague - dawley rats .
in order to mimic the traditional method of extraction , 2.8 kg of sample were blended with boiled distilled water .
the mixture , covered with water , was left to stand overnight in a water bath maintained at 60c and the watery portion ( infusion ) was filtered off using 0.45 m millipore cellulose ester filters and freeze dried to obtain m. oleifera extract ( moe ) .
leaves of the moringa oleifera species were collected from accra , separated from other plant parts , washed and dried in the shade for five days at room temperature , ground into fine powder , sieved ( 212 m mesh size ) to obtain very fine samples , and kept in separate containers .
this was labelled sample b. the loose / powdery nature of samples ( plant extracts a and leaf powder b ) required that they were pelleted before analysis . before pelleting , 4
g of each sample was weighed and 0.9 g of the binder fluxana ( h elektronic bm-0002 - 1 ( licowax c micropowder pm - hoechstwax ) ) was added and homogenized for 3 minutes .
the mixture was pressed at 20 t or 2 minutes into pellets of 32 mm in diameter for the subsequent xrf measurements .
three separate pellets were prepared from each of samples a and b. energy dispersive x - ray ( ed xrf ) was used for simultaneous analysis and measurement of the elemental content of the samples .
the procedure , which used three - axial geometry , reduced background noise due to radiation polarization .
the monochromatic radiations emitted from the x - ray tube were applied to excite the atoms of the sample .
male sprague - dawley rats ( 150 g180 g body weight ) were purchased from the centre for scientific research into plant medicine , mampong .
the rats were housed in plastic cages with stainless steel tops in the animal care facility of the university of ghana medical school and kept under standard 12 h light and 12 h dark schedule where room temperature , humidity , and ventilation were controlled during the acclimatization period of seven ( 7 ) days .
the reconstituted powdered moe mixture was prepared using distilled water as the vehicle and administered as a single dose ( 5,000 mg / kg ) by oral gavage for the acute toxicity studies .
after the administration , animals were observed every hour for the first 6 hours then daily for the next 13 days . in the subacute studies , five groups ( 15 ) of male sprague - dawley rats ( eight weeks old , seven animals / group ) of mean weight 150 g were prepared .
animals in each of the four groups were administered moe extract over a dose range of 0 mg / kg to 1000/kg by oral gavage daily for 14 days .
for the subacute studies , after the14 days of moe administration of moe , each animal was observed every hour for six hours daily for the next 3 days and subsequently every day for 10 days .
animals were fed ad libitum with standard chow diet ( ain-93 g formulation obtained from gafco - ghana ) .
in addition , animals were observed daily for clinical signs of excitability , twitching , salivation , morbidity , miosis , mydriasis , rising fur , sluggish movement , draping , tremors , and so forth . under chloroform
samples of the blood collected were aliquoted into edta-2k tubes and plain tubes , respectively .
the edta blood was immediately analysed for haematological parameters using the sysmex haematology autoanalyser [ kobe , japan ] while serum prepared from blood in the plain tubes was used for biochemical examinations .
the heart , kidney , liver , and gastrointestinal tract were prepared for histopathological examinations .
all experimental procedures and assays were conducted in accordance with the international guidelines for evaluating the safety of herbal medicines [ 2123 ] .
results are presented as means sem and analysis for statistical differences was done using one - way anova followed by bonferroni post - hoc test .
the ed - xrf analyses led to the detection of a total of thirty - five ( 35 ) elements comprising eleven ( 11 ) major elements and twenty - four ( 24 ) minor elements .
the major elements detected included na , mg , al , si , p , s , cl , k , ca , mn , and fe and other heavy metals . tables 1 and 2 show details of the elements detected and their concentrations . in the acute toxicity studies as well as the subacute studies at all the dose levels , grooming , repetitive circling with arched - back posture were observed in all the rats except those in the control group .
rats given high doses of moe ( 1000 mg / kg and 5000 mg / kg ) showed more excitability , twitching , and salivation .
these results of haematological investigations are presented in table 3 ( acute toxicity ) and table 4 ( subacute toxicity studies ) . in the acute toxicity studies ,
similarly , platelets levels dropped significantly ( 62.5% ) following treatment with moe . in the subacute studies ,
wbc increased by 73.75% and 67% ( p < 0.0001 ) at dose levels of 40 mg / kg and 80 mg / kg , respectively .
mcv dropped significantly at the 200 mg / kg and 1000 mg / kg dose levels .
similarly , platelets levels rose significantly over baseline level at the 80 mg / kg dose level .
figure 1 shows results obtained from the biochemical parameters used as markers for renal function .
the figure shows that blood urea levels were elevated at dose levels of 80 mg / kg and 1000 mg / kg . creatinine levels , however , significantly decreased at all dose levels compared to the controls .
figures 2 and 3 show results for the biochemical test for liver function and lipid profiles , respectively .
there were increases in the levels of liver enzymes during moe administration in a non - dose dependent manner .
ast levels , however , reduced at all dose levels except the 80 mg / kg group .
for the lipid profiles , moe administration generally led to a decrease in total cholesterol and triglycerides as well as ldl and , to some extent , hdl cholesterol .
photomicrographs of tissues prepared from control and animals treated with moe at different dose levels are presented in figures 58 .
there were no observable histological lesions in the sinusoids and central vein of the liver ( figure 8) .
normally , herbal preparations are considered relatively safe and devoid of numerous adverse effects probably because they are considered to be natural .
this study attempted to analyse the elemental content of moringa oleifera and to ascertain if a 14-day dosing could have any observable adverse effects .
this is in view of the widespread use of moringa leaf powder as a food supplement and treatment for various disease conditions .
x - ray fluorescence ( xrf ) is a fast , accurate , and nondestructive analytical technique used for the elemental and chemical analysis of powdered , solid , and liquid samples .
analyses of the samples produced a total of 35 elements ( 14 macroelements and 21 microelements ) in m. oleifera .
the concentrations of macroelements in the powdered leaf samples are shown in figure 4 and included s , ca , k , mg , na , p , si , cl , al , fe , and mn .
the minor elements in the decreasing order were v , ba , cr , y , ba , zn , rb , ce , la , cu , cs , sn , hf , co , ni , nb , ta , u , mo , pb , bi , ga , th , as , and zr . all concentrations were within the recommended daily allowance ( rda ) limits .
rda , the average daily dietary intake level , is expected to be sufficient to meet the nutrient requirements of all healthy individuals [ 2426 ] .
these results , therefore , would suggest that consumption of moringa leaf powder can provide users with some of the essential minerals that the human body requires for optimum function .
blood parameter analysis is relevant to risk evaluation as the haematological system has a higher predictive value for toxicity in humans ( 91% ) .
moringa oleifera extract administration was accompanied by a reduction in the haematopoietic system ( tables 3 and 4 ) at a dosage of 40 mg / kg which is evidenced by the levels of rbc , hct , hgb , wbc , and lymphocytes .
the slightly reduced hgb , hct , rbc , and total protein in the rats suggest potential interference with the haematopoietic system that would require further investigations .
the high rbc could also be due to hypoxic conditions that might have resulted due to the high doses of moe administered .
the absence of a major significant change in haematological parameters is consistent with observations by awodele et al . .
the statistically significant increase in lymphocytes at moe doses of 4080 mg / kg and the 5,000 mg / kg bw in the animals might suggest the presence of infection / stress or a potential immune boosting effect of moe at the stated dose levels .
since the animals were specific pathogen - free and were kept in a barrier system , the observed effects are likely to be due to potential immune boosting effects of the extract [ 13 , 14 , 28 ] .
toxic injury to the liver leads to elevation in levels of all these liver enzymes .
thus , the observed rise in alt and alp following administration of moe ( tables 5 and 6 ) would suggest a potential adverse effect of moe on the liver .
since alt is localized primarily in the cytosol of hepatocytes , it is a more sensitive marker of hepatocellular damage than ast and alp [ 27 , 28 ] .
withinlimits , these can provide a quantitative assessment of the degree of damage sustained by the liver .
histopathological examinations , however , did not reveal anyhistological lesionsin the sinusoids or central vein ( figure 8) .
the copper component in the extract ( table 2 ) which is a component of a number of enzymes that are involved in reducing molecular oxygen , metabolizing substances such as histamine , serotonin , epinephrine , norepinephrine , and dopamine could pose a threat during abnormal consumption of the extract .
copper deficiency , although rare , results in hypochromic anaemia ; possible side - effects may include liver damage and wilson 's syndrome .
direct , indirect , and total bilirubin reflects the liver 's ability to take up , process , and secrete bilirubin into bile and can also be considered as a true test of liver function [ 27 , 28 ] .
these were all high at the different doses , especially at the 5,000 mg / kg single oral high dose compared to the controls ( tables 5 and 6 ) . however , total protein , globulin , and albumin remained relatively unchanged or slightly increased in all the groups in comparison with the controls . these were not entirely dose dependent and significant but require further monitoring in subchronic studies . it must also be noted that gross pathological examination of the treated animals did not reveal any abnormalities such as presence of lesions or changes in colour of internal organs and relative organ weights as compared to the controls .
the kidneys are concerned with the elimination of drugs from the body and are likely to be affected during such toxicity studies at high doses . in the treated male rats ,
creatinine levels were reduced compared to the controls ( p < 0.05 ) while urea levels were inconsistent among the groups .
also , histopathological examinations did not reveal any observable histological lesions in the glomeruli and the tubules ( figure 7 ) .
however , the reduction in creatinine levels may be deemed to be positive effect of moe on the renal system , which could be exploited therapeutically .
ingestion of chemicals substances including those of plant origin in excess and especially over long periods may adversely affect major organs like the heart , kidney , liver , and even the gastrointestinal system .
thus , the evaluation of histopathological changes in organs remains a cornerstone in assessing the safety of medicines and other substances [ 30 , 31 ] .
histopathological examinations of the heart and stomach did not reveal any observable cardiomyopathies ( figure 5 ) and ulcerations of the epithelial cells in the male rats ( figure 6 ) .
it might be expected that at high doses of moe may result in accumulation of moe - derived iron .
this mineral , though an important component of haemoglobin and other proteins and enzymes , may cause gastrointestinal distress , hemochromatosis , and so forth [ 22 , 23 ] .
the absence of these effects may be explained by the fact that the various elements were within normal limits .
it is important to note that the effects of chemicals produced in laboratory animals when properly conducted provide a useful indication of safety in humans .
thus , from the above results , it would be expedient to conduct subchronic or chronic toxicity studies with regard to the hematopoietic , renal , hepatic , and reproductive changes because moe is used as a food supplement and is used over a long period of time by consumers .
moreover , the contamination of moringa leaves by heavy metals like lead ( pb ) and arsenic ( as ) may pose a threat to humans because they are not biodegradable . with reference to the rda limits and other studies conducted regarding the presence of heavy metals in the leaves of moe [ 34 , 35 ] ,
it is imperative that prevention of any possible cumulative toxicity of some of the elements is most desirable during prolonged use of moe .
there is , thus , the need for toxicological studies to be conducted on moringa from different locations in order to give an indication of their comparative safety .
it must , however , be stated that , because plants may absorb elements from the soil and environment , some of which may be toxic to humans , plant nutrition , climate , and soil conditions and locations could also determine the elemental contents in the leaves [ 19 , 34 , 35 ] .
this study has provided some evidence that moringa oleifera that was collected in accra , ghana , in west africa is reasonably safe for consumption taking into account the elemental composition when administered to rats .
the acute studies showed that the median lethal dose ( ld50 ) could be greater than 5000 mg / kg as all the animals survived at his dose level .
it is also important to monitor the concentration of its elemental composition as a possible reason of serious health alterations .
based on the levels of these minerals and the permissible amount in the human body , it is recommended that the consumption of moringa oleifera leaves be limited to a maximum of 70 grams per day in order to prevent excessive consumption and subsequent accumulation of some of these essential elements . at 70 grams per day , most of these elements in the leaves could be found in high quantities approaching the rda limit . | moringa oleifera is a multipurpose plant used in ghana and most parts of africa .
its high mineral , protein , and vitamins content has enabled its use as a nutraceutical and panacea for various diseases .
this study aimed at measuring the micro- and macroelements content of dried moringa oleifera leaves using energy dispersive x - ray fluorescence spectroscopic ( edxrf ) and assessing its toxicological effect in rats .
acute toxicity ( 5000 mg / kg ) and a subacute toxicity studies of the leaf ( 40 mg / kg to 1000 mg / kg ) extract were conducted in rats .
blood samples were assessed for biochemical and haematological parameters .
results showed significant levels of thirty - five ( 35 ) elements ( 14 macroelements and 21 microelements ) in m. oleifera extract .
there were no observed overt adverse reactions in the acute and subacute studies .
although there were observed elevations in liver enzymes alt and alp ( p < 0.001 ) and lower creatinine levels in the extract treated groups , no adverse histopathological findings were found .
moringa oleifera dried leaf extract may , therefore , be reasonably safe for consumption .
however , the consumption of moringa oleifera leaves should not exceed a maximum of 70 grams per day to prevent cumulative toxicity of these essential elements over long periods . |
chronic hepatitis b ( chb ) virus ( hbv ) infection is the principal cause of cirrhosis and hepatocellular carcinoma ( hcc ) . the pathogenesis of hbv - related chronic liver disease is not well understood .
however , it is clear that the immune mechanisms associated with the antiviral response are responsible for chb outcome [ 24 ] .
this concept stems from the observation that , in chronic hepatitis b , t - cells can potentially participate both in the immune clearance of hbv - infected cells and in the pathogenesis of hepatocellular injury .
furthermore the numbers of b lymphocytes and plasma cells are significantly higher in patients with liver cirrhosis than of those with inactive chronic hepatitis [ 7 , 8 ] .
enormous intrahepatic b - cells with massive production of igm and igg and infiltrating plasma cells into the hepatic lobules have also been shown in hbv - associated chronic active hepatitis .
b - cells contribute to immune responses through the secretion of effector cytokines and it has been suggested that naive and memory b - cell subsets preferentially produce different effector cytokines [ 10 , 11 ] .
nave b - cells undergo maturation by somatic hypermutation in immunoglobulin variable region of the b - cell receptor ( bcr ) genes following contact with a specific protein accessible on dendritic cells .
then the high affinity antigen receptors which normally consist of two isotypes membranes igm and igd continue to mature to either ig - secreting plasma cells or memory b - cells . in comparison to antigen primary response ,
immunological memory presents the capacity to increase a faster and more vigorous humoral response subsequent to antigen re - exposure .
although antibody associated mechanisms targeting hepatitis b core antigen ( hbcag ) was reported in earlier studies , few data exist on b lymphocytes population in the liver of patients with chb .
cd20 is a b - cell specific surface antigen that is expressed in all stages of b - cell development except on either early pro - b - cells or plasma cells and plays an important role in b - cell activation and proliferation . to elucidate the role of intrahepatic b - cells in the pathogenesis of chronic hepatitis b
, we investigated the expression of cd20 marker on b - cells in liver biopsy of these patients by immunohistochemistry .
liver biopsy specimens from 57 patients with hbv - associated chronic liver disease without liver neoplasm attending the hepatitis clinic of shariati hospital , tehran university of medical sciences , during the years of 2008 to 2011 were studied .
hbv infection was diagnosed by the positivity for hepatitis b surface antigen ( hbsag ) in the patients ' sera .
all the patients were hbeag negative and had a history of familial hbv infection , without coinfection with human immunedeficiency virus ( hiv ) or other hepatitis viruses .
the patients ' clinical data at the time of liver biopsy were acquired from their medical records .
the presence of chb , stage of fibrosis , and histological activity were evaluated by modified histologic activity index ( hai ) scoring system on the liver sections stained with hematoxylin - eosin and sirius red .
commercially available primary monoclonal antibody against cd20 ( clone ucht1 , dako ) was used to stain 3.0 m sections of liver biopsies after deparaffinization by routine microwave antigen retrieval and horseradish peroxidase ( diaminobenzidine ) kit ( dako , carpinteria , ca ) , following manufacturer 's instructions .
immunohistochemical scoring ranged from 1 to 5 score ( matched for 010% , 1025% , 2550% , 5075% , and > 75% cd20 positive cells ) with higher scores indicating a greater proportion of positive cells ( as determined from the positive control ) . for enumeration of cd20 positive b - cells , image acquisition was done with the + 10 , + 20 , and + 40 objectives , but stained cells were counted in the entire field of + 40 corresponding to a tissue area of 30 mm .
positive stained cells appeared with no clear plasmacytoid morphology , but with granular cytoplasm and lobated nucleus of b - cells .
correlations between variables were analyzed using the pearson correlation coefficient ( r ) or spearman rank correlation coefficient ( r ) , as appropriate .
the baseline demographics are shown in table 1 . the mean sd age of patients was 33 9 years and 40 ( 70% ) were male .
the mean sd of total hai score for fibrosis and necroinflammation of patients is shown in table 1 .
nine patients ( 15.7% ) had significant fibrosis more than or equal to 3 . to analyze the impact of b - cell population in chb patients , the cd20 expression score , age , gender , log viral load , serum alt , and hai total score of necroinflammation and fibrosis were included in a multivariable - adjusted logistic regression model .
there was a significant association between log hbv dna with both alt and hai total score ( r = 0.36 , p = 0.006 , and r = 0.3 , p = 0.02 ) , respectively .
serum alt also showed positive correlation with total score of hai though which was not significant ( r = 0.25 , p = 0.06 ) .
there were no lines of evidence of interactions and multicollinearity between variables age and sex with log hbv dna and alt .
no significant correlation was observed between expression of cd20 b - cell marker expression with serum alt and hbv dna level .
the liver was diffusely infiltrated by cd20 positive b - cells , distributed both as aggregates in the portal areas and as single cells within the lobules .
the cd20 positive b - cells were expressed in all 57 liver biopsies with a submembranous and membranous staining pattern ( figure 1 ) .
the expression of cd20 was significantly associated with hai total score ( r = 0.32 , p = 0.01 ) and stage of fibrosis ( r = 0.31 , p = 0.02 ) .
fifteen cases had score 1 , thirty one had score 2 , ten had score 3 , and one had score 4 cd20 positive b - cells .
in this study , the role of intrahepatic b - cells in the pathogenesis of chb infection has been investigated .
the immunohistochemical findings of liver biopsies in this study indicate a major role for b - cells in the pathogenesis of chb which is driven by increased cd20 positive b - cells population .
our evidence showed that cd20 positive b - cells clustered in the portal areas or as single cells within the lobule and correlated with necroinflammation and fibrosis which was consistent with previous studies .
indeed our histological findings resembled the histological picture seen in patients with acute hepatitis b induced liver failure with a reflective b - cell response that could be responsible for huge expression of antibody to hbv core protein ( hbcab ) .
our study indicates that b - cells are involved in inflammatory reactions for destruction of infected cells .
thus , they play an important role in the immune response of chb infection . unlike hbsag and hbeag ,
the immunogenicity of hbcag is able to directly activate b - cells to produce specific antibodies in an extralymphatic situation and in the absence of t - cells .
moreover , it has been shown that the activation of b - cells , in a t - cell independent pathway , produced immunoglobulins and cytokines in the liver that changed fibrotic responses [ 11 , 16 ] .
our recent study showed the association of igd positive b - cells with the fibrosis stage in chronic hepatitis b .
the intrahepatic lymphocytes are recognized as long - lived recirculating b - cells and are similar to splenic b2 cells that produce cytokines il-2 , il-4 , tnf , and il-6 with proinflammatory function and contribute to disease pathogenesis in an antibody - independent fashion , perhaps by modulating t cell responses [ 16 , 18 ] .
earlier studies revealed that b - cells evidently contribute to the development of liver fibrosis by the production of profibrotic cytokine il-6 that induces the differentiation of hepatic stellate cells into myofibroblasts and raise collagen synthesis [ 16 , 18 , 19 ] .
both mature b lymphocytes in liver that belong to b2 subset and resting b - cells typically have cd20 receptor . therefore , we could suggest cd20 marker , as a representative marker of both naive and preswitch memory b - cells .
evaluation of intrahepatic b - cells population is important to determine how b - cells would respond to hbv infection . in this
setting , evidence from the current study indicates that exposure to viral antigens can increase the proportion of b - cells in the liver to carry an exhausted phenotype and therefore to dysfunction .
our present data also support the property of liver as an immune system , which may potentially affect the pathogenesis of various liver diseases .
the authors declare that there is no conflict of interests regarding the publication of this paper . | humoral immunity constitutes major defense mechanism against viral infections .
however , the association of hepatic injury and b - cells population in chronic hepatitis b virus ( hbv ) carriers has not been studied well . in this study ,
fifty seven hepatitis b surface antigen ( hbsag ) positive and hbeag negative patients were studied to determine the expression of cd20 , a cell surface marker expressed on b - cells , in liver biopsy sections using immunohistochemistry .
the patients ' clinical data at the time of liver biopsy were acquired from their medical records . there was a significant association between log hbv dna and both alt ( r = 0.36 , p = 0.006 ) and histologic activity index ( hai ) total score ( r = 0.3 , p = 0.02 ) , respectively .
the cd20 was expressed in all 57 liver biopsy samples with a submembranous and membranous staining pattern and its expression was significantly associated with hai total score ( r = 0.32 , p = 0.01 ) and stage of fibrosis ( r = 0.31 , p = 0.02 ) .
the susceptible b lymphocytes to hepatitis b virus might be implicated in the development of immune mediated inflammation of hbv - induced hepatic injury .
the present data also support that the liver is potentially one of the secondary lymphoid organs . |
its raw or processed roots are the only organ source for clinical uses of traditional chinese medicine , and the leaves are not utilized at all .
however , r. glutinosa leaves have the pharmacological effects and can nourish yin , tonify qi and kidney , and promote blood circulation . in the folk , the fresh leaves are often applied externally to treat malignant sore and tinea manus and pedis . as a perennial herb
, r. glutinosa has to be annually cultivated and harvested since its fleshy roots easily decay during the winter dormancy or are consumed when new plantlets come out from them in the next season . due to its severe continuous cropping obstacle , however , r. glutinosa plants could not grow well on the same land after the first cropping , decreasing the root yield greatly .
thus , exploitation of r. glutinosa leaves not only provides another potential medicinal source for extraction of important bioactive compounds present in its roots but also complements the shortage of r. glutinosa roots due to the limited land and continuous cropping obstacle .
a lot of pharmacologically bioactive secondary metabolites [ 4 , 5 ] including iridoid glycosides , phenylethanoid glycosides , and polysaccharides have been isolated in r. glutinosa , of which iridoid glycosides are thought to be main bioactive constituents . till now , more than thirty iridoid glycosides have been separated and identified [ 46 ] , including catalpol , aucubin , geniposidic acid ( gpa ) , rehmanniosides a , b , c , and d , and rehmaglutosides a k .
for example , as the main active iridoid glycoside in r. glutinosa , catalpol was found to play the important roles in treatment of many diseases including kidney diseases , neurodegenerative diseases , and diabetes [ 9 , 10 ] .
it is revealed that aucubin had pharmacological effects such as antifungals , anti - inflammation and antioxidation , and hepatoprotection .
similarly , gpa had therapeutic effects in anti - inflammation , liver disorders , and antinociception [ 14 , 15 ] . among these three compounds ,
only catalpol was assayed in detail to reveal its spatiotemporal expression profiling among different developmental stages of r. glutinosa leaves and between leaf and root [ 16 , 17 ] .
it is shown that catalpol content varied among different developmental stages of r. glutinosa leaves and within the whole growth stage .
aucubin and gpa were found to accumulate in roots of r. glutinosa [ 18 , 19 ] , and only the former was quantified and much lower than catalpol in the root [ 18 , 20 ] . therefore , in this paper , we aimed to determine the content of these three iridoid glycosides of two r. glutinosa cultivars in different developmental stages of leaves using hplc - uv method and compared their changing trend .
1 , were grown in around mid - april in 2014 at the implad ( institute of medicinal plant development ) , beijing , china .
normal field management was applied during the growth period . on 11th september , 2014 ,
the top eight positions of leaves from each plant were collected separately , washed to remove the surface soil , and then stored at 80c till use .
hplc grade acetonitrile ( acn ) and formic acid were purchased from thermo fisher ( usa ) .
the standard catalpol ( 98% ) was obtained from the national institute for food and drug control , china ( http://www.nifdc.org.cn/ )
. standards of aucubin ( gr-133 - 140104 ) and geniposidic acid ( gr-133 - 140423 ) were purchased from nanjing guangrun biotechnology co. ltd . , china ( http://www.grbiology.com/ ) .
the concentrations of catalpol , aucubin , and geniposidic acid in the mixed working stock standard solution were , separately , 0.5025 mg , 0.0375 mg , and 0.0020 mg in 1 ml of purified water . to make calibration curves , six series concentrations , ranging from 0.01570~0.5025 mg / ml for ca , 0.001172~0.0375 mg / ml for au , and 6.281e 05~0.002010 mg / ml for gpa ,
calibration curves were constructed by plotting the logarithm of the hplc - uv peak areas versus the logarithm concentration of each standard .
the frozen samples were homogenized with liquid nitrogen and one gram of each sample powder was extracted twice with 25 ml of 30% methanol in an ultrasonic water bath at 25c for 20 min .
the supernatants from two cycles of extraction were combined and evaporated to dryness in a rotary evaporator at 50c under the reduced pressure , redissolved in 50 ml of pure water , and then filtered with a 0.22 m millipore membrane filter prior to hplc analysis . for hplc - uv quantification , ten microliters of filtered extracts and standards
was run at 30c on a waters 600e system equipped with a phenomenex kinetex c18 column ( 4.6 mm 100 mm , 2.6 m ) , waters 2487 dual wavelength detector , and 2707 autosampler ( usa ) .
the isocratic mobile phase contained acetonitrile ( 5% ) and 0.1% formic acid in water ( 95% ) and was delivered at 0.4 ml / min .
catalpol and aucubin were monitored at 210 nm and geniposidic acid was monitored at 240 nm .
the presence of the three iridoid glycosides in r. glutinosa leaves was determined by comparing both retention time and spectral data with those of their corresponding authentic standards .
concentrations of three iridoid glycosides in the samples were determined from the below linear standard calibration curves .
are not heat - stable , the batch of leaf samples was used to calculate the dry weight of each stage leaf .
hplc - uv method has been successfully applied to quantify catalpol ( ca ) , aucubin ( au ) , and gpa in medicinal plants [ 16 , 17 , 2022 ] .
the hplc conditions that researchers used varied in terms of the columns , mobile phase system , and detection wavelengths .
therefore , we optimized hplc - uv conditions in terms of column type , uv wavelength , mobile phase composition , addition of formic acid in aqueous phase , and ratio of acn and water in our early work ( table 1 ) .
several uv wavelengths ( e.g. , 203 nm , 205 nm , 206 nm , and 210 nm ) were used to detect the presence and content of catalpol and ( or ) aucubin .
we found that there was no significant difference in detecting these two iridoid glycosides under 203~210 nm and thus chose 210 nm as the detection wavelength , which was used in china pharmacopoeia ( 2010 ) .
there was no much difference of the wavelengths ( 230~240 nm ) in peak shape and separation of gpa except that less miscellaneous peaks and stable baselines were produced at 240 nm .
as ji et al . revealed , adding or not adding formic acid ( fa ) in the mobile phase did not obviously influence the peak shape and separation of catalpol and aucubin , except for gpa .
while investigating the difference of different concentrations of acn in the mobile phase system on the chromatogram , we found that the elution times of the analytes decreased with acn concentration increased ( table 2 ) . when acn was higher than 5% , catalpol peak was overlapped with the solvent peak .
however , when less than 5% of acn was used in the mobile phase , more late au and gpa were eluted .
all the three analytes can be eluted within 10 minutes under 5% of acn . in order to shorten the running time for compounds behind them
the final hplc conditions that we adopted to determine ca , au , and gpa in our study were summarized in table 1 . using the optimized conditions
, we further tested our hplc - uv conditions by checking its linearity , precision , stability , and recovery ( table 3 ) .
figure 1 showed that standards of ca , au , and gpa were eluted separately at 3.412 min , 4.857 min , and 9.850 min .
their corresponding compounds in the sample extract were detected at 3.436 min , 4.941 min , and 9.813 min , respectively .
the linear regression equations obtained were as follows : ca , y = 0.9707x + 6.7723 , r = 0.9997 ; au , y = 0.9952x + 6.8458 , r = 0.9999 ; and gpa , y = 1.0136x + 7.5797 , r = 0.9996 .
the precisions were evaluated as the relative standard deviation ( % , rsd ) and , calculated for ca , au , and pga , were 0.14% , 2.65% , and 2.05% , respectively .
25% , 3.08% , and 2.41% , respectively , which indicated that our extraction method was reproducible . to check the stability of extracts , the same sample extract was assayed separately at 0 , 2 , 4 , 8 , 12 , 16 , and 24 h after extraction .
the rsd values for ca , au , and pga were 1.46% , 2.38% , and 0.43% , respectively , indicating that our sample extract remained stable at least till 24 hours .
the extraction recovery was determined by comparing the content of the compound extracted from the samples with the content of compound from nonextracted standard solutions at equivalent concentrations . to test the sample extraction recovery , one ml of the standard solution containing 4.824 mg of ca , 0.225 mg of au , and 0.0134 mg of gpa , respectively ,
the recovery rates for standards ca , au , and pga were 102.59% , 98.29% , and 101.96% , respectively .
the calculated lod ( limit of detection ) at s / n > 3 was , separately , 1.3333e 04 mg / ml for ca , 1.8170e 04 mg / ml for au , and 4.9926e 05 mg / ml for gpa .
the leaf biomass of r. glutinosa cultivars increased greatly from the seedling stage till 120 dap ( days after plantation ) and then deceased after mid - september ( 140 dap ) , partially due to ongoing senescence of bottom leaves ( figure 2 ) .
however , the top eight positions of leaves still stayed green and varied in leaf size , which can represent the different developmental stages of leaves ( figure 3 ) .
the 8th leaves , counted downwards from the uppermost leaves , looked similarly in their size and appearance status as the lower positions of unsenescent leaves . however , their dry weight ( dry weight per gram fresh weight ) changed dynamically with leaf development ( figure 4 ) .
overall , leaf dry weight decreased with the leaf development and increased for l5 and then decreased in the rest of older leaves .
l1l8 had about 0.140.18 grams of dry biomass per gram fresh biomass . in this paper
, we just focused on the contents of ca , au , and gpa in the top eight positions of leaves and investigated their relationship with the leaf developmental stages .
although the contents of ca , au , and gpa were somehow different between wen 85 - 5 and beijing no .
1 , the changing trends of these three iridoid glycosides with the leaf developmental stage were very similar between the two cultivars , indicating no genetic difference in developmental regulation of these three iridoid glycosides ( figures 5 and 6 ) .
generally , catalpol , a major bioactive iridoid glycoside of r. glutinosa , was higher in younger leaves and decreased with the leaf development , which was consistent with the result of ji et al . .
likewise in r. glutinosa root , aucubin in the leaf was still much lower than catalpol , which was found by pitczak et al . .
the changing trend of aucubin among different developmental stages of r. glutinosa leaves was distinct from catalpol ; that is , the older the leaves , the higher the aucubin in them . it was found that aucubin was one of the intermediates for catalpol biosynthesis in scutellaria albida and paulownia tomentosa .
the opposite metabolic profiling of aucubin and catalpol in r. glutinosa leaf indicated that catalpol might not be synthesized via aucubin ; however , this needs to be further investigated .
gpa was detected to be present in r. glutinosa leaves and the lowest analyte in all the developmental stages of r. glutinosa leaves . like aucubin , gpa was increased with the leaf development and higher in older leaves .
in this paper , we determined contents of catalpol , aucubin , and geniposidic acid in different developmental stages of r. glutinosa leaves using the optimized hplc - uv conditions and further compared their changing trend with the leaf development .
our results showed that aucubin and gpa in r. glutinosa leaves were much lower than catalpol and had the increasing trend with the leaf development , which was different from catalpol .
this work provided an important basis for future exploitation of r. glutinosa leaves such as isolation of interesting metabolites , for example , the high concentration compound present at 4.16 , which was further identified by hplc - lc - ms method .
it also provided a model to study the relationship of aucubin and catalpol metabolisms in r. glutinosa . | although r. glutinosa roots are currently the only organ source in clinics , its leaves are a potential supplement for the roots especially in extraction of some important bioactive compounds .
our early work found that the contents of catalpol and total iridoid glycosides varied among different developmental stages of r. glutinosa leaves .
aucubin and geniposidic acid , the abundant major bioactive compounds in eucommia ulmoides and gardenia jasminoides , respectively , were found present in r. glutinosa roots , however , and have not been analyzed in its leaves . in this paper , we aimed to determine contents of these three iridoid glycosides in different developmental stages of r. glutinosa leaves using the optimized hplc - uv conditions .
our results showed that aucubin and gpa in r. glutinosa leaves were much lower than catalpol and showed the increasing trend with the leaf development , which was different from catalpol .
this work provided the important information for future exploitation of r. glutinosa leaves as a potential supplement for its roots in extraction of some important bioactive compounds and studying the relationship of aucubin and catalpol metabolism . |
leiomyoma , a benign smooth muscle neoplasm is classified by world health organization on the basis of histopathology into three types : 1 ) solid , 2 ) vascular ( angiomyoma , angioleiomyoma ) and 3 ) epithelioid .
leiomyoma is a common neoplasm of uterus and gastrointestinal tract mostly occurring in solid variant . in the head and neck region ,
leiomyoma is rare and in oral cavity , it accounts for only 0.4% of all soft tissue neoplasms and 0.06% of all leiomyomas .
common site of occurrence in oral cavity is lip , tongue , cheek and palate .
usually , it is slow growing , asymptomatic , firm nodular mass and occasionally painful .
we report a rare case of large mandibular gingival angioleiomyoma showing predominantly cavernous - type histopathological growth pattern .
a female patient of 45 years of age reported to a private oral and maxillofacial surgeon with a huge sessile soft tissue mass measuring about 3 3 cm diameter , with an erythematous hue and associated tooth displacement in lower left mandibular posterior gingiva .
the lesion occupied the whole left buccal vestibular space leading to extraoral left side midfacial enlargement [ figure 1 ] .
panoramic radiograph revealed only erosion of underlying bone and no definite osseous component along with displacement of associated premolars and molars [ figure 2 ] .
a presumptive diagnosis of pyogenic granuloma due to its clinical appearance was made and surgical excision was done .
massive bleeding was encountered during excision and was controlled using multiple ligation , cautery and packs .
grossly , a soft tissue nodular mass of approximately 3 3 cm , firm in consistency with grayish - white appearance and two associated mandibular premolars were submitted [ figure 3 ] .
soft tissue gingival mass in lower left posterior region occupying vestibular space as well as lingual sulcus orthopantomogram reveals no underlying bony origin and displacement of premolar and molars along with bony resorption on left mandibular jaw excised gross specimen along with extracted teeth .
the hematoxylin and eosin - stained section showed multiple small to large dilated vascular channels with some of them showing thick vascular wall .
inter - vascular areas showed spindle cells with eosinophilic cytoplasm and elongated blunt - ended cigar - shaped nuclei with perinuclear vacuole .
many vessels showed thin - dilated wall and myxoid inter - vascular stroma [ figure 4a d ] .
a diagnosis of angioleiomyoma was given and immunohistochemistry ( ihc ) for smooth muscle actin ( sma ) showed diffuse intense positivity in vascular wall and intervascular stroma confirming their smooth muscle nature [ figures 5 and 6 ] .
( a ) surface epithelium with underlying connective tissue showing variably sized vessels in eosinophilic spindle cell stromal population ( h&e stain , 40 ) .
( b ) large thick - walled vessels associated with intersecting fascicles of spindle shaped smooth muscle cells ( h&e stain , 100 ) . ( c )
thick vessel wall composed of smooth muscle cells mingling with intervascular stroma ( h&e stain , 100 ) .
( d ) spindle smooth muscle cells with eosinophilic cytoplasm and cigar - shaped bluntended nuclei with perinuclear vacuole ( h&e stain , 400 ) ihc for sma shows diffuse positivity in tumor stroma ( ihc stain , 100 ) intense cytoplasmic sma staining of tumor smooth muscle cells ( ihc stain , 400 )
oral leiomyoma is rare due to paucity of smooth muscles in this region and whatever is found is associated with blood vessels ( tunica media ) , circumvallate papillae ( ductus lingualis ) and heterotopic smooth muscle .
angioleiomyoma is the most common variant encountered in oral cavity followed by solid and rarely by epithelioid variant of leiomyoma .
angioleiomyoma mostly involves lower lip , tongue and palate followed by other rare mucosal sites .
gingiva is a rare location and few cases have been reported ( less than 20 cases ) in literature .
trauma , topical estrogen , hormonal imbalance , infection , arteriovenous malformation , venous stasis and genetic translocation have been implicated as etiology but without any clarity .
oral angioleiomyoma typically presents as slow growing , firm , sessile and nodular soft tissue mass of 5 mm to 2 cm size .
bluish or reddish discoloration with rare history of pain have been reported , but cases associated with extremities are mostly painful .
term angioleiomyoma was coined by stout in 1937 and is circumscribed with distinct eosinophilic spindle cells with blunt - ended cigar - shaped nuclei and perinuclear vacuoles arranged in intersecting fascicles at right angle with abundant vascular channels having varying patterns . according to the predominant vascular pattern angioleiomyoma is classified into three variants , namely:- 1 ) solid ( capillary ) angioleiomyoma composed of narrow , slit - like vascular channels with surrounding densely packed intersecting smooth muscle fascicles .
2 ) venous angioleiomyoma composed of thick vessels and smooth muscle of vessel wall blending with intervascular smooth muscle bundles giving a net - like appearance to vessel wall .
3 ) cavernous angioleiomyoma has dilated vascular channels with sparse smooth muscle and muscular wall of these vessels are difficult to distinguish from intervascular smooth muscle bundles .
the efficacy of imaging system is restricted to know the nature and extent of lesion only .
ultrasound or color flow imaging reveals the tumor as a vascular lesion , so massive bleeding during excision is always a possibility . to be on safer approach , preoperative embolization of tumor
malignant transformation is rare and follow up should be done periodically as a case of leiomyosarcoma shows areas of angioleiomyoma .
histopathologically , angioleiomyoma can mimic cellular benign fibrous histiocytoma , myofibroma and leiomyosarcoma and closely it mimics angioleiomyoma - like myopericytoma .
cellular benign fibrous histiocytoma shows less eosinophilic spindle cells arranged in mostly fascicular pattern or less commonly storiform pattern admixed with histiocytoid cells and is only positive for vimentin .
myofibroma shows overlapping similarity with leiomyoma , at periphery it shows positivity for sma and desmin negativity but central zone shows primitive appearing round cells arranged about hemangiopericytoma component with sma negativity .
myopericytoma is characterized by round to oval cells with eosinophilic cytoplasm arranged circumferentially in layers around vascular lumina .
angioleiomyoma usually lacks this characteristic perivascular concentric growth of myoid tumor cells . however , matsuyama et al .
, has compared angioleiomyoma and myopericytoma on immunohistochemical basis and suggested both to be closely related tumors . however , watanabe et al . , has suggested h - caldesmon to be specific for smooth muscle cell and tumors originating from it .
he showed consistent expression of h - caldesmon in normal visceral and vascular smooth muscle cells and also myoepithelial cells but not in myofibroblasts and vascular pericytes .
the most commonly encountered are especially seen in young female patients and include pyogenic granuloma , peripheral giant cell granuloma , peripheral ossifying fibroma , traumatic fibroma etc .
clinically it mimics vascular lesions like pyogenic granuloma , peripheral giant cell granuloma and hemangiomas . owing to vascular nature ,
a preoperative diagnosis should be confirmed by incisional biopsies , although most of small lesions are excised completely and submitted for histopathological examination .
recent definite marker for smooth muscle h - caldesmon can be used as a specific marker for smooth muscle tumors and also can be used to differentiate among angioleiomyoma and myopericytoma . | gingival angioleiomyoma are rarely encountered lesions and most of these lesions are excised in toto in view of reactive gingival epulitic lesions . however , due to the vascular nature of these lesions , many a times surgeons experience unexpected hemorrhage and require extensive hemostatic management .
a preoperative diagnosis is a must for these lesions and at least color imaging techniques can be utilized to know its vascular nature and preoperative tumor embolization can be done to be on the safer side .
also histopathological dilemma can occur due to the varied types of vascular arrangement in angioleiomyoma or due to simulation of other vascular lesion like hemangiomas , as well as due to similarity with other spindle cell neoplasms .
we advocate the use of immunohistochemistry ( ihc ) to establish the exact diagnosis in such cases . also due to infrequent reports of angioleiomyoma in such unusual location , we want to record this case for further reference in future . |
a 36-year - old woman presented at our institution ( gachon university gil hospital ) with erythematous confluent maculopatches on her face , trunk , and palms ; jaundice ; fatigue ; pruritus ; and aggravation of skin lesions over the previous week .
she had been previously diagnosed as having erythema multiforme 2 weeks earlier at a private dermatology clinic and had also been administered dapsone with oral antihistamines ( mequitazine and cetirizine ) and topical corticosteroid for 8 months . on admission ,
her vital signs were a blood pressure of 110/70 mm hg , heart rate of 78 beats / min , respiratory rate of 18 breaths / min , and body temperature of 38.8. she reported no night sweats , loss of weight , or arthralgia .
a physical examination conducted at presentation revealed bilateral , multiple enlarged lymph nodes on both sides of the neck with splenomegaly .
all of the nodes were non - tender , firm , and measured 0.5 cm to 1.0 cm in diameter .
laboratory tests results were as follows : hb 11.2 g / dl , white blood cell 13,070/mm ( segmented neutrophils 24% , lymphocytes 66.3% , and eosinophils 0.1% ) , platelets 245,000/mm , total bilirubin 5.4 mg / dl , direct bilirubin 3.8 mg / dl , aspartate aminotransferase / alanine aminotransferase ( ast / alt ) 233/366 u / l , lactate dehydrogenase 1,610
iu / l ( normal range , 200 to 485 iu / l ) , and an international normalized ratio of 1.25 .
she was negative for hepatitis b surface antigen , anti - hepatitis c virus ab , and anti - human immunodeficiency ab , but positive for hepatitis b surface antibody ; and positive for anti - nuclear ab , but negative for rheumatoid factor , anti - neutrophil cytoplasmic ab , anti - ro ( ss - a , b ) ab , and anti - smith ab .
serology tests showed no evidence of active epstein - barr virus ( ebv ) infection and no bacterial growth was found during a blood culture study .
an excisional biopsy of a 1.0 cm cervical node showed diffuse polymorphic lymphoid hyperplasia with effacement of the normal architecture and abundant high endothelial venules ; the cellular infiltrate was polymorphic , with abundant transformed lymphocytes and plasma cells ( fig .
immunohistochemically , the majority of infiltrating atypical lymphocytes were cd3 + t - cells ( fig .
2a , b ) , and some activated large cells were reactive for cd30 ( fig . 2c ) .
2d ) , and infiltrating atypical t - cells were negative for cd10 , bcl-6 , and ebv by in situ hybridization . for a definitive diagnosis , a molecular study on the rearrangement of the t - cell receptor (
an abdomino - pelvic computed tomography ( ct ) scan revealed splenomegaly ( 14 cm ) , an enlarged portocaval lymph node ( 1.7 cm ) , and gallbladder wall edema ( fig .
no evidence of lymphomatous involvement was found by chest ct or a bone marrow trephine biopsy .
because aggravation of the skin eruption persisted and the liver function test remained abnormal , the possibility of dhs ( exfoliative dermatitis , acute hepatitis , and multiple lymphadenopathy ) was raised .
the findings of a transjugular liver biopsy were compatible with drug - induced hepatitis manifesting as granulomatous inflammation ( fig . 5 ) .
no evidence of lymphomatous hepatic involvement was found . in view of her skin lesions , medication history , and age
accordingly , low dose prednisolone ( 30 mg / day ) per os was started after transjugular liver biopsy to relieve the dapsone - induced skin lesions and hepatitis .
her total bilirubin , ast , and alt values also normalized after 3 weeks of prednisolone , which was then tapered over a month .
follow - up ct scans conducted 3 and 6 months after presentation showed that the size of the cervical lymph nodes and spleen had normalized , and produced no other findings suggestive of lymphoma progression ( fig .
it was first identified in the early 1940s following the introduction of hydantoin and its derivatives for the treatment of convulsive disorders.3 several drugs other than hydantoins , such as tamoxifen , amlodipine , carbamazepine , and valsartan , have also been reported to cause pseudolymphoma.4 - 7 pathologically , lymph nodes of pseudolymphoma show obliteration of the normal architecture , along with hyperplasia of the reticulum cells and other elements , with frequent mitoses .
they also show eosinophilic leukocyte infiltration , focal necroses , and phagocytosis , but no reed - sternberg cells.8 clinically , systemic reactions are usually combined with pseudolymphoma , fever , skin eruption , hepatitis , and less frequently with hepatosplenomegaly.8 the differential diagnosis of multisystem illness in our patient included : drug reaction with eosinophilia and systemic symptom ( dress ) syndrome and its variants , vasculitis ( churg - strauss syndrome ) , hypereosinophilic syndrome , toxic epidermal necrolysis syndrome ( tens ) and stevens - johnson syndrome .
churg - strauss syndrome is a medium and small vessel autoimmune vasculitis , which leads to necrosis , and mainly involves the blood vessels of the lungs , gastrointestinal system , and peripheral nerves , though it can also affect the heart , skin , and kidneys .
hypereosinophilic syndrome is characterized by a persistently elevated eosinophil count ( 1,500 eosinophils / mm ) in the blood for at least six months without any recognizable cause .
tens and stevens - johnson syndrome are characterized by diffuse erythematous or purpuric macules with involvement of more than 30% of the body surface area with epidermal necrosis with mucosal membrane involvement .
the incidence of dhs ranges from 0.5% to 3% , and the median latency before symptom onset can be as little as 2 to 6 hours in previously sensitized patients , to as late as 6 months.9 the mechanism of dhs has not been clearly defined.9 however , a few mechanisms have been proposed , for example , dhs might be a combination of type i , type iv , and perhaps type iii gel and coombs hypersensitivity reactions,10 or alternately , it could be a modified form of graft - versus - host disease mediated by activated t - lymphocytes.10 it is worth noting that although dapsone hepatotoxicity is a dose - dependent effect , dhs is not.10 the classic triad of dhs consists of fever , eruption , and internal organ involvement , although hepatitis , exfoliative dermatitis , lymphadenopathy , and hemolytic anemia might be observed in varying combinations and sequences.11 in addition , cholangitis has also been described as a component of dhs.11 cutaneous lesions can range from erythematous papules , as in our patient , to plaques , pustules , and eczematous lesions .
some patients may also develop severe dermatitis and complications , such as stevens - johnson syndrome or toxic epidermal necrolysis.12 however , the severity of cutaneous changes is not correlated with the severity or extent of internal organ involvement.12 the histologic findings of the cervical lymph node were compatible with angioimmunoblastic t - cell lymphoma , and without clinical information , it is difficult to exclude this possibility .
however , a diagnosis of dapsone - associated lymphadenopathy was made because of a definite medication history capable of causing a systemic hypersensitivity reaction , and because the lymphadenopathy occurred only after the systemic hypersensitivity reaction . moreover , both tcr- and igh gene rearrangement studies showed polyclonality and infiltrating atypical t - cells and no aberrant expression of bcl-6 or cd10 . for polymerase chain reaction ( pcr ) amplification of the tcr- locus
pcr followed by single - stranded conformational polymorphism analysis were performed.13 thus , in view of the clinical and pathologic characteristics , we concluded that the manifestations were compatible with dhs .
furthermore , we presumed that the hepatitis with hepatosplenomegaly was due to a reactive change induced by dhs rather than lymphomatous involvement , and the lymphadenopathy was actually a pseudolymphoma rather than true angioimmunoblastic t - cell lymphoma ( aitl ) , which is commonly encountered in the seventh decade of life.14 in fact , the findings of a transjugular liver biopsy were compatible with drug - induced hepatitis .
the pathologic findings of the liver biopsy were granulomatous inflammation , which is observed in a large number of drug - associated injury cases.15 in the case described here , had we adopted a combination chemotherapy , such as cyclophosphamide , adriamycin , vincristine and prednisolone ( chop ) for aitl , the unnecessary treatment may have caused further hepatic impairment . to our knowledge , this is the first report of dapsone - induced lymphadenopathy mimicking aitl .
our experience of this case cautions physicians that lymph node biopsy results suggestive of lymphoma in the situation of drug - induced systemic hypersensitivity reaction should be an interpretation of the biopsy before deciding on the optimal treatment . | a 36-year - old woman presented with erythematous confluent macules on her whole body with fever and chills associated with jaundice after 8 months of dapsone therapy .
her symptoms had developed progressively , and a physical examination revealed bilateral cervical lymphadenopathy and splenomegaly .
excisional biopsy of a cervical lymph node showed effacement of the normal architecture with atypical lymphoid hyperplasia and proliferation of high endothelial venules compatible with angioimmunoblastic t - cell lymphoma .
however , it was assumed that the cervical lymphadenopathy was a clinical manifestation of a systemic hypersensitivity reaction because her clinical course was reminiscent of dapsone - induced hypersensitivity syndrome .
a liver biopsy revealed drug - induced hepatitis with no evidence of lymphomatous involvement .
intravenous glucocorticoid was immediately initiated and her symptoms and clinical disease dramatically improved .
the authors present an unusual case of cervical lymphadenopathy mimicking angioimmunoblastic t - cell lymphoma as an adverse reaction to dapsone . |
we report a case of a female patient on aspirin and warfarin who developed a retropharyngeal haematoma following minor blunt head and neck trauma .
an 84 year old woman had a mechanical fall at ground level resulting in neck hyperextension .
a rigid cervical collar was applied and anterior neck and chest swelling and bruising were noted beneath this .
a massive retropharyngeal haematoma was observed extending between c2 and t4 vertebral body levels with displacement and compression of the trachea ( figure 1,2 ) .
there was no evidence of carotid , vertebral or internal jugular vein injury and no cervical fracture .
contrast enhanced axial ct image taken at the level of the hyoid bone shows a large retropharyngeal haematoma ( star ) with active intravenous contrast extravasation ( arrow ) contrast enhanced sagittal ct image shows a large retropharyngeal haematoma ( star ) extending from the c2 vertebral level to below the level of the sternal notch with tracheal compression ( arrow ) nine hours from the time of injury the patient developed stridor .
although significant subglottic swelling was evident at intubation , a 6.5 mm diameter endotracheal tube was successfully and atraumatically positioned into the distal trachea .
a four vessel angiogram was then obtained which revealed an actively bleeding branch of the right inferior thyroid artery .
the patient was managed non - operatively with intravenous antibiotics , steroids and inr reversal . over the coming days her neck swelling decreased and serial ct showed a reduction in size of the haematoma .
dysphagia necessitated nasogastric feeding for a further week and mild dysphonia persisted for two weeks .
the patient otherwise made a good recovery , returning to her previous level of function by six weeks .
aetiological factors implicated in retropharyngeal haematoma include blunt head and neck trauma , cervical spine injury , anticoagulation , bleeding diatheses and tumors .
most cases are thought to be due to bleeding from vessels covering the anterior longitudinal ligament.(2 ) less commonly bleeding has been reported to originate from the thyrocervical trunk ( 3 ) as was likely the situation in this case .
the classic clinical presentation comprises capp s triad of airway compression , displacement of the trachea anteriorly and bruising of the neck and chest.(1 ) however , the clinical presentation is highly variable and may range from mild sore throat to hoarseness , dysphagia , odynophagia , dyspnoea or stridor.(15 ) an asymptomatic interval between time of injury and onset of stridor has been described(4 ) as in this case .
the first step is to secure the airway if there is any evidence of airway obstruction .
the haematoma may distort the anatomy of the upper airway making visualization of the cords difficult .
a surgical airway is advocated by some authors as the preferred method because endosopic intubation may be difficult and carry a higher risk of perforating the haematoma.(3 ) we elected to trial an awake endoscopic intubation method with tracheostomy equipment available if required .
this intubation method allowed direct visualization of the laryngeal anatomy with minimal manipulation of the haematoma whilst maintaining cervical spine alignment . with the airway secured ,
most patients are successfully managed conservatively with observation , supportive treatment and monitoring with serial ct .
the haematoma usually resolves but can take four weeks or more.(5 ) surgical evacuation or transoral aspiration is generally reserved for a haematoma that is rapidly expanding , impeding mechanical ventilation or failing to resolve . although surgery may lead to an earlier extubation and recovery , it does carry an increased risk of infection .
( 1 ) we have presented a case of a large retropharyngeal haematoma in an elderly anticoagulated patient following minor blunt head and neck trauma . the patient presented insidiously with capp s triad and developed delayed airway obstruction necessitating emergency fibreoptic endoscopic intubation .
conservative management may be appropriate in patients with non expanding haematomas that show improvement on serial ct . | retropharyngeal haematoma is a rare and potentially fatal cause of airway obstruction .
the treatment of retropharyngeal haematoma is contentious .
we report a case of an 84 year old woman on aspirin and warfarin who developed a retropharyngeal haematoma following minor blunt head and neck trauma .
the patient presented insidiously with capp s triad and developed delayed airway obstruction necessitating emergency fibreoptic endoscopic intubation .
both tracheostomy and surgical drainage were avoided and she recovered well . |
this type of infection is related , among others , to the type of the transplanted organ .
invasive candidiasis occurs more often in liver recipients , and invasive aspergillosis in lung recipients [ 13 ] .
invasive candidiasis is one of the main post transplant complications , presenting a high risk of death .
there are three phases , depending on the time since transplant : early ( 1 month after transplant ) , intermediate ( 2 - 6 months ) , and late ( > 6 months )
. there might be different types of complications , because of different risk factors depending on how long ago the transplant was performed .
invasive candidiasis is a risk in the early , intermediate and late phase , however , each phase is characterised by different predisposing factors [ 1 , 3 ] .
rapid and precise fungal infection diagnosis and appropriate antifungal treatment administration are crucial in post transplant patients .
tests confirming diagnosis are being developed because of the difficulty in detecting fungal infections , because of scarce and unspecific symptoms , and because of insufficient sensitivity and specificity of classical methods [ 1 , 4 ] . besides classical / culture methods , new ones to detect yeast infections , such as highly immunogenic components of yeast cell walls
mannan [ platelia candida ag plus ( biorad , marne - la- coquette , france ) ] , and also ( 1 - 3)--d - glucan ( such as fungitec- g , glucatell fungitell ) , are being researched [ 2 , 5 , 6 ] .
rapid serological tests seem useful to monitor the physical condition of patients at high risk of fungal infections , and also make it possible to quickly detect them [ 6 , 7 ] .
cell wall components are transient and rapidly taken up by endocytosis or by the forming immune complexes in invasive infections . for practical reasons , it is advisable to regularly monitor the levels of those markers in body fluids in high - risk patients .
the simple detection of yeast cell wall components does not provide any information on strain antimycotic susceptibility .
with mannan , a polysaccharide antigen with strong immunogenic and immunomodulatory properties , it was possible to come up with a new strategy of invasive candidiasis detection .
it focuses on simultaneously monitoring the levels of circulating mannan and anti - mannan antibodies in patients at risk of invasive candidiasis .
this strategy increases the chances of detecting invasive infections , since a high mannan level is related to a decrease in anti- mannan antibodies .
study , where a single identification of mannan or anti - mannan antibodies detected the infection in 40 - 50% of individuals .
simultaneous testing for these two markers resulted in an 80% increase in sensitivity and 93% increase in specificity in the same patients . as it was proven by sendid et al .
, simultaneous testing for mannanaemia and anti - mannan antibodies in diagnosing invasive candidiasis , especially in immunocompetent patients , increased the chances of detecting an infection [ 7 , 8 ] .
sendid et al . investigated their correlation in patients presenting invasive candidiasis symptoms , but no neutropenia symptoms [ 2 , 4 , 7 ] . despite quite thorough descriptions of the importance of testing for mannan and anti - mannan antibodies in bone - marrow transplant recipients ,
there is still little information about the usefulness of these tests in surgical and transplant patients , especially because of the administered immunosuppression .
it could become a helpful parameter in diagnosing invasive candidiasis in recipients with stable organ transplants and without additional aggravations .
however , these tests are most sensitive with candida albicans , c. glabrata , and c. tropicalis infections . for c. krusei , c. parapsilosis , and c. guillermondii infections the sensitivity is only of 40 - 50% [ 6 , 7 , 9 ] . since so little is known about the usefulness of mannan antigen and anti - mannan antibodies testing , the idea was to try assessing the usefulness of this test in surgical and transplant patients .
the present study was to determine the usefulness of mannan antigen and anti - mannan antibody testing in diagnosing invasive candidiasis in liver / kidney transplant recipients .
twenty - five patients were selected and had serum samples collected at least twice , as part of routine diagnostic procedures , and additionally were examined for clinical symptoms of fungal infections , had their fungal prevention routines assessed , and fungi were cultured out of various clinical materials , including blood .
mycological tests were performed on 66 clinical samples from 25 patients hospitalised at the warsaw teaching hospital in 2011 - 2012 .
additionally , 35 clinical samples collected from those patients were analysed during that period of time as part of routine mycological diagnostic procedures , including blood ( 4 ) , bal ( 4 ) , sputum ( 5 ) , and urine ( 4 ) ; other materials included : throat swab , fluid after liver re - transplantation , aortic abscess , wounds , drains , samples of tissues , peritoneal fluid , anal swabs , stool samples , and 66 blood serum samples .
the clinical material was collected during routine diagnostic procedures from patients hospitalised at the warsaw medical university institute of transplantology in 2011 - 2012 .
strains were cultured on sabouraud agar with chloramphenicol for 24 - 48 hours at 30c , and then on chromagar candida media ( graso biotech , starogard gdanski , poland ) .
strain identification was performed using biochemical traits with the automated i d 32 c test ( bio- meriux , marcy l'etoile , france ) according to manufacturer 's guidelines .
all collected serum samples were stored at 20c before testing , according to test manufacturer 's guidelines .
the levels of mannan and anti - mannan antibodies were assessed with platelia candida ag plus , and platelia candida ab plus ( biorad , marne - la - coquette , france ) commercial tests , according to manufacturer 's guidelines .
result interpretation followed manufacturer 's mannan cutoffs : negative < 62.5 pg / ml ; positive > 125 pg / ml , for anti - mannan antibodies : negative < 5 au / ml ; positive > 10 au / ml .
after analysing 66 serums from 25 patients ( 9 liver transplant recipients , 7 kidney transplant recipients , and 9 patients prepared for a kidney transplant ) , 29 serum samples from 15 patients tested positive for mannan antigen .
thirty - one serum samples from 14 patients presented a high level of anti - mannan antibodies .
eleven serum samples from 7 patients presented a high level of both soluble mannan antigen and anti - mannan antibodies . in those patients , candida spp .
was also cultured in different clinical material from 11 patients with a high mannan antigen or anti - mannan antibody levels .
the biggest percentage of fungal infections occurs in bone marrow transplants , more rarely in liver transplants , most rarely in kidney transplants .
mycological tests , including sample microscopy analysis , culture on different media , and identification using biochemical traits , are still insufficient to detect fungal infections [ 3 , 10 ] . according to eortc / ificg guidelines , there are three types of fungal infections in high - risk patients : confirmed , probable and possible . positive blood cultures , which enable identification of systemic mycoses with manifesting clinical signs , indicating organ invasion , are one of the most important diagnostic criteria ; furthermore , additional tests confirm fungal presence in tissues .
additional tests , such as direct microscopic examination confirm the presence of fungi in tissues .
probable invasive mycosis is diagnosed in the presence of clinical signs of infection and the detection of biomarkers ( fungal antigen ) in at least two serum samples .
fungal infections in high - risk patients , not presenting any clinical signs , but with positive cultures in material collected from various sites but not blood , should be considered possible [ 11 , 12 ] .
elisa , the used serological methods , detecting highly immunogenic cell wall components mannan antigens , and also anti - mannan antibodies , are more and more often used in early detection of fungal infections .
serological monitoring of mannan antigen and anti - mannan antibody level in high - risk patients might be a key in detecting or ruling out invasive mycoses .
testing for mannan and anti - mannan antibodies in systemic candidiasis may be performed about six days earlier to positive blood cultures .
many studies emphasise the fact that fungal antigen detection in blood serum in patients under immunosuppression , especially with neutropenia , suggests that adequate antifungal treatment should be administered .
different treatment strategies are used , depending on patient condition and laboratory results . preventive empirical treatment , used in patients with prolonged neutropenia and fever lasting over five days despite antibiotic treatment , and also with mucosal candidiasis , or with inflammatory lung lesions ,
is administered in high - risk patients in order to reduce the risk of invasive fungal infections .
patients with positive biomarker results undergo preemptive treatment if fungal biomarkers in their blood serum are regularly monitored [ 15 , 16 ] .
targeted therapy is used in patients with a diagnosed fungal infection according to the antimicrobial resistance profile of the pathogen [ 11 , 14 , 17 ] .
treatment in patients with suspected invasive candidiasis as well as in those with a diagnosed infection is difficult and depends on patient condition , infection localisation , and infectious strain .
the infectious diseases society of america ( idsa ) recommends the following preventive antifungal drugs : fluconazole , itraconazole , and amphotericin b [ 18 , 19 ] .
echinocandins ( caspofungin , anidulafungin , micafungin ) , drugs with a proven high effectiveness in treating candida spp .
infections , are also often used in empirical treatment , besides fluconazole and amphotericin b .
rescue treatment , consisting of a combination of antimycotics or non - standard doses , is used in patients with advanced fungal infections not responding to standard treatments .
a thorough assessment of study results demonstrates that serological tests may be used to confirm or rule out invasive candidiasis in high - risk patients .
the findings of the present study were analogous to those of foreign research centres [ 10 , 20 ] .
n n401 042738 ) . project manager : e. swoboda - kope , md , phd | solid organ transplant recipients are at high risk of fungal infections , because of ongoing immunosuppressive treatment .
there are three post organ transplant phases : early , intermediate , and late , all of them at risk of candida infections .
since conventional tests are insufficient , specific secondary diagnostic tests are still being explored .
serological tests are currently the most common choice .
the present study was to determine the usefulness of mannan antigen and anti - mannan antibody detection in diagnosing invasive candidiasis in liver or kidney transplant recipients .
the levels of mannan and anti - mannan antibodies were assessed with platelia candida ag plus , and platelia candida ab plus ( biorad , marne - la - coquette , france ) commercial tests , according to manufacturer 's guidelines .
sixty six serum samples were obtained from 25 patients ( 9 liver transplant recipients , 7 kidney transplant recipients , and 9 patients prepared for a kidney transplant ) , 29 serum samples from 15 patients tested positive for mannan antigen .
serum samples were obtained from 14 patients tested positive for anti- mannan antibodies .
fungal antigen detection in blood serum in patients under immunosuppression , especially with neutropenia , suggests that antifungal treatment should be administered .
serological tests , especially mannan and anti - mannan ones , are very useful for confirmation or exclusion of invasive candidiasis in high - risk patients . |
asthma is a disease that is caused by inflammation in the lung and bronchus and is affected by genetic and environmental influences .
aspirin - exacerbated respiratory disease ( aerd ) , which was first reported in 1922 , is a type of asthma .
aerd is characterized by the following three symptoms : bronchial asthma , aspirin sensitivity , and nasal polyposis [ 1 , 2 , 3 ] .
it has been reported that 10 - 20% of asthma patients have aspirin sensitivity , whereas 1 - 2% of the non - asthma population shows aspirin sensitivity [ 4 , 5 ] .
although the mechanisms of aerd pathogenesis are still not fully understood , inflammatory responses by overproduction of leukotrienes are regarded as the main pathogenesis of aerd .
aspirin is a commonly used medication , which belongs to the non - steroidal anti - inflammatory drugs . despite being a widely used medication
, aspirin intake also causes various side effects , including manifested gastrointestinal ulcer , stomach bleeding , and tinnitus , especially in higher doses . although the side effects of aspirin are not common , these effects have been reported in about 10% of adult asthmatics . in aerd
pathogenesis , aspirin inhibits the activation of cyclooxygenase-1 enzyme , leading to block of production of prostaglandin and thromboxane .
this mechanism causes overproduction of leukotrienes , such as leukotriene b4 , leukotriene c4 , leukotriene d4 , and leukotriene e4 [ 6 , 7 ] .
the tyrosine - protein kinase tec ( tec ) is a member of the non - receptor tyrosine kinases and has critical roles in cell signaling transmission , calcium mobilization , gene expression , and transformation .
it has been known that tec family kinases are associated with various intracellular signaling mechanisms , such as cytokine receptors and lymphocyte surface antigens [ 9 , 10 ] . in particular , it was demonstrated that the tec family proteins are involved in regulation of leukotriene secretion via the mast cell signaling pathway .
therefore , we hypothesized that tec polymorphisms might be involved in aerd pathogenesis . in the present study , 38 tec single nucleotide polymorphisms ( snps )
were genotyped in a total of 592 subjects , which comprised 163 aerd cases and 429 aspirin - tolerant asthma ( ata ) controls , to examine the associations between tec polymorphisms and aerd susceptibility .
subjects in this study were recruited from the asthma genome research center , comprising the hospitals of soonchunhyang , chunnam , chungbuk , seoul national , and chung - ang universities in korea .
all subjects provided written informed consents , and the study protocols were approved by the institutional review board of each hospital .
we also performed aspirin challenge in subjects with a history of aspirin hypersensitivity , presence of urticaria , nasal polyp , and sinusitis .
the aerd case group included patients with 20% or greater decreases in forced expiratory volume in 1 second ( fev1 ) or 15% to 19% decreases in fev1 with naso - ocular or cutaneous reactions , whereas subjects showing a rate of fev1 decline less than 15% without extrabronchial nasal or skin symptoms were included in the ata group .
the clinical diagnostic factors for the present study are summarized in table 1 . to investigate the associations between tec polymorphisms and the risk of aerd , we selected candidate snps based on allele frequencies in the asian population , linkage disequilibrium ( ld ) status , and national center for biotechnology information .
genotyping of 38 tec polymorphisms was performed in a total of 592 subjects , including 163 aerd cases and 429 ata controls .
genotyping was carried out with 20 ng of genomic dna by taqman assay using abi prism 7900ht sequence detection system software version 2.3 ( applied biosystems , foster city , ca , usa ) in all subjects .
assay ids of all snps used in taqman assay are listed in supplementary table 1 .
we applied a widely used measure of linkage disequilibrium to all pairs of biallelic loci : lewontin 's d ' ( |d'| ) and r. haplotypes of each individual were inferred using the phase algorithm ( ver .
linear regression analysis was performed to examine the differences in the rates of decline in fev1 following aspirin challenge among the genotypes and major haplotypes .
the data were managed and analyzed using statistical analysis system , version 9.2 ( sas inc . ,
associations for aerd under the logistic model were adjusted by smoking status , atopy , body mass index ( bmi ) , age , and sex ( male = 0 , female = 1 ) .
subjects in this study were recruited from the asthma genome research center , comprising the hospitals of soonchunhyang , chunnam , chungbuk , seoul national , and chung - ang universities in korea .
all subjects provided written informed consents , and the study protocols were approved by the institutional review board of each hospital .
we also performed aspirin challenge in subjects with a history of aspirin hypersensitivity , presence of urticaria , nasal polyp , and sinusitis .
the aerd case group included patients with 20% or greater decreases in forced expiratory volume in 1 second ( fev1 ) or 15% to 19% decreases in fev1 with naso - ocular or cutaneous reactions , whereas subjects showing a rate of fev1 decline less than 15% without extrabronchial nasal or skin symptoms were included in the ata group .
to investigate the associations between tec polymorphisms and the risk of aerd , we selected candidate snps based on allele frequencies in the asian population , linkage disequilibrium ( ld ) status , and national center for biotechnology information .
genotyping of 38 tec polymorphisms was performed in a total of 592 subjects , including 163 aerd cases and 429 ata controls .
genotyping was carried out with 20 ng of genomic dna by taqman assay using abi prism 7900ht sequence detection system software version 2.3 ( applied biosystems , foster city , ca , usa ) in all subjects .
assay ids of all snps used in taqman assay are listed in supplementary table 1 .
we applied a widely used measure of linkage disequilibrium to all pairs of biallelic loci : lewontin 's d ' ( |d'| ) and r. haplotypes of each individual were inferred using the phase algorithm ( ver .
linear regression analysis was performed to examine the differences in the rates of decline in fev1 following aspirin challenge among the genotypes and major haplotypes .
the data were managed and analyzed using statistical analysis system , version 9.2 ( sas inc . ,
associations for aerd under the logistic model were adjusted by smoking status , atopy , body mass index ( bmi ) , age , and sex ( male = 0 , female = 1 ) .
we recruited a total of 592 subjects , which consisted of 163 aerd cases and 429 ata controls , for the present study . according to the results , four clinical characteristics showed significant differences between the case and control groups ( table 1 ) .
the fall rate by aspirin provocation was significantly higher in aerd subjects than ata controls ( 24.63 16.11 vs. 3.54 4.85 ; p = 0.0001 ) .
percentage of predicted fev1 in the aerd subjects showed decreased lung function than in ata subjects ( 87.58 16.94 vs. 91.66 16.87 ; p = 0.009 ) .
also , age of first medical examination and bmi were lower in aerd cases than ata controls .
the other diagnostic factors showed no significant differences between the case and control group . in the present study ,
locations of the polymorphisms are shown in a genetic map of tec with their ld status ( fig .
1a ) . minor allele frequencies ( mafs ) of the snps in korean subjects are displayed in supplementary table 2 with their allele change , position , and p - value for hardy - weinberg equilibrium .
the ld blocks were obtained by using haploview software , and the haplotypes of each ld block were calculated by phase software ( fig . 1b and 1c ) .
the minor haplotypes that had frequencies lower than 0.05 were merged and presented as ' others . ' to compare genetic differences among ethnicities , we obtained mafs of caucasians , han chinese , japanese , and africans from the ncbi database ( dbsnp ) ( supplementary table 2 ) and calculated ld blocks using genotype data from the international hapmap database ( supplementary fig .
logistic analyses were performed to investigate the associations between tec polymorphisms and the risk of aerd . the result from analyses revealed that all snps and major haplotypes were not associated with the risk of aerd ( table 2 ) . on the other hand , two snps ( rs7664091 , p = 0.04 and rs12500534 , p = 0.03 ) and one major haplotype in ld block 2 ( tec_bl2_ht4 , p = 0.03 ) showed marginal associations in the regression analysis with the decline rate of fev1 ( supplementary table 3 ) . however , the association signals of these polymorphisms disappeared after performing corrections for multiple testing ( data not shown ) .
taken together , these results indicate that the tec polymorphisms are not associated with aerd susceptibility , at least in the korean population .
the lack of associations in this study suggests that although tec plays an important role in immune responses , tec variants do not directly affect decreased pulmonary function by aspirin uptake .
however , considering the fact that the difference in frequency of polymorphisms showed different effects in various populations , replication studies in aerd subjects in other ethnicities are recommended .
however , the rare condition of aerd made it difficult to recruit subjects in the korean asthma cohort .
second , snps in coding sequence were not selected , due to very low frequencies , although non - synonymous snps ( nssnp ) in exonic regions could affect risk of the disease .
further studies are required to examine the molecular role of nssnps in tec . in conclusion
however , our results showed that 38 tec polymorphisms and 12 major haplotypes were not associated with the risk of aerd .
although further studies are required to investigate the exact role of the tec snps in immune responses , the preliminary results of the present study may provide useful information for aerd pathogenesis .
supplementary data including three tables and one figure can be found with this article online at http://www.genominfo.org/src/sm/gni-12-58-s001.pdf .
assay ids of 38 single nucleotide polymorphisms in tec gene allele distribution of tec polymorphisms regression analysis of tec polymorphisms and haplotypes with the fall rate of fev1 by aspirin provocation in a korean population linkage disequilibrium ( ld ) plots for tec gene in five ethnicities . | the tyrosine - protein kinase tec ( tec ) is a member of non - receptor tyrosine kinases and has critical roles in cell signaling transmission , calcium mobilization , gene expression , and transformation .
tec is also involved in various immune responses , such as mast cell activation .
therefore , we hypothesized that tec polymorphisms might be involved in aspirin - exacerbated respiratory disease ( aerd ) pathogenesis .
we genotyped 38 tec single nucleotide polymorphisms in a total of 592 subjects , which comprised 163 aerd cases and 429 aspirin - tolerant asthma controls .
logistic regression analysis was performed to examine the associations between tec polymorphisms and the risk of aerd in a korean population .
the results revealed that tec polymorphisms and major haplotypes were not associated with the risk of aerd . in another regression analysis for the fall rate of forced expiratory volume in 1 second ( fev1 ) by aspirin provocation , two variations ( rs7664091 and rs12500534 ) and one haplotype ( tec_bl2_ht4 ) showed nominal associations with fev1 decline ( p = 0.03 - 0.04 ) .
however , the association signals were not retained after performing corrections for multiple testing . despite tec playing an important role in immune responses , the results from the present study suggest that tec polymorphisms do not affect aerd susceptibility .
findings from the present study might contribute to the genetic etiology of aerd pathogenesis . |
the dengue virus strains sequenced for this study as well as sequences obtained from genbank for this study are listed in table 1 .
the virus isolates were obtained from the centers for disease control and prevention , dengue branch , puerto rico , and division of vector - borne infectious diseases , ft .
collins , colorado ; medical research institute , colombo , sri lanka ; school of public health , berkeley , california ; walter reed army institute for research , washington , d.c . ; and university of massachusetts medical center , worcester , massachusetts .
includes original identifier for strain , year of isolation , taxa name used in this paper , dengue virus 3 subtype , source of viral sequence , and genbank accession numbers .
qiaamp viral rna mini kit ( qiagen , valencia , ca ) was used to extract viral rna from both the mosquito grind supernatants and infected tissue culture media following the manufacturer s protocol .
extracted rna was stored at 70c or immediately subjected to reverse transcription polymerase chain reaction ( rt - pcr ) . denv-3
primers were designed to amplify and sequence a 966-bp fragment from positions 1791,144 , encompassing part of capsid , all of prem , and part of the e gene sequences . the reverse primer ( den3/735 ) hybridized to positions 1,1891,171 ( 5-ctcctcaggcaaaaccgct-3 ) and the forward primer ( d1 consensus ) hybridized to positions 132159 ( 5-tcaatatgctgaaacgcgcgagaaaccg-3 ) .
the reverse primer den3/735 was added to extracted rna , incubated at 85c for 90 s , and allowed to cool to room temperature .
rt was carried out for 4560 min in 20 l of reaction mix containing 25 u avian myeloblastosis virus reverse transcriptase ( roche , nutley , nj ) , deoxynucleoside tripophosphate , mgcl2 , and rt buffer .
pcr was performed by adding a 30-l cocktail containing d1 consensus primer , pcr buffer , and expand polymerase ( roche ) to the 20-l rt reaction .
pcr conditions were 4 min at 94c , 3035 cycles of 94c for 30 s , 54c for 30 s , and 72c for 90 s with 5 s / cycle added to elongation step after the first 10 cycles .
we separated 5 l of the reaction products on 2% agarose gels and visualized it by ethidium bromide staining .
when necessary , target bands were excised and purified by using the qiagen qiaquick gel extraction kit ( qiagen ) following manufacturer s instructions .
all remaining pcr reaction products were purified by using the qiagen pcr purification kit following the manufacturer s protocol .
purified pcr products were sent to the automated dna sequencing facility at the university of north carolina , chapel hill , nc .
the denv-3 sequences used in this manuscript included 40 newly determined sequences , which have been submitted to genbank ( accession nos .
overlapping individual nucleic acid sequences were assembled with the aid of vector nti contigexpress ( informax , inc . ,
sequences were aligned and analyzed by using the following software : clustal x ( available from : url : http://inn-prot.weizmann.ac.il/software/clustalx.html ) , paup * ( available from : url : http://www.sinauer.com ) , phylip ( available from : url : http://evolution.genetics.washington.edu/phylip.html ) , and mega ii ( available from : url : http://www.megasoftware.net ) .
genetic distances were calculated by using tamura - nei distance algorithm with 1,000 bootstrap replicates ; the trees were generated by using the minimum evolution method .
the phylogenetic tree in figure 1 is based on a 708-base segment , positions 4371,145 , spanning pre - m / m and a portion of the e gene .
the phylogenetic tree presented in figure 2 is based on 966-base region spanning positions 1791,145 on the viral genome , capturing a portion of the c gene , all of pre - m / m gene , and a portion of the e gene .
phylogenetic tree of established dengue virus 3 subtypes ( 23 ) and the relationship of sri lanka pre and post dengue hemorrhagic fever dengue virus 3 ( denv-3 ) isolates to the established subtypes .
this tree is based on a 708-base segment , positions 437 to 1145 , spanning pre - m / m and a portion of the e gene .
. numbers at nodes indicate bootstrap support values for the branch of the tree inferred at that node . the origin of the viruses and sequences used are listed in table 1 .
positions are numbered sequentially from the first position in the pre - m / m protein .
phylogenetic tree of dengue virus 3 , subtype iii group a , group b , east africa , and latin america . tree is based on 966-base region spanning positions 1791,145 on the viral genome , capturing a portion of the c gene , all of pre - m / m gene and a portion of the e gene .
nucleotide substitutions conserved within each dengue virus 3 , subtype iii group ( group a , b , east africa , and latin america ) are listed in table 3 .
positions are numbered sequentially from the first nucleotide position at the 5 end of the genome .
the dengue virus strains sequenced for this study as well as sequences obtained from genbank for this study are listed in table 1 .
the virus isolates were obtained from the centers for disease control and prevention , dengue branch , puerto rico , and division of vector - borne infectious diseases , ft .
collins , colorado ; medical research institute , colombo , sri lanka ; school of public health , berkeley , california ; walter reed army institute for research , washington , d.c . ; and university of massachusetts medical center , worcester , massachusetts .
includes original identifier for strain , year of isolation , taxa name used in this paper , dengue virus 3 subtype , source of viral sequence , and genbank accession numbers .
qiaamp viral rna mini kit ( qiagen , valencia , ca ) was used to extract viral rna from both the mosquito grind supernatants and infected tissue culture media following the manufacturer s protocol .
extracted rna was stored at 70c or immediately subjected to reverse transcription polymerase chain reaction ( rt - pcr ) .
primers were designed to amplify and sequence a 966-bp fragment from positions 1791,144 , encompassing part of capsid , all of prem , and part of the e gene sequences . the reverse primer ( den3/735 ) hybridized to positions 1,1891,171 ( 5-ctcctcaggcaaaaccgct-3 ) and the forward primer ( d1 consensus ) hybridized to positions 132159 ( 5-tcaatatgctgaaacgcgcgagaaaccg-3 ) .
the reverse primer den3/735 was added to extracted rna , incubated at 85c for 90 s , and allowed to cool to room temperature .
rt was carried out for 4560 min in 20 l of reaction mix containing 25 u avian myeloblastosis virus reverse transcriptase ( roche , nutley , nj ) , deoxynucleoside tripophosphate , mgcl2 , and rt buffer .
pcr was performed by adding a 30-l cocktail containing d1 consensus primer , pcr buffer , and expand polymerase ( roche ) to the 20-l rt reaction .
pcr conditions were 4 min at 94c , 3035 cycles of 94c for 30 s , 54c for 30 s , and 72c for 90 s with 5 s / cycle added to elongation step after the first 10 cycles .
we separated 5 l of the reaction products on 2% agarose gels and visualized it by ethidium bromide staining .
when necessary , target bands were excised and purified by using the qiagen qiaquick gel extraction kit ( qiagen ) following manufacturer s instructions .
all remaining pcr reaction products were purified by using the qiagen pcr purification kit following the manufacturer s protocol .
purified pcr products were sent to the automated dna sequencing facility at the university of north carolina , chapel hill , nc .
the denv-3 sequences used in this manuscript included 40 newly determined sequences , which have been submitted to genbank ( accession nos .
overlapping individual nucleic acid sequences were assembled with the aid of vector nti contigexpress ( informax , inc . , bethesda , md ) .
sequences were aligned and analyzed by using the following software : clustal x ( available from : url : http://inn-prot.weizmann.ac.il/software/clustalx.html ) , paup * ( available from : url : http://www.sinauer.com ) , phylip ( available from : url : http://evolution.genetics.washington.edu/phylip.html ) , and mega ii ( available from : url : http://www.megasoftware.net ) .
genetic distances were calculated by using tamura - nei distance algorithm with 1,000 bootstrap replicates ; the trees were generated by using the minimum evolution method .
the phylogenetic tree in figure 1 is based on a 708-base segment , positions 4371,145 , spanning pre - m / m and a portion of the e gene .
the phylogenetic tree presented in figure 2 is based on 966-base region spanning positions 1791,145 on the viral genome , capturing a portion of the c gene , all of pre - m / m gene , and a portion of the e gene .
phylogenetic tree of established dengue virus 3 subtypes ( 23 ) and the relationship of sri lanka pre and post dengue hemorrhagic fever dengue virus 3 ( denv-3 ) isolates to the established subtypes .
this tree is based on a 708-base segment , positions 437 to 1145 , spanning pre - m / m and a portion of the e gene .
. numbers at nodes indicate bootstrap support values for the branch of the tree inferred at that node .
positions are numbered sequentially from the first position in the pre - m / m protein .
phylogenetic tree of dengue virus 3 , subtype iii group a , group b , east africa , and latin america . tree is based on 966-base region spanning positions 1791,145 on the viral genome , capturing a portion of the c gene , all of pre - m / m gene and a portion of the e gene .
nucleotide substitutions conserved within each dengue virus 3 , subtype iii group ( group a , b , east africa , and latin america ) are listed in table 3 .
positions are numbered sequentially from the first nucleotide position at the 5 end of the genome .
many investigators have used viral nucleotide sequence data and phylogenetic methods to understand genetic relationships between viruses , as well as the epidemiology of viral disease .
phylogenetic studies have shown that dengue viruses can move long distances between continents ( 24 ) as well as short distances between neighboring countries ( 25 ) .
our goal was to use a phylogenetic approach to understand recent dhf outbreaks caused by denv-3 infections in the indian subcontinent and latin america .
previous phylogenetic analysis of denv-3 has principally relied on complete or partial sequences of the pre - m / m and e genes ( 13,16,17,23 ) .
our analysis used a 708-base segment , positions 4371,145 , spanning pre - m / m and a portion of the e gene , coding for 236 amino acids .
this region was selected because it both conserved the original phylogenetic relationship identified by lanciotti et al . and , in preliminary analysis with previously established sequences , captured 44% of the variable sites within denv-3 , subtype iii sri lankan sequences .
a total of 40 denv-3 sequences , including 21 sequences available from genbank and 19 newly determined indian subcontinent and african sequences ( table 1 ) were compared .
dates of isolation ranged from 1963 to 1998 . with the exception of 63puertor , all sequences were from low - passage ( < 4 ) virus cultures . several approaches to phylogenetic analysis , including maximum likelihood , parsimony , and distance methods ,
results presented here used the tamura - nei algorithm to calculate genetic distances and the minimum evolution method to create the trees ( figure 1 ) .
the tree identifies four distinct lineages that correspond to the region of isolation , reproducing the same evolutionary relationship first described by lanciotti , et al .
subtype i includes isolates from southeast asia and the south pacific islands ; subtype ii consists of isolates from thailand ; subtype iii is comprised of isolates from the indian subcontinent , east africa , and a single isolate from samoa ; and subtype iv includes puerto rico and tahiti .
similarity within subtypes was high , with subtype iii showing the greatest mean similarity ( 98.4% ) , followed by subtypes i , ii , and iv ( table 4 ) .
all 24 sri lankan , indian , and east african strains fell into subtype iii ( figure 1 ) .
the circulating virus genotypes within this region have remained closely related over the relatively long period of 18 years ( 19811998 ) , indicating that countries bordering the western indian ocean form a geographically distinct region with regard to denv-3 viruses .
denv-2 viruses in the regions also form a subtype with a similar geographic distribution ( 26,27 ) .
frequent trade between east africa , western indian ocean islands , and the indian subcontinent may have been responsible for the movement of dengue viruses throughout the region ( 26,28 ) .
rico - hesse , for example , demonstrated the introduction of denv-2 to africa from islands in the indian ocean ( 26 ) .
the earliest subtype iii virus on record is an isolate from india in 1966 ; this virus occupies a node that is ancestral to all the subsequent asian and african isolates ( r.s .
lanciotti , pers . comm . ) , suggesting that the denv-3 , subtype iii viruses have their origin in the indian subcontinent and have subsequently spread out of the region . in sri lanka , regular epidemics of dhf have been observed only since 1988 .
denv-3 is responsible for many of the infections that progress to dhf ( 20,22 ) .
denv-3 isolates obtained before and after the emergence of dhf are very closely related and belong to subtype iii , indicating that the emergence of dhf on the island is not due to the introduction of a new subtype from outside the region .
however , within subtype iii , most sri lankan isolates ( except for 93srilan1 ) from before and after the emergence of dhf segregated into two distinct clades , designated groups a and b ( figure 1 ) .
group a , with nine isolates from 1981 to 1989 , consists of viruses collected up to the year epidemic dhf emerged in sri lanka but contains no isolates from later than 1989 .
temporally , the two groups are continuous , by virtue of sharing isolates in 1989 .
group a includes isolate 89srilan2 , while group b contains 89srilan1 , 89srilan3 , and 89srilana .
however , the groups do not form a continuous lineage ; they share a common ancestor only at the node for subtype iii ( figure 1 ) .
group b shares ancestral nodes with isolates from india and east africa . because the indian and east african isolates overlap temporally with group a ( all isolates are from the 1980s ) ,
group a and group b lineages likely diverged sometime before 1981 and followed distinct evolutionary pathways .
we propose two likely scenarios that led to the emergence of group b viruses in sri lanka .
one possibility is that the group b viruses were introduced from india or east africa into sri lanka because indian and east african isolates from the mid-1980s are closely related to sri lankan group b viruses ( figure 1 ) .
of the two regions , india is the more likely source because of geographic proximity to sri lanka , although the east african viruses could be the direct ancestors of the group b viruses .
another possibility is that both groups co - circulated in sri lanka in the early 1980s , with group b being a minor population .
some selective force operating in the late 1980s may have shifted the balance in favor of group b viruses . in either case , group b viruses emerged in sri lanka because a subtype iii variant already established in the greater region became more common in sri lanka and not because a novel virus evolved and emerged de novo on the island .
denv-3 , subtype iii was detected in the americas during df and dhf outbreaks in nicaragua and panama in 1994 ( 11 ) .
subsequently , the virus has spread to many countries in latin america , and denv-3associated dhf was confirmed in several countries ( 13,14,16,2931 ) ( figure 3 ) . to establish the relationship of recent latin american denv-3 isolates to each other and to the previously identified indian subcontinent and east african subtype iii isolates , we sequenced and analyzed a 966-base region spanning positions 1791,145 on the viral genome , capturing a portion of the c gene , all of pre - m / m gene , and a portion of the e gene .
this region adds 288 positions to the 5 end of the sequences initially presented in this study .
map of the spread of dengue virus 3 ( denv-3 ) , subtype iii through latin america and the caribbean .
the introduction of denv-3 , subtype iii was first reported in november 1994 in nicaragua and panama .
this virus strain has been isolated , identified , and reported in at least 16 other countries in the region .
these countries are nicaragua in 1994 and 1998 , brazil and venezuela in 2001 ( pan american health organization , unpub .
forty - three isolates were sequenced ( 21 from mexico and central and south america , 16 from sri lanka , 1 from india , and 5 from east africa ) ( table 1 ) .
years of isolation ranged from 1983 to 2001 , an 18-year span . except for the d3h-87 outgroup ,
most of the nucleotide mutations were silent : only 12 amino acid positions showed any variability and only 2 positions showed variability in more than one isolate . consequently
, the evolutionary relationships observed in this analysis likely reflect the results of genetic drift and are unlikely to have been influenced by host - specific selection events on this portion of the genome ( 26 ) .
all sequences included in this analysis fell within subtype iii ( data not shown ) .
several approaches to phylogenetic analysis were compared , and all approaches yielded identical or nearly identical topologies .
we used the tamura - nei algorithm to calculate genetic distances and the minimum evolution method to create the trees ( figure 2 ) .
bootstrap values are shown at critical nodes . despite the high overall similarity of the isolates in this analysis ,
the first consists of group a viruses isolated from 1981 to 1989 in sri lanka .
the second is composed of sri lankan group b , indian , east african , and all of the isolates from mexico and central and south america .
within group a , members are closely related , with a nucleotide mean similarity of 99.4% ( table 5 ) . within the expanded group b and related viruses ,
three distinct clades exist : a group of closely related sri lankan isolates from 1989 to 1998 , 5 east african isolates from 1985 to 1993 , and 21 isolates from 1994 to 2001 from latin america . isolates 84india and 93srilan1 are less closely related to the other geographically distinct isolates in the larger second lineage .
the isolates from latin america all emerge from a common node on the tree , suggesting a single introduction of a virus and the subsequent diversification of the virus population from the founding strain .
the denv-3 , subtype iii isolates from nicaragua , panama , and costa rica are closest to the latin american group s originating node , with the more recent isolates found farther from that node , reflecting the viral population s ongoing evolution after the point source introduction .
the internal branch from the latin american group shares a common node with the isolates from east africa .
the common hypothetical ancestor for latin america and east africa then shares a common node with the sri lankan group b virus isolates .
both on the phylogenetic tree and in pair - wise comparisons ( table 5 ) , the latin american group was more closely related to the isolates from east africa than to the group b sri lankan isolates .
furthermore , the east african isolates pre - date the earliest latin american isolates by 9 years , while the less closely related sri lankan group b isolates are nearly contemporaneous with the latin american isolates .
therefore , the point source denv-3 introduction into latin america is most likely to have its origins in east africa and not the indian subcontinent ( figure 4 ) .
global spread of dengue virus 3 ( denv-3 ) , subtype iii , which has been continuously circulating in the indian subcontinent from the 1960s to the present .
the virus was first isolated from east africa in 1985 in mozambique and subsequently from kenya ( 1991 ) and somalia ( 1993 ) ( 32,33 ) .
denv-3 subtype iii was first detected in the american continent in 1994 ( nicaragua and panama ) and the virus has subsequently spread through most of latin america ( 13,14,16,29,30 ) .
the arrows depict the most likely directions of spread based on the phylogenetic relationships between the viruses ( see text for details ) .
denv-3 was first detected on the african continent in 1984 to 1985 during an outbreak in mozambique ( 32 ) .
troops in africa and the persian gulf suggested that denv-3 is endemic in those regions but largely undetected ( 33 ) .
the fact that denv-3 was only first isolated from east africa in 1985 , whereas the viruses were present in the indian subcontinent at least as far back as 1966 ( r.s .
, suggests that denv-3 , subtype iii was introduced from the indian subcontinent into east africa in or before 1984 ( figure 4 ) .
this introduction led to the establishment of a stable east african group of denv-3 , subtype iii because all the isolates from mozambique , kenya , and somalia isolated from 1985 to 1993 form a distinct clade within subtype iii ( figure 3 ) .
the denv-3 , subtype iii viruses introduced into latin america are most closely related to subtype iii viruses in east africa ( figure 3 ) . although we can only speculate about the exact mode of transport of denv-3 into latin america
, we propose that panama , with its canal that attracts goods as well as civilians and military personnel from other parts of the world , may have been the point of introduction of subtype iii into the americas .
similarly , the introduction of denv-2 in 1981 into cuba may be attributable to cuban military personnel traveling between southeast asia and cuba ( 24,34 ) .
epidemiologic and clinical studies on dengue in indonesia in the 1970s pointed to strain differences between denv-3 viruses contributing to transmission and disease severity ( 35,36 ) . despite their overall similarity at the nucleotide level
, the denv-3 , subtype iii isolates examined in this study have been associated with severe or mild disease outbreaks ( figure 3 ) .
sri lankan group a viruses were isolated during a time of little to no dhf , while group b viruses were isolated after the emergence of dhf in sri lanka .
the emergence of dhf in sri lanka was not accompanied by a change in dengue transmission or the abundance of any particular serotype ( 21 ) . implicating denv-3 directly as the cause of dhf in sri lanka has been difficult because few virus isolates are available from dhf patients in sri lanka . however , during dengue surveillance studies in 1997 , only denv-3 was isolated from hospitalized dengue cases , whereas denv-1 , denv-2 , and denv-3 were isolated from patients visiting outpatient clinics ( 22 ) .
further studies are required to better establish the relative contribution of denv-3 to severe disease in sri lanka .
we found that the population of denv-3 viruses associated with dhf in sri lanka did not appear to be direct descendants of the group a viruses that were circulating before dhf emerged in that country .
the sri lankan 19891997 isolates are more closely related to the isolates from east africa and the isolates from the americas than they are to the isolates from 1981 to 1989 in sri lanka ( figure 3 ) .
all three groups of subtype iii viruses ( sri lankan group b , east african group , and latin american group ) associated with dhf are more closely related to each other than they are to the pre dhf group a viruses from sri lanka ( figure 3 ) .
thus , all the viruses within subtype iii are closely related ( mean 98.4% identity at the nucleotide level ) , yet they form distinct phylogenetic groups associated with mild or severe disease . the sri lankan group b viruses may be associated with severe disease unlike group a viruses because the group b viruses are inherently more virulent .
alternatively , the ability of preexisting dengue antibody to neutralize group a viruses and enhance group b viruses may account for the observed associations with severe and mild disease . in a recent study , antibodies against american denv-1 viruses neutralized the native american denv-2 genotype better than the southeast asian denv-2 genotype that is currently circulating in the americas and causing dhf ( 37 ) .
this study lends support to the idea that asian denv-2 may produce a more severe disease not because of inherent virulence properties but because persons with previous primary denv-1 infections may enhance infection with this genotype and neutralize infections with the native american denv-2 genotype .
similarly , denv-2 and -3 are the common serotypes in sri lanka , and persons with previous primary denv-2 infections could neutralize the denv-3 group a viruses better than the group b viruses .
this difference may explain the unexpected emergence of dhf associated with group b. further comparative studies with group a and b viruses are needed to understand their association with mild and severe disease , respectively . | over the past two decades , dengue virus serotype 3 ( denv-3 ) has caused unexpected epidemics of dengue hemorrhagic fever ( dhf ) in sri lanka , east africa , and latin america .
we used a phylogenetic approach to evaluate the roles of virus evolution and transport in the emergence of these outbreaks .
isolates from these geographically distant epidemics are closely related and belong to denv-3 , subtype iii , which originated in the indian subcontinent .
the emergence of dhf in sri lanka in 1989 correlated with the appearance there of a new denv-3 , subtype iii variant .
this variant likely spread from the indian subcontinent into africa in the 1980s and from africa into latin america in the mid-1990s .
denv-3 , subtype iii isolates from mild and severe disease outbreaks formed genetically distinct groups , which suggests a role for viral genetics in dhf . |
adult attention deficit hyperactivity disorder ( adhd ) is an extremely debilitating neurodevelopmental disorder that often persists beyond childhood , affecting 2.5%5% of adults in the general population.1 it is one of the most frequent psychiatric disorders that affect the childhood population , and recent data suggest that the diagnosis persists in the adult age in 15%65% of the cases.2 in the national comorbidity survey , the prevalence rate of adhd in 3,199 respondents of age 1844 years was reported to be 4.4%.3 similarly , in the study of who that was carried out in ten countries in the us , europe , and the middle east with 11,422 patients who were 1844 years old , estimates of adhd prevalence averaged 3.4% ( range 1.2%7.3%).4 in turkey , epidemiological studies about the prevalence of adhd are limited and mostly conducted with psychiatry outpatients or special populations . in a study carried out at istanbul university , the prevalence of adhd in adult psychiatric outpatient clinic was found to be at least 1.6% among 850 adult outpatients.5 most adults with adhd predominantly exhibit problems with distraction , which manifest as forgetfulness , disorganization , difficulty in planning , task completion , and time management .
these symptoms have great impact on functioning in daily life , with substantial impairments in work , social life , and relationships.6,7 the results of previous studies suggest that adhd in adults is associated with relatively specific risks for disruptive behavior disorders , school and job performance problems , and driving risks .
individuals with adhd miss significantly more days of work and are more likely to be fired , change jobs , and have worse job performance evaluations than those without adhd .
these situations can adversely affect productivity as measured by income loss and cost to the economy.8,9 adults with adhd are also known to be at increased risk for antisocial behavior , substance abuse , and criminal activity .
furthermore , they often have comorbid psychiatric conditions such as depression , anxiety , and bipolar disorder.1012 the impact of adhd on everyday life may diminish with age , with many patients developing effective coping mechanisms ; nevertheless , adhd often remains an impairing condition and it still continues to be an underdiagnosed disorder in adults.13 because the current diagnostic criteria for adhd were developed when the disease was assumed to be exclusively a childhood disorder , the criteria may not be applicable to the adult manifestation of adhd .
additionally , adults frequently suffer from a range of comorbid conditions , which may further complicate the diagnosis.14 although awareness and recognition of adhd in children , adolescents , and adults have improved in recent years , there are still concerns that an interruption in the clinical management of this disorder occurs while transitioning patients into adult mental health services.15 according to the outcomes of a survey study , adhd in the adult age is a less defined clinical entity the diagnosis of which is not as clear when compared to that occurring in childhood and adolescence , and adult adhd is less adequately treated than in childhood and adolescence .
the results of the survey were interpreted as suggesting that professionals may be more reluctant to diagnose and treat adhd in the adult age.16 despite the presence of a rapidly expanding literature on the importance of diagnosing and treating adhd in adulthood , clinical experience and attitudes of the specialists in the context of adult adhd and transition of the patients into adult mental health services in turkey are not well studied.17 the aim of this study was to provide data about the presentation of adult adhd in clinical practice in turkey and the treatment strategies of turkish adult psychiatrists based on their personal clinical experience in different hospital settings .
a cross - sectional online survey to be filled out by turkish adult psychiatrists was designed in may 2014 . with the aim of reaching respondents in a large geographical area , an internet - based
, self - report survey was administered utilizing software developed by survey monkey ( http://www.surveymonkey.com/ ) .
turkish adult psychiatrists were invited to participate in the survey via an email that was administered through turkish psychiatry association mail group that covers > 90% of turkish adult psychiatrists population all over the country .
online survey link was attached to the survey invitation mail , and adult psychiatrists who accepted the invitation filled out the online survey .
the survey had three main parts with 13 questions , focusing on the treatment environment , patterns of patient applications and transition , treatment strategies , and medication management for adults with adhd .
the first part of the survey refers to the clinicians working place , the number of patients treated daily in total , and the number of adult patients with adhd in follow up .
the second part of the survey involves questions about applications and referrals of patients with adhd to adult mental health services .
the third part of the survey includes questions about comorbidity and different aspects of treatment ( the clinician s preference on treatment , follow up duration , and treatment adherence ) .
one question specifically refers to the clinician s opinion about his / her self - competence to treat adult adhd in terms of their knowledge and clinical practice . due to the characteristics of the study , evaluation by the ethics committee of medical park gaziosmanpasa hospital
first - part questions on working environment of the physicians were single - select multiple choice questions and respondents selected one answer from the list of answer choices .
questions about the referrals of patients , comorbidity , and treatment were comparison and frequency ranking question scales .
descriptive analysis was conducted with the results of the qualitative variables in the form of frequency and percentages .
pearson s correlation coefficient and multiple linear regression analysis were used to examine the relationship between the selected variables .
a cross - sectional online survey to be filled out by turkish adult psychiatrists was designed in may 2014 . with the aim of reaching respondents in a large geographical area , an internet - based
, self - report survey was administered utilizing software developed by survey monkey ( http://www.surveymonkey.com/ ) .
turkish adult psychiatrists were invited to participate in the survey via an email that was administered through turkish psychiatry association mail group that covers > 90% of turkish adult psychiatrists population all over the country .
online survey link was attached to the survey invitation mail , and adult psychiatrists who accepted the invitation filled out the online survey .
the survey had three main parts with 13 questions , focusing on the treatment environment , patterns of patient applications and transition , treatment strategies , and medication management for adults with adhd .
the first part of the survey refers to the clinicians working place , the number of patients treated daily in total , and the number of adult patients with adhd in follow up .
the second part of the survey involves questions about applications and referrals of patients with adhd to adult mental health services .
the third part of the survey includes questions about comorbidity and different aspects of treatment ( the clinician s preference on treatment , follow up duration , and treatment adherence ) .
one question specifically refers to the clinician s opinion about his / her self - competence to treat adult adhd in terms of their knowledge and clinical practice . due to the characteristics of the study ,
first - part questions on working environment of the physicians were single - select multiple choice questions and respondents selected one answer from the list of answer choices .
questions about the referrals of patients , comorbidity , and treatment were comparison and frequency ranking question scales .
descriptive analysis was conducted with the results of the qualitative variables in the form of frequency and percentages .
pearson s correlation coefficient and multiple linear regression analysis were used to examine the relationship between the selected variables .
a total of 124 adult psychiatrists across all regions of turkey personally filled out the online survey from different treatment centers . regarding the institution , 34.6% of the psychiatrists worked in university hospitals , 15.3% at research and training hospitals , 29% at state hospitals , and 20.9% at private practice .
the daily number of treated patients in total has been asked in order to understand the clinical environment and workload of psychiatrists .
the results showed that only 30% of respondents treated 15 or less patients per day , but on the contrary 53% reported that they treated > 21 patients in a day ( figure 1 ) .
significant differences were observed in relation to the question of the number of adult patients with adhd in follow up .
approximately 58% of the clinics were following less than ten adult adhd patients at the time the survey was conducted , whereas only 16% of clinics were following > 40 adult patients with adhd ( figure 2 ) .
results of the pearson correlation coefficients analyses showed no correlation between the number of adult patients with adhd in follow up and the psychiatrists workload measured by the daily treated patient ( rp=0.14 and p=0.12 ) . on the other hand , there was a significant positive correlation between number of adult patients with adhd in follow up and the level of self - competence to treat adult adhd ( rp=0.52 and p<0.00001 ) .
the relationship between the working environment and number of adult patients with adhd in follow up was evaluated with multiple linear regression analysis but no significant relationship was found between the treatment centers and number of adult patients with adhd in follow up ( p=0.08 ) .
when the data about the admission ways of adult patients with adhd for treatment were examined , three of the ways came into prominence : guidance of the patient s family or social milieu , individual application by gaining awareness from media channels , and referral of child and adolescent psychiatrists ( figure 3 ) .
the survey results were significant in that 85% of the respondents stated that they have not received any information about their adult patients with adhd from the child and adolescent psychiatrists who followed up the patients during childhood .
in addition , only 18% of the adult psychiatrists stated that > 50% of their patients have been treated for adhd in childhood ( table 1 ) . in relation to the question about treatment choice
, there were significant differences in attitudes about the treatment of adult adhd ( table 2 ) .
rating questions were asked to survey respondents to compare treatment strategies in order of frequency .
a total of 62.6% of the participants reported that they always prefer psychoeducation in addition to medication treatment , whereas only 9.59% of the participants reported that they always combine psychotherapy with medication in the treatment of adult adhd .
always and 57.4% reported as using it frequently ; table 3 ) .
the other frequently preferred medications were antidepressants ( 56.6% ) and nonstimulants ( 37.4% ) .
when considering the reasons of discontinuation to treatment , there were significant differences : most of the participants were classified as nonadherent to medical treatment , which is the main reason of discontinuity and also the negative feedback from the patient s social milieu , patients thoughts that the treatment is unnecessary , and prejudices about stimulant medication were other reasons .
anxiety disorders have been reported as the most common comorbid disorder with adult adhd ( 40.4% ) , followed by alcohol / substance abuse disorders ( 29.7% ) , and depression ( 15.9% ; figure 4 ) .
results of the question that evaluated the self - competence to treat adult adhd in terms of knowledge and clinical practice , showed 85% of the clinicians surveyed did not define themselves competent enough to treat and follow up adult patients with adhd .
this study sought to understand the clinical experience and the attitudes of the specialists in the context of adult adhd in turkey .
given the impact of adhd in adult life , it is vital that adolescents with adhd are able to access continuing support from mental health services as they transition into adulthood.18 it turned out in our study that there are significant differences between clinics in relation to the number of adult patients with adhd in follow up in turkey .
although the daily number of patients treated in outpatient clinics is really high , adhd diagnosis is not encountered as much as we expected .
a previous study from turkey showed that 75% of the adult psychiatrists were reticent or unwilling to screen for adhd in patients with depression / anxiety and 20% of them never screen for adhd.15 adult adhd seems to be a clinical entity with a lower degree of awareness even among psychiatrists compared with adhd in childhood and adolescence .
the literature shows that treatment is prematurely discontinued in some young adults in whom symptoms persist .
there are also many adults with adhd who were never diagnosed or treated for adhd when they were children.19 the psychiatrists may be omitting the diagnosis in adults due to the age - dependent changes in the presentation of adhd symptoms .
the more overtly impairing symptoms in childhood , hyperactivity and impulsivity , often become less obvious in adulthood , shifting the problem to more subtle symptoms such as inner restlessness , inattention , disorganization , and to other executive function difficulties in the impairments related to the demands of adult life .
adhd in adults should therefore be judged with reference to developmentally appropriate norms.20 furthermore , adults may be more successful at hiding or compensating their symptoms in order to escape the stigma of having a psychiatric disorder than children .
a diagnosis based only on self - report can also lead to underdiagnosis of adult adhd ; retrospective recall of childhood symptoms may be compromised in adults with adhd due to difficulties with accurate recall.21 additionally , comorbid psychiatric disorders such as mood , anxiety , sleep , substance use disorders , and personality disorders may also complicate the diagnosis .
there is also a solid reality that in turkey psychiatrists working on adult adhd are still few and there is a lack of confidence in dealing with adult adhd .
our results showed that 85% of the clinicians surveyed themselves competent enough to treat and follow up adult patients with adhd .
the psychiatrists working on adult adhd may perceive themselves as not equipped enough to diagnose and treat adhd due to the lack of standardized training programs and clear guidelines for adults .
while several national psychiatry organizations , including the canadian adhd resource alliance , the national institutes of health , and the british association for psychopharmacology , have developed practice guidelines for the assessment and treatment of adults with adhd , none of the turkish psychiatry associations , including turkish psychiatry association , turkish psychopharmacology association , developed a treatment guideline for adult adhd treatment . until recent years
, adhd has been accepted as a childhood disorder , which can be another reason for adult psychiatrists not to think about the diagnosis .
adult psychiatrists may be reluctant to prescribe the medications because of the perceived risk of abuse and addiction of especially stimulant drugs.2,15 despite concerns , research shows that stimulant treatment does not increase the risk of substance use disorders in adolescents or adults with adhd but rather effective treatment of adhd may reduce the development of substance use disorders.2224 this study is the first to evaluate the admission ways and transition of adult patients with adhd into adult mental health services in turkey .
our findings indicate that in turkey the media and the restraint of social milieu turned up to be effective factors for treatment application in adults .
as we know , there is no published research about the transition of adhd patients into adult services in turkey . in a recent study that audited the transition of adhd patients in england , it was found that a high percentage of young people eligible for transition ( 73% ) were discharged or lost during follow up.25 similarly , our results show that a very low percentage of patients who were diagnosed at childhood are being referred to adult psychiatrists without any informative instruction and unfortunately there is no transition policy formed between child and adolescent psychiatrists and adult psychiatrists . in addition , national electronic health record systems are still under development and there is lack of available and usable patient data for clinicians in turkey .
this situation is also limiting the follow up of the adhd patients in health care system .
furthermore , adhd medications licensed for adults are involved neither in government mandatory reimbursement formulary nor in private insurance companies reimbursement formulary in turkey . as a result of this
, young patients may have difficulty to reach the fundamental treatment and follow up . in terms of treatment , stimulants are by far the best - studied and most effective treatment for adhd , and the nice guidelines recommend stimulants as the usual first - line treatment for adults with adhd.26 our findings indicate that stimulants and antidepressants are the most preferred choice of medication in daily clinic of participant clinicians .
psychological treatments should have an important place in the treatment of adults considering how the priorities , responsibilities , and interpersonal relations change within the daily life of an adult .
our results showed that psychoeducation , which is an important part of the treatment that helps the patient and his / her family to give meaning to symptoms and get awareness about the disorder and its impact , seems to be a more embraced treatment than psychotherapy in treatment attitudes of adult psychiatrists .
psychotherapy is suggested as an adjunctive treatment for adult adhd , targeting the impairments associated with adhd such as comorbidities , low self - esteem , relationship , and work problems.21 in a recent review , improvements in adhd symptomatology and associated symptoms have been reported after psychotherapeutic treatment.27 this study has some limitations that have to be pointed out .
the low response rate ( 6.4% of psychiatrists practicing in turkey ) and the self - reported online survey nature of the study allow us to discuss attitudes of the psychiatrist instead of general adhd treatment strategies .
although treatment environment was included in the survey , educational level or specialization information of the psychiatrists was not included .
however , educational level for adhd management has been evaluated by self - competence while there is no official subspecialization of psychiatrists in turkey .
adhd is a psychiatric disorder that causes significant levels of impairment in multiple domains of an adult s life .
because of this , early recognition , diagnosis , sufficient treatment , and follow up are important for adult adhd .
the findings from this survey indicate the need for standardized training programs to increase the knowledge , skills , and awareness of adult psychiatrists about adult adhd .
it is also clear that a more collaborative working relationship between child and adolescent psychiatrists and adult psychiatrists with a definite transition policy is required in order to help young people reach health services more effectively . | objectivealthough adult attention deficit hyperactivity disorder ( adhd ) often persists beyond childhood , daily clinical practices and transition of adult patients with adhd into adult mental health services in turkey are not well studied .
the aim of this study was to provide data about the presentation of adult patients with adhd and evaluate the treatment strategies of turkish adult psychiatrists based on their personal clinical experience in different hospital settings.methodsa cross - sectional online survey to be filled out by turkish adult psychiatrists was designed and administered in may 2014 .
the survey focused on the treatment environment , patterns of patient applications and transition , treatment strategies , and medication management for adults with adhd.resultssignificant differences were observed in the number of adult patients with adhd in follow up , and a significant positive correlation was found between number of adult patients with adhd in follow up and the clinician s opinion about their level of self - competence to treat adult adhd .
a significant portion of adult psychiatrists have not received any information about their adult adhd patients treatment during childhood .
the most preferred medical treatment was stimulants and the majority of the participants always preferred psychoeducation in addition to medication treatment .
a majority of participants did not define themselves competent enough to treat and follow up adult patients with adhd.conclusionthe findings of this study indicate the need to increase the knowledge , skills , and awareness of adult psychiatrists about adult adhd .
in addition , a more collaborative working relationship between child and adolescent psychiatrists and adult psychiatrists with a definite transition policy is required in order to help patients with adhd more effectively . |
with estimated 80,000 deaths among children in 2013 , tuberculosis ( tb ) continues to be major infectious disease among children . low- and middle - income countries bear disproportionate burden with more than 50% of the global cases occurring in southeast asia and the western pacific .
india alone accounts for 27% of the global burden of paediatric tuberculosis and nearly five percent of the new cases in india are reported among children .
it is equally valuable in prevention of multidrug - resistant tuberculosis ( mdr - tb ) , which affects nearly 32000 children globally .
the private general practitioner is usually the first point of contact for patients in india .
majority of tb patients receive care from a large variety of providers outside of the national tb programme despite the high costs and varying quality .
even those registered by india 's revised national tb control programme ( rntcp ) reach the programme after going through cycles of consultation with private practitioners ( both qualified and nonqualified ) .
management of childhood tb in the private sector , especially adherence to standard treatment guidelines , is crucial for limiting effects of the disease among children .
the first study in india investigating management practices of tb in private sector revealed high magnitude of inappropriate prescribing practices .
the studies highlight high variance in clinical practice and most of the practices being inappropriate ones in india [ 912 ] .
lack of knowledge of tb treatment regimen and inappropriate prescriptions have been reported across multiple countries [ 1316 ] . in 2006 ,
tuberculosis coalition for technical assistance ( tbcta ) developed international standards for tuberculosis care ( istc ) with the purpose of laying down a widely accepted level of care that all practitioners , public and private , should seek to achieve in managing patients who have , or are suspected of having , tuberculosis .
evidence from india shows that practices in private sector often deviate from the standards laid down in istc [ 1820 ] .
most of these studies have focused on general practitioners ( with no post graduate qualification in medicine ) who manage tuberculosis among adults . compared with this
appropriate treatment of tb is essential to prevent death , drug - resistance , and adverse effects among affected children .
it is therefore pertinent to explore diagnostic and treatment practices in private sector and difficulties they face while providing care to children infected with tb .
many guidelines exist for tuberculosis diagnosis , treatment , and control globally and in india .
the world health organisation ( who ) guidance for national tuberculosis programmes on the management of tb in children , the indian guidelines of the rntcp , who 's rapid advice - treatment for tuberculosis in children , istc , and the consensus statement formed by the indian academy of paediatrics ( iap ) are but a few of these . while the first two are more applicable to national health programme that presume it to be the end provider , istc and iap guidelines are more relevant for the private sector that manages tb in its own setting .
the healthcare providers in private sector range from nonqualified medical practitioners , pharmacists , and qualified practitioners without training in paediatrics to paediatricians .
there is , however , increasing tendency to seek care from paediatricians for a sick child in the city of mumbai .
this study was conducted among paediatricians in mumbai who treat tb patients with objective of comparing practices in diagnosis and treatment of tb and drug - resistant tuberculosis to standards set in the istc ( 2009 ) .
mumbai is the second largest urban agglomeration in india with a population of over 18 million , more than 1.7 million of whom are children under the age of six . those who dwell in mumbai face various environmental hazards including inadequate water and sanitation , pollution , and poor ventilation with almost half the population living in slums where many families live in a single room , making them vulnerable to diseases such as tuberculosis .
a diverse healthcare system exists with both public and private sectors and has a range of healthcare service providers including traditional healers , nonqualified medical practitioners , qualified medical practitioners in primary care , and specialists who provide specific services and includes paediatricians .
allopathy , ayurveda , and homeopathy are the main medical disciplines that are offered to patients . while most primary care providers ( also referred to as family physicians ) practice at single or two locations , most specialists including paediatricians provide their services in multiple healthcare facilities which may include public hospitals , trust hospitals , private - for - profit hospitals , many small size hospitals , and clinics in private sector .
patients diagnosed with tb in private sector may either be treated in private sector itself ( most common ) or referred to public sector for further management .
participation in public private partnership schemes is poor and nearly half of tb patients of mumbai continue to take treatment in private sector .
it was carried out from december 2012 to february 2014 , with data collection period from may 2013 to october 2013 .
the study tool was a self - administered questionnaire , which was developed by the researcher .
the researcher has graduate training in medicine and at time of developing study tool had more than two years of experience in managing health interventions in the field of tb and hiv through a nongovernmental organisation in india .
the researcher was pursuing master 's degree in public health at time of conducting this research . during preparation of the tool
another researcher with postgraduate training in community medicine and five years of field experience in management of tuberculosis reviewed the questionnaire .
after incorporating comments from second researcher , questionnaire was pilot tested by administering it to a private paediatrician working in a tertiary private hospital in mumbai .
istc ( 2009 ) includes 21 standards covering four broad areas , namely , standards for diagnosis , standards for treatment , standards for addressing hiv infection and other comorbid conditions , and standards for public health . since the objective was to assess diagnostic and treatment practices of childhood pulmonary tb including drug - resistant tb among paediatricians , eight standards pertaining to diagnosis and treatment , with components relevant to the study ,
international standards for tuberculosis care ( istc ) for diagnosis and treatment of tuberculosis ( source : istc 2009 )
standards for diagnosis
standard 1 : all persons with otherwise unexplained productive cough lasting two - three weeks or more should be evaluated for tuberculosis .
standard 2 : all patients , including children who are capable of producing sputum , suspected of having pulmonary tuberculosis should have at least 2 sputum specimens obtained for microscopic examination .
standard 4 : all persons with chest radiographic findings suggestive of tuberculosis should have sputum specimens submitted for microbiological examination .
standard 6 : children suspected with intrathoracic tb should have sputum examined ( by expectoration , gastric washings , or induced sputum ) for smear microscopy and culture . in children with negative sputum smears diagnosis should be based on the finding of chest radiographic abnormalities consistent with tb , a history of exposure to an infectious case or evidence of tb infection ( positive tst or igra ) and clinical findings .
standard 1 : all persons with otherwise unexplained productive cough lasting two - three weeks or more should be evaluated for tuberculosis .
standard 2 : all patients , including children who are capable of producing sputum , suspected of having pulmonary tuberculosis should have at least 2 sputum specimens obtained for microscopic examination .
standard 4 : all persons with chest radiographic findings suggestive of tuberculosis should have sputum specimens submitted for microbiological examination .
standard 6 : children suspected with intrathoracic tb should have sputum examined ( by expectoration , gastric washings , or induced sputum ) for smear microscopy and culture . in children with negative sputum smears diagnosis should be based on the finding of chest radiographic abnormalities consistent with tb , a history of exposure to an infectious case or evidence of tb infection ( positive tst or igra ) and clinical findings .
standards for treatment
standard 8 : all patients who have not been treated previously should receive an internationally accepted first - line treatment regimen using drugs of known bioavailability .
the initial phase should consist of 2 months of isoniazid , rifampicin , pyrazinamide , and ethambutol and continuation phase consists of h and r for 4 months , with doses conforming to international recommendations .
standard 10 : patients with pulmonary tb should be monitored for response to therapy by follow - up sputum microscopy ( 2 specimens ) at the time of completion of initial phase of treatment ( 2 months ) .
if smear positive at this stage , smears should be repeated at 3 months , and if positive , culture and dst should be performed . in children ,
standard 11 : patients should be assessed for dr - tb based on history of prior treatment , exposure to a possible source case of dr - tb , and community prevalence of dr .
dst should be conducted at start of therapy for previously treated patients with assessment for dr - tb in all those who remain sputum smear positive at completion of 3 months of treatment and those who have failed , defaulted , or relapsed following one or more courses of treatment .
culture and dst plus resistance to at least isoniazid and rifampicin should be performed in patients in whom dr is likely .
standard 12 : those likely to have tb caused by dr organisms should be treated with specialized regimens containing second - line antituberculosis drugs ; regimen should be standardized or based on suspected or confirmed ds patterns .
at least 4 drugs to which the organisms are known to be susceptible , including an injectable agent , should be used for at least 1824 months beyond culture conversion .
standard 8 : all patients who have not been treated previously should receive an internationally accepted first - line treatment regimen using drugs of known bioavailability .
the initial phase should consist of 2 months of isoniazid , rifampicin , pyrazinamide , and ethambutol and continuation phase consists of h and r for 4 months , with doses conforming to international recommendations .
standard 10 : patients with pulmonary tb should be monitored for response to therapy by follow - up sputum microscopy ( 2 specimens ) at the time of completion of initial phase of treatment ( 2 months ) .
if smear positive at this stage , smears should be repeated at 3 months , and if positive , culture and dst should be performed . in children ,
standard 11 : patients should be assessed for dr - tb based on history of prior treatment , exposure to a possible source case of dr - tb , and community prevalence of dr .
dst should be conducted at start of therapy for previously treated patients with assessment for dr - tb in all those who remain sputum smear positive at completion of 3 months of treatment and those who have failed , defaulted , or relapsed following one or more courses of treatment .
culture and dst plus resistance to at least isoniazid and rifampicin should be performed in patients in whom dr is likely .
standard 12 : those likely to have tb caused by dr organisms should be treated with specialized regimens containing second - line antituberculosis drugs ; regimen should be standardized or based on suspected or confirmed ds patterns .
at least 4 drugs to which the organisms are known to be susceptible , including an injectable agent , should be used for at least 1824 months beyond culture conversion . for assessing compliance to these standards , suitable items and indicators
cough listed as one of the symptoms that would lead to suspicion of tb , advising sputum smear / culture for tb suspects , those with positive chest x - rays , advising gastric lavage or bronchoalveolar lavage if child is not able to bring out sputum , and whether chest x - ray and/or tuberculin skin test were advised along with sputum smear / culture .
the indicators related to treatment included whether regimen prescribed is two months of four drugs ( isoniazid , rifampicin , pyrazinamide and ethambutol ) followed by four months of two drugs
drug - resistance suspected at beginning of treatment in cases of previous treatment with anti - tubercular drugs and/or history of contact with a known case of multidrug - resistant tb , whether those who are smear positive at three months are tested for sensitivity to rifampicin , and whether the regimen prescribed for mdr - tb includes four new drugs with one injectable and one fluoroquinolone .
standard 11 states that culture and drug sensitivity tests with at least those for isoniazid and rifampicin should be performed for patients with drug - resistance
. however , traditional cultures are associated with delay in diagnosis and rapid molecular tests like xpert mtb / rif are recommended by who . since xpert
mtb / rif does not involve culture and tests for only rifampicin resistance , we measured
these descriptive variables included paediatricians ' characteristics including age , sex , duration of clinical practice , and average number of new tb patients treated every month and the guideline referred by them for managing tb in children .
with respect to diagnosis , respondents were asked to list signs that may suggest tuberculosis and challenges faced ( if any ) in diagnosis . with respect to first regimen offered by paediatricians , we also asked about dosages of each drug prescribed , frequency of administration of drugs ( daily or intermittent ) , and challenges in treatment of childhood pulmonary tb . for assessing practices related to monitoring progress of treatment
apart from the primary outcomes listed in previous paragraph , these included usual duration of therapy after which non - response to treatment is first suspected by the practitioner , action taken for such patients ,
usual duration of therapy after which drug - resistance is suspected , basis of diagnosis of mdr - tb ( bacteriological , clinical , radiological , or combination of these ) , type of bacteriological test advised to diagnose drug - resistance ( culture or molecular techniques or both ) , and
laboratory where the investigation is carried out ( accredited or not accredited ) . for patients diagnosed with drug - resistant tb ,
further line of management was enquired into ( treat the patient or refer to another practitioner or health facility ) .
the inclusion criteria for participants were postgraduate qualification in paediatrics ( allopathy stream ) practicing in private sector in mumbai and those who treated children with pulmonary tuberculosis .
an initial list of paediatricians was prepared by approaching indian academy of paediatricians ( iap ) , online local directory websites , and names suggested by study participants .
paediatricians were approached in person at their respective healthcare facilities and at a conference or by e - mail .
e - mail provided link to an open source website harbouring an online version of the questionnaire developed for the study .
paediatricians who consented to participate were provided with either the hard copy or soft copy of the questionnaire .
the questionnaire included some closed ended questions ( e.g. , whether you treat or refer mdr - tb patients ) .
other items were open ended ( e.g. , list investigations advised when you suspect a child to be suffering from pulmonary tb ) . for open ended items , responses were listed and categorised ( e.g. , in line with istc or not ) and codes were assigned .
data was entered in microsoft excel version 14.0.0 and analysis involved calculation of frequencies and percentages of practices for variables mentioned above .
although it was not primary aim of the study , the authors also referred to guidelines of iap and compared the first regimen advised by the practitioners with iap guidelines .
ethical norms were followed with an introduction to paediatricians about the nature and purpose of the study .
informed consent process highlighted confidentiality of respondents ' identity and the filled questionnaires ( hard copy ) were kept under lock and key .
the online filled questionnaires were accessible only to the researcher and access was protected by a password to the online account .
doctors ' doubts regarding funding by a pharmaceutical company were cleared with the assurance that the study was for academic purposes only and was self - funded with no attachments or conflict of interests . written consent was taken and complete volunteerism was assured before collecting data .
the doctors had choice of dissent at any point of answering the questionnaire or after .
a total of 644 doctors were approached : 197 ( 30.6% ) in person and 447 ( 69.4% ) by e - mail .
the 197 paediatricians were reached by visits to seventeen tertiary hospitals , seven children hospitals , twelve small size hospitals , three polyclinics , 31 exclusive paediatric clinics , and one conference venue .
only 58 of the 197 ( 29.4% ) participated in the study ; response to e - mail questionnaire was poorer with six participants responding online .
more than a third of paediatricians were treating three or more cases of childhood tb every month . out of the 52 doctors who responded that they referred to a guideline , 17 ( 33% ) referred only to guidelines of the iap , while another 17 ( 33% ) referred to iap along with other guidelines , including 13 ( 25% ) referring to iap as well as rntcp guidelines .
other guidelines they had listed included that of the who and center for disease control ( cdc ) .
almost all paediatricians suspected tuberculosis in a child with fever and most suspected the disease in a child with cough of more than two - week duration or loss of weight ( table 3 ) .
next common symptoms included child in contact with a case of tb and loss of appetite .
other symptoms ( not shown in table ) raising suspicion of tb included failure to thrive or no weight gain ( reported by 19 ; 30% ) , swellings in the body ( 14 ; 22% ) , no improvement with general antibiotics ( 5 ; 7% ) , malaise ( 3 ; 4.5% ) , headache and convulsions ( 3 ; 4.5% ) , pain in abdomen ( 2 ; 3% ) , night sweats ( 2 ; 3% ) , postmeasles respiratory case ( 1 ; 1.5% ) , diarrhoea ( 1 ; 1.5% ) , and a history of residing in an overpopulated area ( 1 ; 1.5% ) . among signs , malnutrition , palpable matted lymph nodes , and respiratory sounds ( crepitations and/or rhonchi )
less commonly reported signs included hepatomegaly ( reported by 12 ; 19% ) , splenomegaly ( 11 ; 17% ) , neck stiffness ( 8 ; 12% ) , fever ( 7 ; 11% ) , clubbing ( 7 ; 11% ) , absence of a bcg scar ( 6 ; 9% ) , ascites ( 4 ; 6% ) , chronic sinus or ulcer ( 4 ; 6% ) , abdominal mass ( 2 ; 3% ) , and phlycten ( 2 ; 3% ) .
almost all paediatricians reported asking for x - ray of chest for diagnosis of pulmonary tb when open ended question of list investigations requested to diagnose pulmonary tb was asked ( table 3 ) .
majority paediatricians reported prescribing investigations including complete blood count ( cbc ) , erythrocyte sedimentation rate ( esr ) , and tuberculin skin test ( tst ) .
more than one - third of the respondents reported advising sputum smears ( and/or cultures ) for presence of acid fast bacilli ( afb ) .
other investigations included immunoglobulins ( reported by 7 ; 11% ) and gamma interferon ( 3 ; 4.7% ) .
all 24 paediatricians ( 38% ) who advise sputum smear or culture also advise for chest x - ray and/or tst .
another 20 paediatricians ( 31.7% ) reported using sputum test only for some patients ; most commonly , test was requested for older children only . among the 24 paediatricians who were prescribing sputum test for all tb suspects , 21 ( 87.5% )
reported that they request inducing of sputum if child is not able to produce sputum sample .
equal number of paediatricians reported doing sputum examination for all patients whose x - ray findings were suggestive of tuberculosis .
more commonly , tst was employed to confirm diagnosis ( table 3 ) ; the three others included interferon gamma release assay ( igra ) , starting treatment without any further investigations and repeat x - ray after a course of antibiotics .
the challenges included patients not able to afford cost of diagnostic tests limiting options with paediatricians ( reported by 10 ; 35.7% ) , absence of one specific test to confirm tb ( 9 ; 32.1% ) , certain diagnostic procedures like inducing sputum being difficult to implement in practice ( 7 ; 25% ) , and availability of certain tests limited to fewer laboratories ( 2 ; 7.1% ) . out of 64 private paediatricians , two doctors ( 3% )
regimens prescribed by remaining 62 practitioners have been presented in table 4 and are compared with istc and iap guidelines .
all respondents who provided information on drug dosages were prescribing istc recommended dosage for rifampicin ( r ) and ethambutol ( e ) .
but , doses of isoniazid ( h ) and pyrazinamide ( z ) were often lower than istc recommendations ( 45% and 68% , resp . ) . combining data on combination , duration , and dosages for all drugs ( data on all three provided by 58 paediatricians ) , only ten ( 17.2% ) practitioners out of 58 were prescribing appropriate regimen with dosages as recommended by istc 2009 . when compared with iap guidelines , dosages of almost all paediatricians
were appropriate for r and h but nearly two - thirds of paediatricians were advising higher and lower dosage for e and z , respectively . a total of 37 paediatricians specified challenges faced by them with paediatric formulations in the treatment of children with tb .
doctors reported that young children found these tablets difficult to ingest ( reported by 9 ; 24.3% ) and the taste unpalatable ( 5 ; 13.5% ) .
other reported difficulties included different drug combinations making treatment confusing ( 8 ; 21.6% ) unavailability of wider choice of drugs ( 6 ; 16.2% ) , adverse effects of drugs ( 1 ; 2.7% ) , and long treatment duration ( 1 ; 2.7% ) .
some paediatricians were not advising any routine follow - up investigation ; 12.7% were not advising any investigation during treatment and 11.9% at end of treatment ( table 5 ) .
these investigations included those for assessing effectiveness of treatment ( chest x - ray , sputum smear / culture , cbc , and esr ) as well as those for identifying adverse effects of drugs ( liver and renal function tests ) .
chest x - ray was the most common investigation used for monitoring response during ( 61.9% ) and at end of treatment ( 84.7% ) .
sputum smear or culture was requested by less than a third of respondents during treatment and only 8.5% were using it at end of treatment .
decision to stop treatment at end of full course of antitubercular drugs was a clinical one for all respondents , aided by x - ray in a third and sputum in 13.8% of cases .
time at which nonresponse to treatment was suspected varied from practitioner to practitioner and was as less as one month to as long as 8 months but most ( 45 out of 50 ; 90% ) were suspecting it early ( within three months of therapy ) ( table 6 ) .
most common actions reported by 58 paediatricians included advising sputum culture and drug sensitivity test ( 35 ; 60.3% ) , suspecting and investigating for hiv infection ( 26 ; 44.8% ) , and prescribing another regimen ( 23 ; 39.7% ) .
since practices related to hiv - tb coinfection are not part of the paper , we have presented actions taken by paediatricians for hiv negative nonresponders in figure 1 .
out of the other remaining 63 , four ( 6.3% ) did not answer the question and 36 ( 57.1% ) said that they investigate for mdr , whereas 23 ( 36.5% ) mentioned that they prescribe another regimen ( details of these regimens are provided subsequently ) .
a total of 50 paediatricians answered question regarding time of suspecting drug - resistant tb , out of which 23 ( 46% ) suspected only after trying second regimen ( table 6 ) . remaining 27 ( 54% ) reported suspecting drug - resistance during first regimen itself , if sputum was positive at end of first ( 3 ; 11.1% ) , second ( 17 ; 63.0% ) , or third month ( 7 ; 25.9% ) of therapy , respectively .
thus , 24 ( 48% ) were suspecting drug - resistance at appropriate time ( 2 to 3 months ) , all of whom were testing for rifampicin resistance .
a total of 31 paediatricians listed conditions when they suspected drug - resistance at beginning of treatment , two - thirds of them listed contact with case of drug - resistant tb , one - third investigated for resistance among hiv positive children , and only four ( 12.9% ) reported suspecting resistance in cases treated with antitubercular drugs in the past .
almost all ( 48 ; 94.1% ) paediatricians reported that basis of diagnosis of drug - resistance was bacteriological ( table 6 ) .
33 of these who specified the type of test conducted reported use of sputum culture and drug sensitivity ( reported by 10 ) or xpert mtb / rif ( 8) or both ( 15 ) .
of the 35 who mentioned name of testing laboratories , 27 ( 77.1% ) named laboratories that were accredited by national programme for drug - resistance testing whereas 8 ( 22.9% ) were sending patients to nonaccredited laboratories .
as stated previously and shown in figure 1 , 23 paediatricians did not investigate for drug - resistance when patient did not respond to first regimen ; they chose to prescribe another regimen .
details of such second regimens offered prior to investigation of mdr - tb are presented in table 7 .
one or no drug was added by nine paediatricians ( 39% ) whereas 14 ( 61% ) reported adding at least two new drugs to previous regimen ( eight adding two drugs , three adding three drugs , and four adding four drugs ) .
three of these 23 ( 4% ) also reported having attempted a third regimen with addition of one new drug ( one paediatrician ) and two new drugs ( two paediatricians ) ( not shown in table ) .
a total of 14 paediatricians reported treating drug - resistant tb and 12 of them ( 86% ) provided details on the type of regimen .
of these 12 paediatricians , five ( 41.7% ) were adding four new drugs including an injectable and a fluoroquinolone ( table 7 ) .
another four ( 33.3% ) said that drugs are chosen on basis of sensitivity test results but did not specify the number of drugs making it not possible to assess compliance with istc standards .
the remaining three were not adding enough number of drugs or had no injectable drug in combination and were not as per istc standards .
this cross - sectional survey reveals that diagnostic and treatment practices in management of childhood pulmonary tuberculosis among paediatricians in mumbai 's private sector deviate from practices recommended by the international standards for tuberculosis care ( istc ) 2009 .
we discuss such deviations in detail and possible reasons of such inappropriate practice and its implications for tuberculosis control in mumbai .
some studies earlier have assessed management practices for childhood tb and found them to be poor . of these , one study assessed practices related to chemoprophylaxis in south africa , another assessing appropriateness of regimens offered in public sector [ 2628 ] .
this is the first study from india that shows that management of childhood tb disease in private sector is also deviating from guidelines .
prompt diagnosis of tb is one of the basic principles in management of this disease . in present study ,
more than a fifth of paediatricians did not mention cough as symptom of tb ( standard 1 of istc ) .
suggested that not all patients with respiratory symptoms receive an adequate evaluation for tuberculosis highlighting also the missed opportunities for earlier detection that could lead to an increased likelihood that tb bacilli will spread among family members and other people in the community . in previous studies of diagnostic practices ,
chest x - rays have been the commonest method used [ 10 , 19 , 20 , 29 , 30 ] and this study showed that it holds true for childhood tb as well .
few paediatricians reported using sputum smear or culture for diagnosis in young children who can not cough up sputum easily ( standard 2 ) along with difficulty of implementing procedures like gastric lavage and bronchoalveolar lavages ; also mentioned by previous investigators [ 30 , 31 ] .
in contrast to this , a recent study in the united kingdom ( uk ) , however , reported that about 77% of respondents use sputum induction for microbiological confirmation .
several systematic reviews questioning reliability of serodiagnostic tests led who to release a negative policy recommending nonuse of such diagnostics , leading to its ban in india in 2012 [ 33 , 34 ] .
however few paediatricians in this study reported using these techniques in 2013 , with possibility of serious epidemiological consequences .
adequate and complete treatment of tb is cornerstone of tb control as it results in better health outcomes for affected patient as well as reduced chances of infection to others .
previous literature on treatment practices exhibits wide variance in practices , with almost every participating physician having a unique regimen to deal with tb [ 8 , 1012 , 29 ] .
although less variance was found in first regimen offered by paediatricians compared to previous literature , more than one - third of paediatricians were following istc recommended regimen . while diagnosing tb in a child
may be faced with practical difficulty as discussed already , there is no such hindrance for prescribing appropriate treatment as shown in a recent study in uk where adherence to istc recommended regimen was universal .
many paediatricians prescribed lower doses of isoniazid ( h ) and pyrazinamide ( z ) than recommended .
such lower dosage may potentiate drug - resistance especially when there are no mechanisms to ensure adherence to treatment .
treatment is only prescribed in private sector ; there is no mechanism to supervise patients [ 8 , 12 ] .
mechanisms to ensure adherence for patients treated in private sector are a priority and need operational research including piloting of interventions .
drug - resistant tb is a bacteriological diagnosis for which sputum culture is considered to be the gold standard .
however , in this study , many practitioners reported the use of clinical and radiological , and in a few cases serological , means to diagnose dr - tb . this could on the one hand imply missing some children with dr - tb not investigated bacteriologically , while on the other hand other children may unnecessarily be on second- and third - line regimens without bacteriological confirmation . in this study , less than half who advised bacteriological diagnosis asked for both rapid and culture tests ; some used culture methods alone which , although accurate , are time consuming and delay diagnosis .
use of rapid molecular techniques like xpert mtb / rif is useful to prevent delay but it only detects resistance to rifampicin [ 35 , 36 ] .
presence of resistance to other antibiotics goes undetected and it does not help in formulating individualised regimen .
the study findings reveal that resistance is not suspected in a child even if she / he has history of prior treatment with antitubercular drugs ; patients not responding to treatment are offered another regimen without investigating for presence of drug - resistance , very few of which are adequate to treat mdr - tb .
the study also shows that prescriptions after diagnosis of mdr - tb are often inadequate .
this is similar to studies not specific to childhood tb [ 12 , 18 ] .
such inadequate regimens will compound monoresistance to mdr to extensively drug - resistant tb ( xdr - tb ) .
such treatment not only will result in failure of treatment in patients but may spread drug - resistant forms to contacts making tb control a very difficult task to accomplish .
there are many differences between diagnostic and treatment standards of istc and the consensus statement formed by the iap [ 23 , 24 ] .
iap recommends use of x - ray for diagnosis of childhood pulmonary tb and similar is practice of many paediatricians .
for patients not responding to treatment , it considers addition of single drug ( streptomycin ) as adequate and appropriate as practised by a few paediatricians in this study . in this study ,
where first regimens offered by only a little over third paediatricians were in line with istc , almost all were appropriate as per iap criteria .
point out that some of the guidelines fail to cover many aspects of management and recommend practices that are sometimes not evidence - based .
the guidelines that not necessarily agree with each other could create confusion rather than lead to best practices and may be an important reason of lack of compliance to istc found in this study .
first , the response rate was poor and many practitioners did not answer all questions .
paediatricians who refused to participate may have practices different from those who did participate in the study .
paediatricians who follow national or international guidelines , those who are following updates in tb management , may be more likely to participate in the study which might have affected generalisability of study .
the method was questionnaire based and did not involve examining records of patients nor interviewing their guardians . even in the presence of these limitations , the deviations from istc can not be denied .
childhood tb is managed by general physicians as well but the study did not cover it due to feasibility issues . there is need to study practices among general physicians as even today they are first point of care for majority of people .
there is less reliance on sputum smear or culture for diagnosis of childhood pulmonary tb .
there is delay in suspecting drug - resistance and second - line drugs are commonly given without confirming drug - resistance .
the study highlights that practices regarding management of childhood pulmonary tb are often not as per international standards for tb care . | majority of children with tuberculosis are treated in private sector in india with no available data on management practices .
the study assessed diagnostic and treatment practices related to childhood pulmonary tuberculosis among paediatricians in mumbai 's private sector in comparison with international standards for tuberculosis care ( istc ) 2009 . in this cross - sectional study , 64 paediatricians from private sector filled self - administered questionnaires .
cough was reported as a symptom of childhood tb by 77.8% of respondents .
38.1% request sputum smear or culture for diagnosis and fewer ( 32.8% ) use it for patients positive on chest radiographs and 32.8% induce sputum for those unable to produce it . sputum negative tb suspect
is always tested with x - ray or tuberculin skin test .
61.4% prescribe regimen as recommended in istc and all monitor progress to treatment clinically .
drug - resistance at beginning of treatment is suspected for child in contact with a drug - resistant patient ( 67.7% ) and with prior history of antitubercular treatment ( 12.9% ) .
about half of them ( 48% ) request drug - resistance test for rifampicin in case of nonresponse after two to three months of therapy and regimen prescribed by 41.7% for multidrug - resistant tb was as per istc .
the study highlights inappropriate diagnostic and treatment practices for managing childhood pulmonary tb among paediatricians in private sector . |
monocytes and macrophages , which are categorized among the leukocytes , play a pivotal role in the innate immune system [ 13 ] .
their origin lies in a common myeloid precursor , while they both play important roles in innate immunity .
macrophages , which are produced from monocytes after their exposure to certain stimuli , circulate in the blood stream ; they are short - lived as they undergo spontaneous apoptosis [ 4 , 5 ] . however , in response to differentiating factors , some monocytes migrate and populate tissues , thereby escaping their apoptotic fate and become macrophages which have a longer lifespan and are found in almost every organ of the body . residing as phagocytic cells in lymphoid and nonlymphoid tissue , they have the ability to recognize a broad range of pathogens and are efficient phagocytes , while they also induce the production of inflammatory cytokines [ 68 ] .
monocytes have the potential to differentiate to dendritic cells ( dcs ) or tissue macrophages , although many dcs and tissue macrophages do not originate from monocytes in a steady state . under certain circumstances , monocytes
do differentiate into dcs during infection that produce inflammatory mediators such as tnf- , nitric oxide ( no ) and reactive oxygen species , as in listeria monocytogenes infection [ 7 , 9 ] .
nevertheless , under other conditions , monocytes differentiate into mucosal macrophages with a different surface phenotype and capability of inflammatory mediators production , as happens in toxoplasma gondii infection .
monocytes and their precursors can either activate or inhibit immune response , depending on local and systemic signals and the pathogen involved .
monocytes acquire capability of differentiation and inflammatory mediators production early in life [ 7 , 11 ] .
nevertheless , there is evidence of immaturity of the innate immune system in human fetuses , including its fundamental component , the mononuclear phagocyte system .
this immaturity is thought to be functional in origin , as the various cell types involved in innate immunity are present in the fetus , although their immunity virtues are in question .
neonates are prone to severe bacterial infections , and the mortality caused by infections is high despite prompt antibiotic therapy : this phenomenon is especially prevalent among preterm neonates . as production of phagocytes and their proper functioning are crucial for an effective bactericidal immune mechanism , defective functioning of the mononuclear system may account for the vulnerability of neonates to infections .
few studies on the fetus and neonate immune system are available and knowledge is sparse as regards the phagocytic and oxidative capacity of macrophages or their adhesion molecule expression [ 1519 ] .
nevertheless , it has been determined that various functions of the fetal innate immune system are essentially different from those observed in term neonates or adults [ 11 , 20 ] .
a considerably diminished phagocytic activity of fetal monocytes has been described , this manifesting in sharp contrast to elevated production of reactive oxygen products . moreover , significant numbers of fetal monocytes are capable of production of proinflammatory cytokines in response to inflammatory stimulation . a difference , however , was noted in the pattern of cytokine production as compared with that demonstrated in more mature individuals ; in other words , there was notable diminishment in the number of il-6 and tumor necrosis factor - positive monocytes .
in contrast to term infant responses , tnf- and il-6 responses of preterm infants ( with a gestation age ( ga ) of < 30 weeks ) were severely impaired due to a diminished proportion of cytokine producing monocytes .
likewise , responses among preterm monocytes with respect to whole cord blood stimulated with live group b streptococci ( gbs ) were found to be almost invariably less robust than those noted in term newborns . in neonates ,
the expression of several adhesion molecules involved in monocyte migration has been found to be appreciably compromised , thereby conducing to a diminishment in neonatal immune response .
the fact that newborn infants , particularly those delivered prematurely , generate comparatively low quantities of g - csf after inflammatory stimulation suggests that this may partially account for their deficient upregulation of both neutrophil production and function during infection .
nevertheless , investigations into preterm neutrophils and preterm rabbit alveolar macrophages have documented reduced bacterial uptake [ 23 , 24 ] , while other bacterium - derived stimuli have indicated that monocytes from preterm neonates possess an intrinsic gestation age - related deficiency in their ability to recognize and respond to gbs [ 12 , 25 ] .
meanwhile , it is well established that decreased tnf- secretion , bactericidal function , and adherence receptor expression in monocytes occur in preterm neonates [ 7 , 11 , 12 ] ( figure 1 ) .
the decreased monocyte function could possibly explain the equivalence of dna content between preterm and full - term monocytes as demonstrated via dna analysis [ 12 , 21 ] .
apart from dna content , equivalence was also shown between secretion of the cytokines il-1 and il-6 by adherent monocytes in both preterm and term neonates .
the same phenomenon is observed with regard to superoxide anion ( o2 ) production and degranulation , which are equivalent or elevated in freshly isolated monocytes from preterm neonates compared to full - term neonates .
the above observations strongly indicate the presence and full functionality of the essential cellular constituents of the bactericidal response in monocytes from preterm neonates .
moreover , monocytes placed in culture show that the processes of adherence and differentiation bring about changes in protein synthesis and cellular metabolism , by which modifications in turn modulate bactericidal activity .
thus , the decreased o2 production and degranulation observed in preterm adherent monocytes appear to be related to regulatory processes rather than defective or absent components of the bactericidal response . on the other hand , one may also hypothesize that slight modifications take place in the adherence capacity of preterm neonates ' monocytes , which would exert effects on signaling pathways regulating tnf- secretion .
it is of note in this regard that production of il-1 and il-6 was seen to be unchanged in preterm cells , this suggesting the functioning of regulatory controls different from those of tnf- .
two main research questions have been posed : is it an anatomy issue , that is , is there a structural defect in preterm monocytes , or does it concern a functional impairment ?
the few available observations in preterm infancy tend to point to the second explanation without furnishing clear - cut elucidation .
findings regarding integrity of monocyte phagocytic mechanism indicate the presence and total functionality of the essential cellular components of the bactericidal activity in monocytes from preterm newborns .
diminished cell count , if present , may conceivably account for the decreased monocyte function . consequently , anatomical impairment may be excluded and functional impairment , at least an intrinsic one , is under question .
when the secretion of il-1 is examined , il-1 response to lipopolysaccharide ( lps ) seems to be intact in newborn human monocytes , while an increased unstimulated activity of these following neonatal complications has also been recorded . assuming that the deficiency in monocytes of the preterm neonates may be an anatomy issue of the innate immune system
, this could come about as a result of allelic mutations at a single locus toll - like receptor 4 ( tlr4 ) gene , since this is the only pathway serving lps .
it is noteworthy in this respect that these mutations of the tlr4 gene are responsible for endotoxin resistance .
the ability of the host to respond to antigens alters as the gene - sequence changes , which means that the differences in lps response in humans are associated with common mutations in the particular gene , this effect also being corroborated by genetic evidence [ 26 , 27 ] .
furthermore , in some populations of high - risk preterm infants , tlr4 genotypes may influence the bpd ( biparietal diameter of the fetus ) severity .
regarding il-6 , data are somewhat conflicting , since equivalent correspondence between secretion of the cytokines il-1 and il-6 by adherent monocytes in preterm , on the one hand , and term neonates , on the other hand , has been determined .
nevertheless , the induced il-6 levels were significantly low in preterm neonates , especially in those born before 30 weeks of gestation .
neither the low serum immunoglobulin g levels accompanied by low serum complement levels in preterm neonates nor the slightly reduced monocytes number provided a sufficient explanation , since the level of il-6 induced by lps ( lps stimulation presumably does not require the opsonization process ) remained lower when the newborns were less than 30 weeks of gestational age .
however , differences in monocyte numbers alone are not large enough to explain the marked differences in il-6 levels produced by preterm and term neonates .
the synthesis of il-6 and g - csf among other cytokines in preterm and term newborns in an ex vivo cord blood culture endotoxin model opsonization appears to be related to immaturity .
the suppressed production of il-6 , along with diminished production of hydrogen peroxide , is considered typical of the so - called endotoxin intolerance .
the neonate cells are capable of responding appropriately to the antigen presentation and the direct microbicidal activity and oxidative burst . in this context
, normally lethal doses of endotoxin can be tolerated if the organism has been previously exposed to sublethal doses of endotoxin . in the clinical setting
, this state of immune tolerance renders the organism vulnerable to nosocomial infections : presentation in neonates of indolent , and persistent chorioamnionitis most probably is a manifestation of this high - risk condition [ 13 , 31 ]
. the published studies to date have some limitations , partly because of the inherent difficulties of studying a crucial part of the immune system separately from the whole system . in this context , the use of isolated mononuclear cells often results in the nonspecific stimulation of these cells to produce various cytokines . on the other hand ,
it has become evident that an ideal platform for assessment of the cellular response to stimulating agents in vitro may be provided via cultures using whole blood or human placenta and myometrium and examining their interactions with the immune system during labor .
the difficulties in studying the innate immune system of human neonates also stems from the fact that release of cytokines and inflammatory mediators may occur in other nonpathological situations as well , such as pregnancy and labor , and not merely in infections . in sum
, the above findings , established in humans , strongly indicate that the same could also arise in preterm neonates , further studies being required to investigate this hypothesis .
however , in vitro studies may not adequately describe in vivo processes , since monocytes in vivo act as part of the whole immune system , under real conditions .
moreover , multiple level interactions with other components of the immune system do happen and in vitro studies may be considered as an oversimplification .
in conclusion , although it is well established that monocytes have a central role in innate immunity and their defective function in preterm neonates is recognized , there is as yet scant knowledge about the pathophysiological mechanisms that account for this functional impairment .
the greatest degree of impairment is most likely to be encountered in those neonates that were born at around 30 weeks of gestational age .
this defective response to pathogens along with endotoxin intolerance may well place neonates at risk for nosocomial infection .
future research should focus on production of specific molecules , such as cytokines and other mediators of inflammation , along with their interaction with other components of the immune system . via precise identification of the mechanism of deficient immune system response in preterm neonates
, it may be possible in the future to successfully address infection in these patients by achieving speedy activation of innate immune mechanisms as well as , if feasible , acceleration of monocyte maturation . | it is well known that the innate immunity system , involving the contribution of monocytes and macrophages , may dysfunction in fetuses and preterm neonates .
monocytes are capable of differentiating into dendritic cells ( dcs ) or into mucosal macrophages during certain infections and of producing inflammatory mediators such as tnf- ( tumor necrosis factor - alpha ) , nitric oxide , and reactive oxygen species .
fetuses as well as neonates are prone to infections as a result of a defective mechanism within the above mononuclear system .
monocyte function in fetuses and preterm neonates depends on the phagocytic and oxidative capacity of macrophages and their antigen - adhesion ability .
functional rather than anatomical impairment is probably the underlying cause , while a defective production of cytokines , such as tnf- , il-6 ( interleukin 6 ) , il-1 ( interleukin 1 beta ) , and g - csf ( granulocyte colony - stimulating factor ) , has also been involved .
the insufficient production of the above inflammatory mediators and the phenomenon of endotoxin intolerance , which latter occurs during entry of any antigen into the premature neonate , place preterm neonates at higher risk for infections .
existing research data are herein presented which , however , are deficient and fragmental , this accounting for the fact that the precise pathophysiology of these disturbances is not yet fully clarified . |
emergence agitation ( ea ) was described at first by eckenhoff et al in the early 1960s .
ea has been described as a dissociated state of consciousness in which the child is inconsolable , irritable , uncooperative , typically thrashing , crying , moaning or incoherent .
nowadays , about 4 million children undergo general anesthesia annually and ea has been identified as a significant problem in children at postanesthetic care unit ( pacu ) with an incidence ranging from 10 to 80% .
ea as a postanesthetic problem interferes with child s recovery and presents a challenging situation for post - anesthesia care provider in terms of assessment and management .
although , several factors have been identified as etiologies of ea , there is no entire description for emergence agitation .
many different causes have been suggested , such as rapid awakening in an unfamiliar environment , painful events like surgical wounds , agitation on induction , airway obstructions , environmental disturbances , the duration of anesthesia , hyperthermia , hypothermia , type and site of operation , premedication , inhaled and intravenous anesthetics and the anesthetic technique . although ea is usually self - limited and occurs within the first 30-minutes of recovery in pacu , but it can last up to 2 days and lead to physical damage , disconnection of intravenous catheters , removing of dressing or drainage tube and monitoring devices . on the other hand , controlling the agitated child needs more nursing care and more post - anesthesia care providers .
in addition , administration of sedative and analgesics is associated with increased recovery time and delayed pacu discharge .
generally , treatment in all cases mentioned above is directed to the correction of causative agents .
although , numerous medications have been studied to prevent or reduce ea in children , no special preventive method has been shown to be highly superior .
so , this cross - sectional descriptive and analytic study had been undertaken to determine the prevalence of ea , evaluate the risk factors associated with and predictive of it , and describe the outcomes related to ea in children aged 3 to 7 years that underwent general anesthesia for elective surgical procedures .
with the approval from the hsr ethics committee of mazandaran university of medical sciences and parental informed consent , this cross - sectional descriptive analytic study was performed on 747 children aged 3 - 7 years with asa physical status i - ii who underwent elective surgery at bou - ali sina hospital in sari , northern iran , between january 2010 and january 2011 .
the children enabled to localize the pain , undergoing regional anesthetic technique , recovery time less than 10 minutes , and history of neurological or cardiovascular disorders were excluded .
all perioperative routine care was performed under supervision of an anesthesiologist or other physicians and care providers .
the data were recorded in a questionnaire including four separated parts ; the first part included the demographic characteristics and the three remaining parts included the behavioral criteria of ea and related factors in pre - anesthetic , during anesthesia , and post- anesthetic period in pacu . at pre - anesthetic period ,
the child s behavior during separation from his parents and at the time of induction of general anesthesia was graded as calm / cooperating , slightly anxious / fearful , or restless / uncooperative and recorded at presence of an anesthesiologist .
the presence or absence of behavior was recorded by yes / no into the questionnaire .
perioperative medications , induction and maintenance of anesthetic agents , duration of anesthesia , time of awakening ( namely , time from anesthetic off to time of initial arousal ) were recorded . during pacu ,
all routine monitoring was performed and all emergence behaviors were observed and recorded by a trained nurse using the reliable and valid pediatric anesthesia emergence delirium ( paed ) scales ( table 1 ) .
then the answers of each item were converted into scores and summed together and considered as paed score ; the higher the score , the more agitated child .
differentiating ea from the symptoms of postoperative pain was done using flacc behavioral scales with five categories including face , legs , activity , cry and consolability .
each of the five categories was scored from 0 - 2 , resulting in a total between 0 and 10 .
the results of flacc scale were interpreted as follows : 0 = relaxed and comfortable ; 1 - 3 = mild discomfort ; 4 - 6 = moderate pain ; 7 - 10 = severe pain or discomfort or both . also , all the pharmacologic and non - pharmacologic interventions , undesired side effects and time of discharge from recovery , and physical disturbances were recorded .
the patients have been discharged according to pacu discharge criteria with routine practice and at the discretion of the pacu nurse .
also , the questionnaire has been shown to have validity in a variety of studies and populations , and reliability of the questionnaire has been approved via a pilot study and cronbach s alpha equal to 0.76 was obtained .
pediatric anesthesia emergence delirium ( paed ) scales data were analyzed using spss software 16 and independent sample t - test , and binary logistic regression .
seven hundred forty- seven children aged 3 - 7 years with asa class i - ii were enrolled over a one year period .
one hundred thirty four children ( 17.9% ) had ea . from total , 479 ( 64.1% ) were males and 268 ( 35.9% ) females .
the mean age of the patients was 4.891.42 years . from 134 patients with ea , 89 ( 11.9% ) were males and 40 ( 6.0% ) females ( p>0.05 ) .
the previous history of surgery and medical diseases were seen in 22 ( 2.9% ) and 8 ( 1.1% ) patients , respectively ( p>0.05 ) .
the incidence of ea was higher in operative procedures less than one hour duration ( p=0.022 ) .
the mean of awakening time from anesthesia in ea and non - ea patients was 6.202.98 and 4.854.06 , respectively .
a significant relationship was observed between mean of awakening time from anesthesia and incidence of ea ( t=3.601 df=742 , p<0.05 ) .
the most frequent surgical procedures were ent 315 ( 42.2% ) , abdominal surgery 177 ( 23.7% ) , orthopedic surgery 137 ( 18.3% ) , urologic surgery , 97 ( 13% ) and ophthalmic surgery 24 ( 3.2% ) , respectively .
otorhino - laryngological surgical procedures were associated with higher rates of ea ( p<0.05 ) ( table 2 ) .
the most sites operated on were the head and neck areas ( 45.6% ) , trunk ( 22.4% ) , extremities ( 18.1% ) and pelvic area ( 13.9% ) .
there was significant statistical relationship between the site of operation , and frequency of ea ( p<0.05 ) .
this relationship was higher in the head and neck surgery ( p<0.05 ) [ or : 0.34 , ci95% ( 0.23 - 0.50 ) ] temper tantrum was seen in 620 children ( 83.1% ) and 99 ( 13.3% ) of them experienced ea ( p=0.003 ) . from 191 children ( 25.6% ) with history of anxiety , 36 ( 4.8% ) had ea ( p > 0.05 ) . among the 94 ( 12.6% ) of children with different degrees of pain at pacu , 61 ( 8.2% )
had presented ea ( p<0.05 ) . also , the incidence of ea was higher in children with flacc scored 1 - 3 ( p<0.05 ) .
there was no relationship between behavioral separation of the patients and ea ( p>0.055 ) , but their induction behavior had significant relationship with ea in pacu ( p<0.005 ) ( table 3 ) .
as shown in the table , isoflurane and atracurium had the highest relationship with incidence of ea ( p<0.05 ) .
the logistic regression analysis of the factors that had a significant effect on the incidence of ea , revealed that two factors ; pain and children s induction behavior were associated with the increased incidence of ea from anesthesia ( table 5 ) .
the relationship between type of surgical operation and presence of ea relationship between children s behavior in separation and induction times and ea
postoperative emergence agitation , also known as emergence delirium in international literature is a well - known clinical phenomenon with an incidence ranging 10 - 80% . recognizing the risk factors for ea is very important to minimize the contributing factors and managing ea appropriately .
we investigated multiple risk factors associated with ea such as the patient and surgical related factors , as well as anesthesia related risk factors such as rapid emergence and type of anesthetics .
the incidence of ea in our study was 17.9% based on paed score of 12 .
the present study showed that the children aged 4 - 6 years had a higher incidence of ea compared to children aged 7 years .
it may be due to less psychological and physiological immaturity of young - aged children compared to school - aged children being less able to cope with rapid awakening in an unknown environment .
similarly , we found thatchildren with agitation during the induction of anesthesia had a higher risk of developing ea .
some early reports have suggested that otorhinolaryngological surgical procedures appeared to exhibit an increased incidence of agitation .
when eckenhoff et al first described the ea in 1961 , attributed the increased incidence among otolaryngologic procedures to the sense of suffocation .
after that , in 2003 , voepel - lewis in a prospective study has shown that the otolaryngologic procedures are independent risk factors for ea . in our study , ea was associated with ent , abdominal and orthopedic surgical procedures .
also , surgical procedures performed on the head and neck was associated with higher risk of ea .
so , not only the type of surgical procedure , but also the surgical site may play a role in the incidence of ea .
the association between anesthetic agents and presence of emergence agitation factors predictive of emergence agitation ( step 1a ) postoperative pain appears to be an aggravating factor , and its behavioral manifestations may be confused with emergence agitation diagnosis .
several studies have discussed that pain during impaired consciousness leads to severe ea in some children . however , a clear relationship has not been established . in differentiating postoperative pain from ea
, we used both flacc and paed tools to facilitate the decision making in involved children .
we found that although ea decreased after adequate pain control , it was not eliminated completely .
the authors believe that flacc may be less reliable in children who experienced ea than in those who experienced pain only .
nevertheless , clinical experience and the review of the literature have suggested that when caring for children in pacu , it may not be necessary to differentiate ea from pain , especially because the treatment is similar for both of them .
some authors have observed a reduction in preoperative anxiety and easier to separate children from parents . also , they observed a reduction in agitation when midazolam was used as premedication followed by sevoflurane as anesthetic .
however , some authors have reported that emergence time was prolonged when midazolam was used . among the inhaled anesthetic agents used in this study ,
isoflurane was associated with 9.8% of ea compared with sevoflurane ( 2.5% ) and halothane ( 0.7% ) .
eeg changes caused by the effects of isoflurane and sevoflurane on the central nervous system have also been suggested as a possible causative agent of agitation .
propofol has been demonstrated to be effective as an adjunct to sevoflurane inhalational general anesthesia in reducing the incidence of ea .
we have also found no relationship between propofol and increased ea . among the intravenous anesthetics used in the present study
conversely , dalens et al showed that the administration of 0.25 mg / kg of ketamine at the end of the mri in children reduced agitation with no delay in discharge .
sufentanil is a potent opioid which is usually used as a standard treatment of ea because it relieves pain without influencing pacu length of stay .
we found a positive association between sufentanil used as opioid and the incidence of ea .
the authors suggest that the co - administration of sufentanil and isoflurane may cause this controversy .
there are controversies about the effect of duration of anesthesia on the incidence of ea . the higher incidence of ea has been observed in short - duration surgery as well as in the long - duration .
it was suggested that ea may be attributed to shorter - duration surgeries due to rapid washout of anesthetics from the body , causing rapid emergence before analgesics have enough time to act and reach their peak effect .
atracurium was another anesthetic agent that we detected related to the incidence of ea in our patients .
although , laudanosine as main metabolite of atracurium can play a role in ea because of its cns excitation effect .
the authors suggest that it can not be the causative phenomenon , because laudanosine can be activated in neuromuscular junction at its non - clinical high concentration . on the other hand , the short duration of most surgical procedures
this study has identified that multiple independent risk factors such as post - operative pain , short time awaking , anesthetic drugs including sufentanil , isoflurane , atracurium , and also ent surgeries , as well as site of operation were the risk factors of ea in children aged 3 - 7 years .
although ea in this sample was relatively of short duration , pharmacologic intervention was required in 6.7% of cases , and ea was associated with a prolonged pacu stay without significant adverse effect only in 2% of all cases .
alam sahbpour , sa mousavi : critical revision of manuscript s. khani and s. shahmohammadi : concept / design , data analysis and manuscript preparation all authors approved of the final version of the article .
| abstractobjectivepostanesthetic emergence agitation is a common problem in pediatric postanesthetic care unit with an incidence ranging from 10 to 80% .
this study was done to determine the prevalence of emergence agitation and associated risk factors in pediatric patients who underwent general anesthesia.methodsthis cross - sectional descriptive and analytic study was performed on 747 pediatric patients aged 3- 7 years that underwent general anesthesia for various elective surgeries at bou - ali sina hospital in sari , iran between january 2010 and january 2011 .
a non - probability quota sampling technique was used .
the presence of emergence agitation was recorded using pediatric anesthesia emergence delirium scale .
the factors linked with emergence agitation were recorded in a questionnaire .
the data were analyzed using spss software 16 and independent sample t - test , 2 and binary logistic regression .
p - values less than 0.05 were considered as significant.findingsone hundred thirty - four ( 17.9% ) children had emergence agitation .
the most frequent surgical procedures were ent surgical procedures 315 ( 42.2% ) , abdominal surgery 177 ( 23.7% ) , orthopedic surgery 137 ( 18.3% ) , urology 97 ( 13% ) and ophthalmic surgery 24 ( 3.2% ) .
otorhinolaryngological surgical procedures ( p=0.001 ) , pain ( p<0.05 ) and induction behavior of children ( p<0.005 ) were associated with higher rates of post anesthetic emergence agitation ( p=0.001).conclusionthis study identified the multiple independent risk factors which are associated with emergence agitation in children . to minimize the incidence of postanesthetic emergence agitation , these risk factors should be considered in the routine care by care providers in postanesthetic care unit . |
the study included 79 obese patients undergoing weight - reducing surgery and 14 lean patients who underwent elective cholecystectomy .
participants were included if they had fasting blood glucose levels < 7.0 mmol / l , no history of diabetes or use of glucose- or lipid - lowering drugs , and no weight changes > 3% during the previous 2 months .
all study participants provided informed consent , and study protocols were approved by the local ethics committee .
adipose tissue biopsies were collected in rnalater ( ambion , austin , tx ) and stored at 80c until rna isolation ( 22 ) .
five - week - old male db / db leptin receptor deficient mice ( bks.cg-dock7 lepr /j ) were obtained from charles river ( sulzfeld , germany ) and maintained in a virus / antibody - free central animal facility of the innsbruck medical university . at 6 weeks of age
body weight , fasting glucose levels , and urinary albumin excretion were measured regularly once a week , and 24-h urine samples were collected in metabolic cages .
mice were treated with either 200 g of an anti - cd25 monoclonal antibody ( mab ; clone pc61 ; bd biosciences , san diego , ca ) or an isotype control antibody administered intraperitoneally the day after uninephrectomy and 28 days thereafter . in a second set of experiments , db / db mice were treated intravenously with either 0.52 10 cd4foxp3gfp or cd4gfp cells on days 4 , 18 , 32 , and 46 after uninephrectomy .
adoptively transferred cells were isolated from pooled spleen and lymph node suspensions ( minced and passed through a 70-m mesh ) of b6.cg-foxp3/j reporter mice ( jackson laboratories , bar harbor , me ) . after staining for cd4 ( bd biosciences )
, cd4foxp3gfp tregs were purified by fluorescence - activated cell sorter ( facs ) using a bd facsaria cell sorter ( bd biosciences ) .
all animal experiments were approved by austrian veterinary authorities and corresponded to the european commission directive 86/609/eec .
single cell suspensions from liver , spleen , mvat , and murine subcutaneous adipose tissue ( msat ) were costained for surface cd4 , cd8 , and cd69 ( all bd biosciences ) and intracellular foxp3 ( ebiosciences , san diego , ca ) strictly adhering to the manufacturer s instructions .
blood glucose levels were measured using onetouch ultra2 ( lifescan , vienna , austria ) , urine creatinine was quantified spectrophotometrically using a picric acid based method ( sigma , st .
louis , mo ) , and urinary albumin was determined by a double - sandwich elisa ( abcam , cambridge , ma ) as reported previously ( 23 ) .
, we injected 0.75 iu / kg body wt ( in the treg - depletion experiments ) and 1.5 iu / kg body wt ( in the treg - treatment experiments ) of human insulin aspartat ( novo nordisk , sorgenfri , denmark ) and measured the blood glucose level prior to and 15 , 30 , 45 , 60 , 75 , and 90 min after insulin application . on the same day , we also measured fasting insulin levels ( crystal chem , downers grove , il ) by elisa .
the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated by using the glucose - insulin product divided by the constant 22.5 .
furthermore , we measured the fat cell diameter in msat and mvat ( epididymal ) and the glomerular diameter at a magnification of 100 using a progres c12 camera and the progres capture pro 2.5 software with the straight line tool ( jenoptik gmbh , jena , germany ) .
at least 200 adipocytes from both msat and mvat or 50 glomeruli were evaluated for each mouse in a blinded fashion .
formalin - fixed renal tissue was embedded in paraffin , cut in 4-m sections , and stained with hematoxylin - eosin and periodic acid - schiff using standard staining techniques .
the three - layer immunoperoxidase technique was used for the detection of macrophages and t cells in frozen kidney sections .
macrophages were stained using rat anti - mouse f4/80 and cd68 antibodies ( serotec , oxford , u.k . ) .
a semiquantitative scoring system was performed as follows : 0 , 0 to 4 cells ; 1 + , 5 to 10 cells ; 2 + , 10 to 50 cells ; 3 + , 50 to 200 cells ; and 4 + , > 200 cells stained positive per low - power field . for the detection of cd4 and cd8 t cells , rat anti - mouse cd4 and cd8- mab ( serotec ) were used .
biotin - conjugated goat anti - rat igg antibody ( jackson immunoresearch laboratories , west grove , pa ) was used as a secondary antibody , followed by incubation with a streptavidin - biotin complex and subsequent development with 0.4% 3-amino-9-ethylcarbazole for 6 min and counterstaining with gill 's hematoxylin
quantification of t cells was done by counting the number of cells in six adjacent high - power fields of renal cortex and medulla .
total rna was isolated from the respective tissue using trizol ( sigma ) according to a standard protocol .
the integrity of rna was detected by rna - gel electrophoresis in stratified random rna samples .
thereafter , 2 g of total rna was reverse transcribed using superscript iii transcription kit ( nitrogen , carlsbad , ca ) and random primers ( roche , basel , switzerland ) .
real - time pcr was performed on a steponeplus real - time instrument ( applied biosystems , foster city , ca ) or a cfx96 real - time system ( biorad , hercules , ca ) .
for amplification of murine foxp3 and tnf- , sybr green master mix ( nitrogen ) and the following primers were used : foxp3 forward 5-tct tgc caa gct gga aga ct-3 , reverse 5-agc tga tgc atg aag tgt gg-3 ; and tnf-
forward 5-gaa ctg gca gaa gag gca ct-3 , reverse 5-agg gtc tgg gcc ata gaa ct-3. the chosen primer pairs span the exon - intron border .
for quantification of human foxp3 , helios and tnf- taqman master mix ( applied biosystems ) and the gene expression assays hs01085834_m1 , hs00212361_m1 , and hs00174128_m1 ( applied biosystems ) were used .
for quantification of murine il-6 , interferon- ( ifn- ) , gata-3 , il-10 , the gene expression assays mm00446190_m1 , mm00901778_m1 , mm00484683_m1 , and mm00439616_m1 ( applied biosystems ) were used . in murine samples ,
, we evaluated the mean expression of three different housekeeping genes , namely , 18s , -actin , and hprt-1 , which was then set as reference .
the coefficient of variation for the intra - assay variance was < 0.0005 and for the interassay variance , < 0.001 .
enhanced green fluorescent protein positive cells were detected using the following primers : forward 5-aag ttc atc tgc acc acc g-3 , reverse 5-tcc ttg aag aag atg gtg cg-3. nested pcr was performed with the following primer pair : 5-gtg tcc ggc gag ggc gag g-3 , reverse 5-cgt gct gct tca tgt ggt cg-3. cdna isolated from spleens of b6.cg-foxp3/j served as positive control .
rt - pcr was carried out using hotstartaq polymerase ( qiagen , hilden , germany ) .
human data were analyzed for normal distribution using kolmogorov - smirnov test with lilliefors correction .
since the data were not normally distributed , the bivariate associations between helios / foxp3 mrna levels , bmi , fasting glucose levels , and homa - ir were calculated using the spearman correlation coefficient . in a case of comparing three groups ,
normal distribution of data were assessed by the kolmogorov - smirnov test with lilliefors correction .
both groups were compared by either nonparametric mann - whitney u test or unpaired t test as appropriate , depending on the distribution of the tested variable .
a two - tailed p < 0.05 was considered statistically significant . for the insulin - sensitivity assay in treg - depleted mice versus controls ,
all statistical analyses were done with spss 15.0 for windows ( spss , chicago , il ) .
the study included 79 obese patients undergoing weight - reducing surgery and 14 lean patients who underwent elective cholecystectomy .
participants were included if they had fasting blood glucose levels < 7.0 mmol / l , no history of diabetes or use of glucose- or lipid - lowering drugs , and no weight changes > 3% during the previous 2 months .
all study participants provided informed consent , and study protocols were approved by the local ethics committee .
adipose tissue biopsies were collected in rnalater ( ambion , austin , tx ) and stored at 80c until rna isolation ( 22 ) .
five - week - old male db / db leptin receptor deficient mice ( bks.cg-dock7 lepr /j ) were obtained from charles river ( sulzfeld , germany ) and maintained in a virus / antibody - free central animal facility of the innsbruck medical university . at 6 weeks of age
body weight , fasting glucose levels , and urinary albumin excretion were measured regularly once a week , and 24-h urine samples were collected in metabolic cages .
mice were treated with either 200 g of an anti - cd25 monoclonal antibody ( mab ; clone pc61 ; bd biosciences , san diego , ca ) or an isotype control antibody administered intraperitoneally the day after uninephrectomy and 28 days thereafter . in a second set of experiments , db
/ db mice were treated intravenously with either 0.52 10 cd4foxp3gfp or cd4gfp cells on days 4 , 18 , 32 , and 46 after uninephrectomy .
adoptively transferred cells were isolated from pooled spleen and lymph node suspensions ( minced and passed through a 70-m mesh ) of b6.cg-foxp3/j reporter mice ( jackson laboratories , bar harbor , me ) . after staining for cd4 ( bd biosciences )
, cd4foxp3gfp tregs were purified by fluorescence - activated cell sorter ( facs ) using a bd facsaria cell sorter ( bd biosciences ) .
all animal experiments were approved by austrian veterinary authorities and corresponded to the european commission directive 86/609/eec .
single cell suspensions from liver , spleen , mvat , and murine subcutaneous adipose tissue ( msat ) were costained for surface cd4 , cd8 , and cd69 ( all bd biosciences ) and intracellular foxp3 ( ebiosciences , san diego , ca ) strictly adhering to the manufacturer s instructions .
blood glucose levels were measured using onetouch ultra2 ( lifescan , vienna , austria ) , urine creatinine was quantified spectrophotometrically using a picric acid based method ( sigma , st .
louis , mo ) , and urinary albumin was determined by a double - sandwich elisa ( abcam , cambridge , ma ) as reported previously ( 23 ) .
, we injected 0.75 iu / kg body wt ( in the treg - depletion experiments ) and 1.5 iu / kg body wt ( in the treg - treatment experiments ) of human insulin aspartat ( novo nordisk , sorgenfri , denmark ) and measured the blood glucose level prior to and 15 , 30 , 45 , 60 , 75 , and 90 min after insulin application .
on the same day , we also measured fasting insulin levels ( crystal chem , downers grove , il ) by elisa .
the homeostasis model assessment of insulin resistance ( homa - ir ) was calculated by using the glucose - insulin product divided by the constant 22.5 .
furthermore , we measured the fat cell diameter in msat and mvat ( epididymal ) and the glomerular diameter at a magnification of 100 using a progres c12 camera and the progres capture pro 2.5 software with the straight line tool ( jenoptik gmbh , jena , germany ) .
at least 200 adipocytes from both msat and mvat or 50 glomeruli were evaluated for each mouse in a blinded fashion .
formalin - fixed renal tissue was embedded in paraffin , cut in 4-m sections , and stained with hematoxylin - eosin and periodic acid - schiff using standard staining techniques .
the three - layer immunoperoxidase technique was used for the detection of macrophages and t cells in frozen kidney sections .
macrophages were stained using rat anti - mouse f4/80 and cd68 antibodies ( serotec , oxford , u.k . ) .
a semiquantitative scoring system was performed as follows : 0 , 0 to 4 cells ; 1 + , 5 to 10 cells ; 2 + , 10 to 50 cells ; 3 + , 50 to 200 cells ; and 4 + , > 200 cells stained positive per low - power field . for the detection of cd4 and cd8 t cells , rat anti - mouse cd4 and cd8- mab ( serotec ) were used .
biotin - conjugated goat anti - rat igg antibody ( jackson immunoresearch laboratories , west grove , pa ) was used as a secondary antibody , followed by incubation with a streptavidin - biotin complex and subsequent development with 0.4% 3-amino-9-ethylcarbazole for 6 min and counterstaining with gill 's hematoxylin no .
quantification of t cells was done by counting the number of cells in six adjacent high - power fields of renal cortex and medulla .
total rna was isolated from the respective tissue using trizol ( sigma ) according to a standard protocol .
the integrity of rna was detected by rna - gel electrophoresis in stratified random rna samples .
thereafter , 2 g of total rna was reverse transcribed using superscript iii transcription kit ( nitrogen , carlsbad , ca ) and random primers ( roche , basel , switzerland ) .
real - time pcr was performed on a steponeplus real - time instrument ( applied biosystems , foster city , ca ) or a cfx96 real - time system ( biorad , hercules , ca ) .
for amplification of murine foxp3 and tnf- , sybr green master mix ( nitrogen ) and the following primers were used : foxp3 forward 5-tct tgc caa gct gga aga ct-3 , reverse 5-agc tga tgc atg aag tgt gg-3 ; and tnf- forward 5-gaa ctg gca gaa gag gca ct-3 , reverse 5-agg gtc tgg gcc ata gaa ct-3. the chosen primer pairs span the exon - intron border .
for quantification of human foxp3 , helios and tnf- taqman master mix ( applied biosystems ) and the gene expression assays hs01085834_m1 , hs00212361_m1 , and hs00174128_m1 ( applied biosystems ) were used .
the human foxp3 gene expression assay detects all human foxp3 splicing variants . for quantification of murine il-6 , interferon- ( ifn- ) , gata-3 , il-10 , the gene expression assays mm00446190_m1 , mm00901778_m1 , mm00484683_m1 , and mm00439616_m1 ( applied biosystems )
, we evaluated the mean expression of three different housekeeping genes , namely , 18s , -actin , and hprt-1 , which was then set as reference .
the coefficient of variation for the intra - assay variance was < 0.0005 and for the interassay variance , < 0.001 .
enhanced green fluorescent protein positive cells were detected using the following primers : forward 5-aag ttc atc tgc acc acc g-3 , reverse 5-tcc ttg aag aag atg gtg cg-3. nested pcr was performed with the following primer pair : 5-gtg tcc ggc gag ggc gag g-3 , reverse 5-cgt gct gct tca tgt ggt cg-3. cdna isolated from spleens of b6.cg-foxp3/j served as positive control .
rt - pcr was carried out using hotstartaq polymerase ( qiagen , hilden , germany ) .
human data were analyzed for normal distribution using kolmogorov - smirnov test with lilliefors correction .
since the data were not normally distributed , the bivariate associations between helios / foxp3 mrna levels , bmi , fasting glucose levels , and homa - ir were calculated using the spearman correlation coefficient . in a case of comparing three groups ,
normal distribution of data were assessed by the kolmogorov - smirnov test with lilliefors correction .
both groups were compared by either nonparametric mann - whitney u test or unpaired t test as appropriate , depending on the distribution of the tested variable .
for the insulin - sensitivity assay in treg - depleted mice versus controls , the fisher exact test was performed .
all statistical analyses were done with spss 15.0 for windows ( spss , chicago , il ) .
to test whether tregs accumulate in visceral obesity , we analyzed hvat from 14 lean control subjects , 39 obese patients without insulin resistance ( homa - ir < 2.5 ) , and 39 obese patients with insulin resistance ( homa - ir > 5 ) .
we evaluated the mrna expression of the thymic treg marker helios as well as of the master regulator of tregs foxp3 in hvat .
we found the mrna expression of helios to be downregulated in both obese patient groups , whereas foxp3 was significantly downregulated only in obese patients without insulin resistance . in obese patients with insulin resistance ,
significance was reached in the correlation with fasting blood glucose level and homa - ir .
obese patients with insulin resistance display significantly decreased helios but increased foxp3 mrna expression in their hvat .
a : hvat of lean control subjects ( white bar , n = 14 ) , obese patients with normal insulin sensitivity ( homa < 2.5 , gray bar , n = 39 ) , and obese patients with impaired insulin sensitivity ( homa > 5 , black bar , n = 39 ) was analyzed for helios and foxp3 mrna expression .
correlation of the helios - to - foxp3 mrna ratio in hvat ( n = 92 ) with bmi ( p = 0.121 ) ( b ) , homa - ir ( p = 0.003 ) ( c ) , and fasting blood glucose levels ( p = 0.045 ) ( d ) was analyzed using spearman . p < 0.05 was considered significant .
since treg lineage plasticity is influenced by insulin resistance , we next studied their pathogenic role in a murine model of type 2 diabetes .
we therefore used a well - established model of murine type 2 diabetes ( i.e. , the leptin - deficient db / db mice ) .
tregs were eliminated in these mice using the treg - depleting anti - cd25 antibody , which was injected intraperitoneally on days 1 and 28 after uninephrectomy .
we chose this application schedule since we could not detect any cd4cd25 t cells in the inguinal lymph nodes 3 weeks after application of a single dose of the antibody ( data not shown ) .
treg - depleted mice and the respective controls had a comparable body weight throughout the observation period of 56 days ( fig .
2a ) ; however , fasting blood glucose levels were significantly increased as early as 21 days after uninephrectomy in treg - depleted db / db mice when compared with vehicle - treated db / db mice ( fig .
, insulin - sensitivity testing revealed that insulin lowered blood glucose levels more efficiently in vehicle - treated db / db mice compared with treg - depleted db / db mice ( table 1 ) .
in addition , treg - depleted db / db mice displayed a significantly increased homa - ir on day 56 as compared with control animals ( fig . 2c ) . to analyze whether these metabolic differences were also reflected by changes in the inflammatory profile within the mvat and msat compartment , real - time pcr for mrna expression of ifn- , il-6 , tnf- , gata-3 , and il-10 was performed .
we could detect a prominent increase of the th1 cytokines ifn- , il-6 , and tnf- in mvat of treg - depleted db / db mice as compared with control animals ( fig .
no difference in the mrna expression of gata-3 and il-10 was detected in mvat ( fig .
furthermore , we did not detect any difference in the expression of the respective transcripts in msat ( fig .
flow cytometry revealed increased numbers of activated cd4cd69 t cells in the mvat of treg - depleted mice ( fig .
3c and d ) , while no difference in the abundance of this cell population was found in msat ( fig .
no difference in the percentage of cd8 t cells and cd11c dendritic cells in mvat was detected ( data not shown ) .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intraperitoneally received an anti - cd25 antibody starting the day of and 4 weeks after uninephrectomy ( and white bar , n = 8) .
control animals received an isotype control antibody ( and black bar , n = 8) . body weight ( a ) and blood glucose levels ( b ) were measured weekly .
treg depletion aggravates insulin resistance db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group received an anti - cd25 antibody intraperitoneally starting the day of and 4 weeks after uninephrectomy ( n = 8) .
insulin - sensitivity testing was performed on day 56 . the number of mice with blood glucose levels > 600 mg / dl at the indicated time points are shown .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intraperitoneally received an anti - cd25 mab the day of and 4 weeks after uninephrectomy ( white bar , n = 8) ; the control group received an isotype control antibody ( black bar , n = 8) .
the cytokine response in vat ( a ) and sat ( b ) was evaluated by real - time pcr .
c : the percentage of cd4cd69 t cells was evaluated in vat and sat by flow cytometry . pooled
treg depletion by anti - cd25 antibody did not compromise endogenous insulin production since mice that received the cd25-depleting antibody had even slightly higher insulin levels as compared with control mice ( control : 3.63 0.62 ng / ml ; treg depleted : 3.93 0.67 ng / ml ) .
the urinary albumin - to - creatinine ratio as a marker of renal impairment was increased in treg - depleted versus nondepleted db / db mice starting on day 28 and reached statistical significance on day 42 after uninephrectomy ( fig .
, the glomerular diameter was found to be significantly increased in treg - depleted mice on day 56 after uninephrectomy ( fig .
semiquantitative assessment of renal inflammatory cell infiltration revealed a profound increase in cd4 and cd8 t cells in the kidneys of treg - depleted db / db mice ( fig .
real - time analysis revealed a significantly increased mrna expression of the th1 cytokine tnf- in the kidneys of treg - depleted db / db mice ( fig .
4e ) . ifn- and il-10 tended to be increased in treg - depleted mice , whereas il-6 and gata-3 were not differentially regulated ( fig .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intraperitoneally received an anti - cd25 antibody on the day of and 4 weeks after uninephrectomy ( white bar , n = 8) .
a : the albumin - to - creatinine ratio was evaluated in the urine every 14 days .
b : after 56 days , kidneys were harvested and the glomerular diameter was measured .
kidney sections were stained for the infiltration of cd4 and cd8 t cells ( c ) as well as f4/80 cells and cd68 macrophages ( d ) .
e : the cytokine response in the kidney was evaluated by real - time pcr . the fold increase to the mean expression of control animals
db / db mice underwent uninephrectomy at the age of 6 weeks . on days 4 , 18 , 32 , and 46 after uninephrectomy , cd4foxp3gfp
( tregs ) or cd4foxp3gfp ( control ) t cells isolated from healthy donor animals were adoptively transferred into db / db mice .
the blood glucose levels were significantly decreased only in the treg - treated mice on day 28 , whereas on the other evaluated time points , no difference between the groups was detectable ( fig .
moreover , significantly improved insulin sensitivity was observed in db / db mice treated with tregs compared with db / db mice injected with control t cells ( fig .
5c ) . a trend toward an improved homa - ir in treg - treated obese animals was detected , but no statistical significance was reached ( fig .
5d ) . in addition , treg - treated db / db mice displayed a significantly decreased mvat cell diameter as compared with controls ( fig .
6a ) , whereas we could not detect any difference in the msat cell diameters ( data not shown ) .
treg transfer increased the expression of the treg marker foxp3 and the th1 cytokine ifn- in mvat , whereas il-6 , tnf- , gata-3 , and il-10 mrna were not differentially regulated ( fig .
it is interesting that the percentage of proinflammatory mvat - infiltrating cd8cd69 effector t cells was significantly decreased in treg - treated db / db mice ( fig .
6c and d ) , whereas no difference of these cells could be detected in msat ( fig .
no difference in the mvat and msat infiltration of cd4cd69 t cells was observed ( fig .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intravenously received facs - sorted cd4foxp3 tregs every 14 days ( gray cubes and bar , n = 8) .
body weight ( a ) and blood glucose levels ( b ) were measured weekly . on day 56 , the insulin - sensitivity testing was performed ( c ) and the homa - ir index was calculated ( d ) . pooled data of two independent experiments are shown . *
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intravenously received facs - sorted cd4foxp3 tregs every 14 days ( gray bar ) , whereas the control group was injected with cd4foxp3 control t cells ( black bar ) .
a : the vat cell diameter was determined by morphological evaluation ( n = 4 per group ) .
b : the cytokine response in vat was quantified by real - time pcr ( n = 8 per group ) .
c : the percentage of cd4cd69 and cd8cd69 t cells was evaluated in vat and sat by flow cytometry ( n = 4 per group ) . pooled or
d : representative dot plots from gated cd8 t cells and from cd8/cd69 double staining in vat .
adoptive transfer of treg significantly decreased urinary albumin - to - creatinine ratio in db / db mice when compared with respective controls on days 42 and 56 after uninephrectomy ( fig .
in addition , the glomerular diameter , which reflects hyperfiltration , was also significantly decreased in kidneys of treg - treated mice ( fig .
cd8 t cells were significantly less abundant in the kidneys of treg - injected db / db mice ( fig .
7c ) , whereas the infiltration of cd4 t cells and macrophages was not affected ( data not shown ) .
despite a trend toward higher expression of the proinflammatory th1 markers ifn- , il-6 , and tnf- in kidneys of treg - treated db / db mice , there was a predominant and statistically significant effect on the expression of the anti - inflammatory markers gata-3 , il-10 , and foxp3 ( fig .
, we were not able to detect adoptively transferred gfp tregs in harvested spleens , livers , kidneys , msat , and mvat of uninephrectomized db / db mice on day 56 ( i.e. , 14 days after the final treg - transfer ) using either flow cytometry or a highly sensitive pcr for gfp ( data not shown ) .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intravenously received facs - sorted cd4foxp3 tregs every 14 days ( gray bar ) , whereas control animals received cd4foxp3 t cells ( black bar ) .
a : the albumin - to - creatinine ratio was evaluated in the urine every 14 days .
b and c : after 56 days , kidneys were harvested and the glomerular diameter was morphologically determined ( n = 4 per group ) ( b ) , and kidney sections were stained for the infiltration of cd8 t cells ( n = 8 per group ) ( c ) .
d : the cytokine response in the kidney was evaluated by real - time pcr ( n = 8 per group ) .
to test whether tregs accumulate in visceral obesity , we analyzed hvat from 14 lean control subjects , 39 obese patients without insulin resistance ( homa - ir < 2.5 ) , and 39 obese patients with insulin resistance ( homa - ir > 5 ) .
we evaluated the mrna expression of the thymic treg marker helios as well as of the master regulator of tregs foxp3 in hvat .
we found the mrna expression of helios to be downregulated in both obese patient groups , whereas foxp3 was significantly downregulated only in obese patients without insulin resistance . in obese patients with insulin resistance ,
significance was reached in the correlation with fasting blood glucose level and homa - ir .
obese patients with insulin resistance display significantly decreased helios but increased foxp3 mrna expression in their hvat .
a : hvat of lean control subjects ( white bar , n = 14 ) , obese patients with normal insulin sensitivity ( homa < 2.5 , gray bar , n = 39 ) , and obese patients with impaired insulin sensitivity ( homa > 5 , black bar , n = 39 ) was analyzed for helios and foxp3 mrna expression .
correlation of the helios - to - foxp3 mrna ratio in hvat ( n = 92 ) with bmi ( p = 0.121 ) ( b ) , homa - ir ( p = 0.003 ) ( c ) , and fasting blood glucose levels ( p = 0.045 ) ( d ) was analyzed using spearman . p < 0.05 was considered significant .
since treg lineage plasticity is influenced by insulin resistance , we next studied their pathogenic role in a murine model of type 2 diabetes .
we therefore used a well - established model of murine type 2 diabetes ( i.e. , the leptin - deficient db / db mice ) .
tregs were eliminated in these mice using the treg - depleting anti - cd25 antibody , which was injected intraperitoneally on days 1 and 28 after uninephrectomy .
we chose this application schedule since we could not detect any cd4cd25 t cells in the inguinal lymph nodes 3 weeks after application of a single dose of the antibody ( data not shown ) .
treg - depleted mice and the respective controls had a comparable body weight throughout the observation period of 56 days ( fig .
2a ) ; however , fasting blood glucose levels were significantly increased as early as 21 days after uninephrectomy in treg - depleted db / db mice when compared with vehicle - treated db / db mice ( fig .
, insulin - sensitivity testing revealed that insulin lowered blood glucose levels more efficiently in vehicle - treated db / db mice compared with treg - depleted db / db mice ( table 1 ) .
in addition , treg - depleted db / db mice displayed a significantly increased homa - ir on day 56 as compared with control animals ( fig . 2c ) . to analyze whether these metabolic differences were also reflected by changes in the inflammatory profile within the mvat and msat compartment , real - time pcr for mrna expression of ifn- , il-6 , tnf- , gata-3 , and il-10 was performed .
we could detect a prominent increase of the th1 cytokines ifn- , il-6 , and tnf- in mvat of treg - depleted db / db mice as compared with control animals ( fig .
no difference in the mrna expression of gata-3 and il-10 was detected in mvat ( fig .
furthermore , we did not detect any difference in the expression of the respective transcripts in msat ( fig .
flow cytometry revealed increased numbers of activated cd4cd69 t cells in the mvat of treg - depleted mice ( fig .
3c and d ) , while no difference in the abundance of this cell population was found in msat ( fig .
no difference in the percentage of cd8 t cells and cd11c dendritic cells in mvat was detected ( data not shown ) .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intraperitoneally received an anti - cd25 antibody starting the day of and 4 weeks after uninephrectomy ( and white bar , n = 8) .
control animals received an isotype control antibody ( and black bar , n = 8) .
body weight ( a ) and blood glucose levels ( b ) were measured weekly .
treg depletion aggravates insulin resistance db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group received an anti - cd25 antibody intraperitoneally starting the day of and 4 weeks after uninephrectomy ( n = 8) .
insulin - sensitivity testing was performed on day 56 . the number of mice with blood glucose levels > 600 mg / dl at the indicated time points are shown . pooled data of two independent experiments are shown .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intraperitoneally received an anti - cd25 mab the day of and 4 weeks after uninephrectomy ( white bar , n = 8) ; the control group received an isotype control antibody ( black bar , n = 8) . the cytokine response in vat ( a ) and sat ( b )
was evaluated by real - time pcr . the fold increase to the mean expression of controls
c : the percentage of cd4cd69 t cells was evaluated in vat and sat by flow cytometry .
treg depletion by anti - cd25 antibody did not compromise endogenous insulin production since mice that received the cd25-depleting antibody had even slightly higher insulin levels as compared with control mice ( control : 3.63 0.62 ng / ml ; treg depleted : 3.93 0.67 ng / ml ) .
the urinary albumin - to - creatinine ratio as a marker of renal impairment was increased in treg - depleted versus nondepleted db / db mice starting on day 28 and reached statistical significance on day 42 after uninephrectomy ( fig .
, the glomerular diameter was found to be significantly increased in treg - depleted mice on day 56 after uninephrectomy ( fig .
semiquantitative assessment of renal inflammatory cell infiltration revealed a profound increase in cd4 and cd8 t cells in the kidneys of treg - depleted db / db mice ( fig .
real - time analysis revealed a significantly increased mrna expression of the th1 cytokine tnf- in the kidneys of treg - depleted db / db mice ( fig .
4e ) . ifn- and il-10 tended to be increased in treg - depleted mice , whereas il-6 and gata-3 were not differentially regulated ( fig .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intraperitoneally received an anti - cd25 antibody on the day of and 4 weeks after uninephrectomy ( white bar , n = 8) .
a : the albumin - to - creatinine ratio was evaluated in the urine every 14 days .
b : after 56 days , kidneys were harvested and the glomerular diameter was measured .
kidney sections were stained for the infiltration of cd4 and cd8 t cells ( c ) as well as f4/80 cells and cd68 macrophages ( d ) .
e : the cytokine response in the kidney was evaluated by real - time pcr . the fold increase to the mean expression of control animals
db / db mice underwent uninephrectomy at the age of 6 weeks . on days 4 , 18 , 32 , and 46 after uninephrectomy , cd4foxp3gfp
( tregs ) or cd4foxp3gfp ( control ) t cells isolated from healthy donor animals were adoptively transferred into db / db mice .
the blood glucose levels were significantly decreased only in the treg - treated mice on day 28 , whereas on the other evaluated time points , no difference between the groups was detectable ( fig .
5b ) . moreover , significantly improved insulin sensitivity was observed in db / db mice treated with tregs compared with db / db mice injected with control t cells ( fig .
a trend toward an improved homa - ir in treg - treated obese animals was detected , but no statistical significance was reached ( fig .
5d ) . in addition , treg - treated db / db mice displayed a significantly decreased mvat cell diameter as compared with controls ( fig .
6a ) , whereas we could not detect any difference in the msat cell diameters ( data not shown ) .
treg transfer increased the expression of the treg marker foxp3 and the th1 cytokine ifn- in mvat , whereas il-6 , tnf- , gata-3 , and il-10 mrna were not differentially regulated ( fig .
it is interesting that the percentage of proinflammatory mvat - infiltrating cd8cd69 effector t cells was significantly decreased in treg - treated db / db mice ( fig .
6c and d ) , whereas no difference of these cells could be detected in msat ( fig .
no difference in the mvat and msat infiltration of cd4cd69 t cells was observed ( fig .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intravenously received facs - sorted cd4foxp3 tregs every 14 days ( gray cubes and bar , n = 8) .
body weight ( a ) and blood glucose levels ( b ) were measured weekly . on day 56 ,
the insulin - sensitivity testing was performed ( c ) and the homa - ir index was calculated ( d ) . pooled data of two independent experiments are shown . * p < 0.05 .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intravenously received facs - sorted cd4foxp3 tregs every 14 days ( gray bar ) , whereas the control group was injected with cd4foxp3 control t cells ( black bar ) .
a : the vat cell diameter was determined by morphological evaluation ( n = 4 per group ) .
b : the cytokine response in vat was quantified by real - time pcr ( n = 8 per group ) .
c : the percentage of cd4cd69 and cd8cd69 t cells was evaluated in vat and sat by flow cytometry ( n = 4 per group ) . pooled or representative
d : representative dot plots from gated cd8 t cells and from cd8/cd69 double staining in vat .
adoptive transfer of treg significantly decreased urinary albumin - to - creatinine ratio in db / db mice when compared with respective controls on days 42 and 56 after uninephrectomy ( fig .
the glomerular diameter , which reflects hyperfiltration , was also significantly decreased in kidneys of treg - treated mice ( fig .
cd8 t cells were significantly less abundant in the kidneys of treg - injected db / db mice ( fig .
7c ) , whereas the infiltration of cd4 t cells and macrophages was not affected ( data not shown ) . despite a trend toward higher expression of the proinflammatory th1 markers ifn- , il-6 , and tnf- in kidneys of treg - treated db / db mice , there was a predominant and statistically significant effect on the expression of the anti - inflammatory markers gata-3 , il-10 , and foxp3 ( fig .
7d ) . of note , we were not able to detect adoptively transferred gfp tregs in harvested spleens , livers , kidneys , msat , and mvat of uninephrectomized db / db mice on day 56 ( i.e. , 14 days after the final treg - transfer ) using either flow cytometry or a highly sensitive pcr for gfp ( data not shown ) .
db / db mice were uninephrectomized at the age of 6 weeks and followed for 56 days .
one group intravenously received facs - sorted cd4foxp3 tregs every 14 days ( gray bar ) , whereas control animals received cd4foxp3 t cells ( black bar ) .
a : the albumin - to - creatinine ratio was evaluated in the urine every 14 days .
b and c : after 56 days , kidneys were harvested and the glomerular diameter was morphologically determined ( n = 4 per group ) ( b ) , and kidney sections were stained for the infiltration of cd8 t cells ( n = 8 per group ) ( c ) .
d : the cytokine response in the kidney was evaluated by real - time pcr ( n = 8 per group ) .
the fold increase to the mean expression of controls is provided . pooled data of two independent experiments
the pandemic of type 2 diabetes is one of the major health burdens in the industrial world today , and diabetic nephropathy due to type 2 diabetes has become the leading cause of end stage renal disease .
are driven by chronic inflammation ( 24 ) where not only macrophages but also t cells are critically involved ( 24 ) . here , we present evidence that tregs are key regulatory cells in the pathogenesis of insulin resistance and that they have the capacity to improve insulin resistance and diabetic nephropathy when transferred in vivo . it is long known that macrophages accumulate in the mvat of obese mice ( 25 ) , but recently winer et al .
, they detected significantly improved insulin resistance in their murine type 2 diabetes model . in a similar approach , ilan et al .
( 16 ) showed that the ratio between th1 effector cells and tregs changed toward tregs as a result of oral administration of anti - cd3 and -glucosylceramide .
the importance of t cells and tregs in insulin resistance was suggested by observational human studies .
nevertheless , published human data on tregs in hvat have been , thus far , conflicting ( 1719 ) .
while zeyda et al . ( 19 ) found t cells together with tregs to accumulate in hvat of obese patients , others detected decreased infiltrating treg numbers ( 17,18 ) .
our patient population consisted of lean , obese insulin - sensitive , and obese insulin - resistant patients .
we detected decreased foxp3 mrna signals only in obese insulin - sensitive patients as compared with lean control subjects .
most interesting , helios a new marker for thymic - derived natural tregs ( 13)differed significantly from our foxp3 data , since helios expression was significantly lowered in hvat of all obese patients irrespective of their insulin resistance .
moreover , peripherally induced adaptive foxp3 tregs seem to accumulate in the inflammatory microenvironment of hvat in obese patients with overt insulin resistance .
limitations for these suggestions come from the technique used throughout our studies since we had no access to fresh hvat to perform foxp3/helios double staining .
since foxp3 cell populations have also been shown to produce il-17 or ifn- ( 26,27 ) , we can not exclude that our foxp3 mrna expression reflects other cd4 t cells infiltrating hvat of obese patients with insulin resistance . to gain further insights in the functional role of treg in insulin resistance , we used db / db mice , which lack signaling capacity of the leptin receptor and therefore develop hyperphagia , diet - induced obesity , and type 2 diabetes ( 20 ) .
after treatment with a cd25-depletion antibody , which has been used for treg depletion in various disease models ( 2830 ) , db / db mice displayed augmented insulin resistance and a more severe diabetic nephropathy . in line with recent reports ( 15,17 ) , we detected significantly increased th1 cytokines and activated t cells in the mvat of treg - depleted db / db mice , whereas no changes were detected in msat .
these data strongly support the idea that vat but not sat is crucial for impaired insulin sensitivity .
independent studies in lean mice demonstrate that treg depletion induced by a genetic approach aggravated insulin resistance ( 17 ) . crossing adipose mouse strains with foxp3-dtr mice
we provide the first direct evidence that foxp3 tregs have the capacity to improve insulin resistance .
nave t cells provide the potential to respond to new pathogens , whereas central - memory and effector - memory t cells can mount rapid and efficient secondary proliferate and effector responses to recall antigens ( 31 ) . in our experimental setup
, cd8 t cells infiltrating mvat mainly expressed the t - cell activation marker cd69 .
since t - cell activation markers such as cd69 and cd25 are expressed only on a small fraction of the two memory subsets ( 32 ) , it can be proposed that treg transfer decreased the mvat infiltration of effector cd8 t cells , which originated from nave cd8 t cells .
this is further supported by the recent observation that tregs dramatically decrease the expansion and differentiation of nave cd8 t cell precursors in an experimental murine model of repopulation of lymphopenic environment ( 33 ) .
cd8 effector t cells are involved in the pathogenesis of obesity and infiltrate adipose tissue ( 34 ) .
nishimura et al . ( 34 ) provided evidence that cd8 t cells lead to the recruitment of macrophages , thereby mediating insulin resistance .
15 ) found cd8 t cells did not influence diet - induced obesity in lymphocyte - free rag1-null mice . in our murine model ,
transfer of cd4foxp3 tregs not only improved insulin resistance but also resulted in less end organ damage , such as diabetic nephropathy . in the kidney , we observed a shift toward a th2 response , reflected by increased mrna expression of gata-3 and il-10 , as well as significantly increased treg numbers .
thus , il-10 produced by tregs might be of crucial importance for the treg - mediated protective effect , but further studies using tregs from il-10 knockout mice are necessary .
the role of il-10 needs further evaluation since renal il-10 mrna expression also tended to be increased in kidneys of treg - depleted mice .
again , we found significantly decreased numbers of cd8 t cells to infiltrate the kidneys , whereas no change in the number of renal cd4 t cells was noted .
it is interesting that no significant difference in the macrophage infiltration of kidneys was detected .
it has been proposed that cd8 t cells improve insulin resistance by recruiting macrophages ( 34 ) , which seems not to be the case in the pathogenesis of diabetic nephropathy .
further studies depleting cd8 t cells in diabetic nephropathy are needed to shed more light on the role of cd8 t cells in diabetic nephropathy . it might be further speculated that tregs , independently from improving insulin resistance , improve diabetic nephropathy by exerting their anti - inflammatory capacities , but our study design does not allow discrimination between the two mechanisms . by transferring foxp3-gfp tregs , we transferred a selective fluorescent treg population .
contrary to the murine model of nephrotoxic nephritis ( 2 ) , we failed to track these fluorescent tregs by using molecular genetic methods and flow cytometry .
the absence of foxp3-gfp tregs in mvat , liver , kidney , and spleen on day 56 is probably due to the long interval of 14 days after the final transfer .
it seems that at the tested time point , all transferred tregs were already apoptotic and that they had previously , in their short lifetime , primed the immune system toward an anti - inflammatory milieu . taken together , we prove that transfer of cd4foxp3 tregs improves insulin resistance and diabetic nephropathy by tipping the balance toward anti - inflammation and inhibiting cd8 effector cell infiltration in vat and kidneys .
our results confirm the importance of tregs in the pathogenesis of insulin resistance and might lead to new therapeutic concepts of type 2 diabetes . | objectiveto assess the potential role of foxp3-expressing regulatory t cells ( tregs ) in reversing obesity - linked insulin resistance and diabetic nephropathy in rodent models and humans.research design and methodsto characterize the role of tregs in insulin resistance , human visceral adipose tissue was first evaluated for treg infiltration and second , the db / db mouse model was evaluated.resultsobese patients with insulin resistance displayed significantly decreased natural tregs but an increase in adaptive tregs in their visceral adipose tissue as compared with lean control subjects . to further evaluate the pathogenic role of tregs in insulin resistance ,
the db / db mouse model was used .
treg depletion using an anti - cd25 monoclonal antibody enhanced insulin resistance as shown by increased fasting blood glucose levels as well as an impaired insulin sensitivity .
moreover , treg - depleted db / db mice developed increased signs of diabetic nephropathy , such as albuminuria and glomerular hyperfiltration .
this was paralleled by a proinflammatory milieu in both murine visceral adipose tissue and the kidney .
conversely , adoptive transfer of cd4+foxp3 + tregs significantly improved insulin sensitivity and diabetic nephropathy .
accordingly , there was increased mrna expression of foxp3 as well as less abundant proinflammatory cd8+cd69 + t cells in visceral adipose tissue and kidneys of treg - treated animals.conclusionsdata suggest a potential therapeutic value of tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the proinflammatory milieu . |
cardiac events are a leading cause of death worldwide and despite our best life support efforts outcomes after cardiac arrest are still poor .
the european resuscitation council ( erc ) cardiopulmonary resuscitation ( cpr ) 2010 guidelines emphasize the importance of minimizing the interruption of chest compression to maximize coronary and cerebral perfusion pressure .
tracheal intubation is still considered the optimal method for maintaining a secure airway according to the erc guidelines 2010 .
these guidelines also suggest that the intubator should be able to secure the airway without interrupting chest compression ( cc ) .
however , intubation during cpr is difficult and requires frequent practice to maintain the skill .
accordingly , the new guidelines for dealing with difficult airway management which will be released in 2015 , of which the most important details are already available in electronic form , assume that medical personnel have access to video - laryngoscopes as well as direct laryngoscopy devices .
the etview vivasight sl ( etview ; etview ltd . , misgav , israel ) is single - lumen airway tube with an integrated high - resolution imaging camera .
the image is transmitted to the display in real time , so that the person performing the intubation is able to view the entrance to the larynx and insert the endotracheal tube into the trachea . in the case of flexible fiberscopes and videolaryngoscopes , visibility can be reduced by blood or other fluids in the throat .
the etview is equipped with an integrated flushing system that allows for rapid and efficient camera lens cleaning in situ .
increasing evidence indicates that the etview may be suitable for tracheal intubation in various clinical settings .
we hypothesized that the etview vivasight sl ( etview ) could be an alternative to standard direct laryngoscopy ( mac ) during intubation of adult manikins while performing cpr . in the present study ,
we compared the effectiveness of the etview and mac intubation in adult resuscitation with and without cc .
this study has been approved by the institutional review board of the international institute of rescue research and education ( approval 5.2014.12.32 , november 3rd , 2014 ) and registered at the clinicaltrials register ( www.clinicaltrials.gov , identifier nct02295618 ) .
one hundred seven internal medicine faculty members who had less than 1-year experience in medicine participated in this study .
participants had limited experience in clinical intubation ( less than 10 intubations ) and none had prior experience with the etview vivasight sl .
voluntary informed consent was obtained from each participant before the study . before the study , all participants received a standardized audio - visual lecture lasting 45 minutes covering relevant aspects of anatomy and different techniques for securing an airway , including the mac and the etview . following the lecture , physicians participated in a practical demonstration , during which intubations with the respective devices were demonstrated by an independent anesthesiologist .
participants were then given 30 minutes to practice intubation using the mac and the etview with a manikin .
two tracheal intubation devices in this trial were used : the etview vivasight - sl ( etview ; size 7.0 i d ) and the macintosh laryngoscope blade no .
3 ( mac ) ( mercury medical , clearwater , fl ; figure 1 ) . in the adult intubation scenario ,
a simman 3 g training manikin ( laerdal , norway ) was used , and a lucas-2 device ( physio - control , redmond , wa ) was used for chest compression ( figure 2 ) .
endotracheal intubations ( eti ) using the macintosh laryngoscope were performed using a size 7.0-mm tracheal tube .
after each device , participants took a 10-minute rest and then performed intubation using the next device .
the participants were not allowed to watch each other so as to avoid learning through observation .
laryngoscopes used for this study were ( a ) etview vivasight sl , ( b ) macintosh laryngoscope .
after the training session , each participant started their intubations using the etview and the mac in adult resuscitation scenarios in a randomized order .
the order of interventions was randomized for each participant by a computer program ( research randomizer [ www.randomizer.org ] ; figure 3 ) .
after completing the eti procedure participants had a 30-minute break before performing intubation using another method .
the primary outcome of the study was time to intubation , defined as the time from insertion of the laryngoscope blade between the teeth to the first manual ventilation of the mannequin 's lungs .
if the oesopharyngeal tube was incorrectly placed or intubation lasted longer than 60 seconds , the airway - management attempt was defined as a failure . in many studies ,
this time is over than 120 seconds , but for the good of the patient , we took a period of 60 seconds as the time required for intubation .
after each attempt , participants were asked to rate the glottic view they had during the attempt using a cormack & lehane grade .
the severity of the potential dental trauma was calculated based on a previously described modified grading scale . to assess subjective opinions about the difficulty of the procedure
, participants were asked to rate it on a visual analogue scale ( vas ) with a score from 1 ( extremely easy ) to 10 ( extremely difficult ) .
in addition , participants were asked which method they would prefer in a real - life resuscitation .
sample size calculations indicated that 107 participants would be sufficient to detect moderate differences in intubation time between the macintosh and the etview ( using 5% level of significance and 80% power ) .
the r statistical package for windows ( version 3.0.0 ) was used for statistical analysis . as data were found not to be normally distributed ,
all results are shown as a number ( % ) , mean and standard deviation ( sd ) , or median and interquartile range ( iqr ) .
sample size calculations indicated that 107 participants would be sufficient to detect moderate differences in intubation time between the macintosh and the etview ( using 5% level of significance and 80% power ) .
the r statistical package for windows ( version 3.0.0 ) was used for statistical analysis . as data were found not to be normally distributed ,
all results are shown as a number ( % ) , mean and standard deviation ( sd ) , or median and interquartile range ( iqr ) . a p < 0.05 was considered to be statistically significant .
one hundred seven novice physicians ( 46 women and 61 men , age 31.2 7.4 years ) participated in the study .
did not significantly increase intubation time with the etview ( 17(iqr , 1519 ) s without chest compression versus 19 ( iqr , 1522 ) s with chest compression ) .
intubation times were significantly longer using the mac when chest compression was being performed ( 27(iqr , 2533 ) s without chest compression versus 38 ( iqr , 3146 ) s with chest compression , p < 0.005 ) .
there was a statistically significant difference in intubation time between etview and mac in both scenarios : without chest compressions ( p < 0.001 ) and with uninterrupted chest compressions ( p < 0.001 ) .
table 1 shows the number of successful intubations for each trial . in the scenario without cc , the overall effectiveness of intubation using mac and etview was 100% .
however , the success rate after the first attempt using the mac and etview varied and amounted to : 83.4% versus 100% , respectively ( p = 0.017 ) . in the chest compression scenario , the success rate after the first attempt using the distinct laryngoscopes varied and amounted to : 46.7% versus 100% ( mac and etview , respectively ) .
there was a statistically significant difference between mac and etview in first intubation attempt effectiveness ( p < 0.001 ) and overall effectiveness ( p < 0.001 ) .
success rates of intubation according to laryngoscopes and intubation attempts dental compression was observed during intubation when the mac was used .
dental compression during mac intubation in cc scenarios was observed in 6.5% of intubation attempts , whereas 12 of 107 participants ( 11.2% ) caused dental compression during chest compression ( p < 0.001 ) .
dental compression was not observed during intubation when the etview was used in scenarios both with and without cc .
glottic view quality was best with the etview , with 100% of participants reporting a quality of glottic view corresponding to a cormack & lehane grade classification of i , regardless of whether chest compressions were being performed .
grade of glottic view according to the cormack & lehane classification that was achieved with the different devices .
data are given in absolute numbers and percentage participants ratings of their subjective opinions about the difficulty of the intubation procedure using the etview and the mac varied and amounted to : 2.1 versus 3.2 points ( p = 0.027 ) for intubation without chest compression , and 2.3 versus 3.9 points ( p = 0.009 ) for eti with uninterrupted chest compression .
when participants were asked which laryngoscope they would prefer in real - life intubation with uninterrupted chest compression , 100% selected the etview .
interruption of chest compression during cpr is associated with reduced cardiac arrest survival rates . ensuring adequate airway management and adequate oxygenation are integral parts of resuscitation .
the european resuscitation council 2010 guidelines for cardiopulmonary resuscitation indicate that tracheal intubation is the gold standard for securing airways and providing effective ventilation .
these guidelines also suggest that skilled operators should be able to secure the airway either without interrupting chest compression , or with only a brief pause to view the vocal cords to allow passage of the tracheal tube .
eti . however , they emphasize that bag - mask ventilation should be used if a person does not have the right skills to perform direct laryngoscopy .
however , it is important to note that in prehospital care all patients should be treated as if they have a full stomach as incompetent ventilation using a bag - mask can cause vomiting .
the conducted study is the first study comparing the efficacy of endotracheal intubation using the etview vivasight sl ( etview ) and the macintosh laryngoscope during simulated adult resuscitation with and without chest compressions .
all participants were able to intubate the trachea with the etview during resuscitation with and without chest compression .
the reason for this outstanding 100% success rate is presumed to be the characteristic tube guide system .
etview is a single - lumen airway tube with an integrated high - resolution imaging camera that provides continuous real - time images of tube position on an lcd screen . even in the resuscitation with an uninterrupted chest compression scenario ,
the median time to complete intubation was 19 ( iqr , 1522 ) s , which is approximately 1 cycle of cpr .
this result suggests that the etview might be able to establish tracheal intubation without interruption of chest compression during cardiopulmonary resuscitation .
this facilitates the minimal interruption in chest compressions suggested by the current 2010 european resuscitation council guidelines for cpr .
there are a few reports showing the effectiveness of etview during different clinical situations , however , none of them refers to intubation using etview during resuscitation . in our study
, the overall effectiveness of intubation using the mac is 100% in scenarios without chest compression and only 68.2% in scenarios with uninterrupted chest compression .
conducting chest compressions significantly reduced the effectiveness of direct laryngoscopy using the macintosh laryngoscope , and increased the statistical significance of the intubation process from 27 ( iqr , 2533 ) s without cc scenario to 38 ( iqr , 3146 ) s for eti during uninterrupted chest compression .
gaszynska and gaszynski reported in their study that the success ratio on the first attempt of eti using mac during uninterrupted chest compressions was 73.3% when performed by third - year paramedic students .
kohama et al in their study concerning the effectiveness of intubation using mac during resuscitation by 18 novice doctors from an anesthesia department reported shorter time of intubation ( 18.9 4.0 s ) ; however , overall effectiveness of intubation during cc was similar to our study and amounted to 66.7% .
overall effectiveness of mac intubation in different studies varied and amounted to 81.3% ( han et al ) to 89.4% ( kim et al ) . as shown in our study , people who previously had no contact with the etview are able with a little training to effectively perform intubation using this device .
the high efficiency of etview during simultaneous chest compressions means there is no need to interrupt the cc for the duration of intubation .
this is particularly important in view of the fact that survival of cardiac arrest victims is directly related to the quality of cpr . among factors that determine this quality
the standard endotracheal tube costs about $ 2 , whereas the etview tube is 15 times more expensive , not including the additional cost of the monitor . however , at many emergency medical services and emergency departments displays are now available which can be connected to an etview via a special cable , and thus can display the image captured by the etview .
the cost of the etview is not high compared with other video - laryngoscopy or intubation fiberoscopy devices .
also , it should be borne in mind that the life and health of the patient are paramount .
however , according to the international liaison committee on resuscitation ( ilcor ) , randomized clinical trial for cases of cardiac arrest is highly unethical ; therefore , cpr mannequins are an important and established tool for training medical professionals in these crucial and life - saving procedures .
some studies have been published with airway simulators , and many concepts for managing the airway were developed and practised on manikins and subsequently transferred to clinical scenarios .
the strengths of this study include the fact that it was a randomized crossover trial that used a highly advanced patient simulator for performing intubation during resuscitation , and the lucas-2 chest compression system was used to standardize chest compressions .
the etview provides a promising device for the management of intubations during resuscitation by novice physicians .
use of this device seems to offer advantages over the mac during cpr , as rates of successful intubation are increased and number of intubation attempts are reduced , thereby improving the cormack & lehane grading and diminishing potential damage . | abstractthe aim of this study was to assess the performance of the etview vivasight sl ( etview ) single - lumen airway tube with an integrated high - resolution imaging camera in a manikin - simulated cardiopulmonary resuscitation scenario with and without chest compression.this was a randomized crossover manikin trial . following a brief training session ,
107 volunteer novice physicians who were inexperienced with airway management attempted to intubate a manikin using a macintosh laryngoscope ( mac ) and an etview , with and without chest compressions .
the participants were instructed to make 3 attempts in each scenario . in this trial , we compared intubation time , intubation success rates , and glottic visibility using a cormack & lehane grade .
dental compression and ease of use of each device were also assessed.median intubation times for the etview and mac without chest compressions were 17 ( iqr , 1519 ) s and 27 ( iqr , 2533 ) s , respectively ( p < 0.001 ) .
the etview proved more successful on the first intubation attempt than the mac , regardless of compressions .
continuation of compressions caused an increase in intubation times for both the etview ( p = 0.27 ) and the mac ( p < 0.005).the etview vivasight sl is an effective tool for endotracheal intubation when used by novice physicians in a manikin - simulated cardiac arrest , both with and without chest compressions.trial registration : clinicaltrials.gov identifier : nct02295618 . |
intacs ( addition technology inc , des plaines , il , usa ) are intrastromal corneal rings consisting of two thin , clear hexagonal polymethyl methacrylate segments .
they are placed in surgically created semicircular channels between the stromal lamella at two - thirds the stromal depth during an outpatient procedure .
intrastromal ring segments work by flattening the central corneal curvature , and are adjustable and reversible .
since receiving approval by the us food and drug administration in 1999 , they have been used to correct low to moderate myopia1 and to treat ectasia following laser in situ keratomileusis.2 asymmetric implantation of intrastromal corneal ring segments in eyes with keratoconus has been demonstrated to improve both uncorrected and best spectacle - corrected visual acuity , and to reduce irregular astigmatism in corneas with and without scarring.37 relatively few infectious complications have been reported with the use of the intrastromal corneal segments ; channel infections have been infrequently documented,1,810 and only a limited number of infectious keratitis cases have been reported following insertion of intacs.8,9,1116 although uncommon , microbial keratitis following intrastromal corneal segments insertion is potentially one of the most serious complications and may be sight - threatening .
appropriate suspicion by the eye care provider along with rapid diagnosis and appropriate management can be critical and result in improved visual recovery .
the purpose of this study was to describe the clinical and pathologic features of candida parapsilosis keratitis , which occurred 8 months following the initially uneventful placement of intacs inserts for keratoconus .
this is the first report in the peer - reviewed literature of c. parapsilosis following intacs insertion and includes molecular confirmation at the species level .
the patient was a 52-year - old female with a history of keratoconus and no known systemic disease or risk factors for infection .
preoperatively , the patient had uncorrected visual acuity of 20/60 od and 20/200 os , and best - corrected visual acuity of 20/30 od and 20/50 os , with a refraction of 3.003.50 170 degrees od and 5.504.50 20 degrees os .
intacs intrastromal corneal ring segments were vertically placed in her left eye for treatment of her keratoconus .
specifically , a 35 ring was inserted superiorly and a 45 ring inferiorly through a 65%70% depth channel .
good visual rehabilitation was obtained following intacs insertion ( uncorrected visual acuity , os 20/40 ; best - corrected visual acuity os 20/25 ) and was uneventful at 4 months post insertion ( figure 1a ) .
spontaneous extrusion of the inferior intrastromal corneal ring segment with no indication of infection was noted 5 months after insertion ( figure 1b ) , at which time it was removed .
eight months post intacs implantation ( 3 months post replacement ) , the location of the lower lateral edge of the intrastromal corneal ring segments showed an epithelial defect and infiltrate on slit - lamp examination ( figure 1c ) .
the size of the epithelial defect and infiltrate was 1 mm and the infiltrate was at a depth of 40% .
the intrastromal corneal ring segments were removed because of suspected infection and the infiltrate was treated for 2 weeks with topical ofloxacin 0.3% every 2 hours and prednisolone acetate 1% once daily .
corneal scrapings cultured on blood agar , chocolate agar , thioglycolate broth , and sabouraud s agar were negative , as was a gram stain . the patient was followed weekly and demonstrated flares and exacerbation one month after treatment .
the infiltrate progressed into the deep cornea at the level of the intrastromal corneal ring segments at approximately 60% thickness ( figure 1d ) .
every 2 hours , vancomycin 10 mg / ml every 2 hours , and clarithromycin 12 mg / ml every 2 hours were prescribed for one week without improvement .
intraoperative amphotericin and vancomycin were dropped open sky in the anterior chamber . a formal in - fixed keratectomy specimen
was histopathologically examined by light microscopy using standard histopathologic techniques , including periodic acid - schiff staining .
the corneal tissue clearly demonstrated insertion channels for the previously removed intrastromal corneal ring segments and a perforating corneal ulcer ( figure 2 ) .
dense mid - stromal infiltrates and a break in descemet s membrane were also noted . as seen in the figure 2 insert , c. parapsilosis yeast forms have darker staining central nuclei and produce broad - neck buds . to confirm the histopathologic findings , the corneal tissue was evaluated using a molecular polymerase chain reaction ( pcr)-based diagnostic approach .
the forward and reverse panfungal pcr primers encompass a highly conserved region of fungal dna and are designed to amplify and detect a broad spectrum of fungal dna without amplifying nonfungal dna.17 the current case report corneal specimen was compared with a previously confirmed ocular case of c. parapsilosis - induced infectious crystalline keratopathy,18 with dna isolated from various species of candida grown in culture ( table 1 ) , and with plasmid pca1 dna that contains a relevant fragment of candida albicans dna.17 formalin - fixed , paraffin - embedded clinical corneal specimens were cut with a microtome at a thickness of 10 m using precautions necessary for pcr analysis .
twelve to 16 microtome sections were placed into sterile microfuge tubes and the dna extracted using a protocol previously described19 and modified.20 overnight cultures of c. parapsilosis , c. albicans , candida guilliermondii , and candida ( yarrowia ) lipolytica were grown as described17 and processed for dna extraction using standard genomic dna methods21 with modifications previously described.17 plasmid pca1 was grown in escherichia coli dh5-alpha cells and purified.17 twenty percent of each dna sample extracted from the clinical specimens was used for pcr analysis .
approximately 1 10 genome equivalents of culture - purified dna from the control candida strains and 4 10 copies of plasmid pca1 were used as positive control templates .
the pcr assay conditions had been previously optimized and described.17,22,23 twenty percent of each pcr assay product was electrophoretically resolved on 1.8% agarose tris - borate - edta gel and visualized using ethidium bromide and ultraviolet excitation .
pcr amplicons were compared in size with a 50 bp ladder ( life technologies , grand island , ny , usa ) .
the current keratoplasty specimen produced a pcr amplicon product of approximately 311 bp , as did the crystalline keratopathy corneal specimen and the dna from cultured c. parapsilosis ( figure 3 ) .
the control dna from cultured c. albicans , c. guilliermondii , and c. lipolytica produced amplicons of 338 bp , 379 bp , and 242 bp , respectively ( figure 3 ) in agreement with the predicted sizes ( table 1 ) .
the plasmid pca1 containing a fragment of c. albicans dna produced the predicted 338 bp pcr product , while the negative control showed no amplification product .
the patient underwent a negative systemic workup , and at one year postoperatively had no signs of recurrence .
intrastromal corneal ring segments are an option in the management of some patients with keratoconus , and their use has been increasing since their efficacy were first reported by colin et al.5 according to multiple case studies , microbial keratitis occurs as a complication in approximately 1.4%6.8% of cases post intrastromal ring segments insertion.15,2426 infectious keratitis following intrastromal corneal ring segments insertion , while uncommon , is potentially one of the most serious complications . reported microbial species in post - intacs keratitis include staphylococcus aureus , staphylococcus epidermidis , staphylococcus viridans , streptococcus pneumoniae , streptococcus mitis , pseudomonas species , nocardia species , klebsiella species , paecylomices species , and clostridium perfringens.8,9,11,12,15 ophthalmologists identified c. parapsilosis as a cause for an epidemic of post - cataract endophthalmitis that was linked to intraocular irrigating solutions.27,28 this report demonstrates the potential for c. parapsilosis to be the etiologic cause of infectious keratitis following insertion of corneal ring segments in the treatment of keratoconus .
in addition to its link with the endophthalmitis outbreak and with the current case , c. parapsilosis has other ophthalmic associations , such as being the most frequently isolated fungal organism from the normal outer eye in a 1969 study in south florida29 and several reports of secondary ophthalmic infections.18,3038 it has also been linked with crystalline keratopathy in a corneal graft18 and suppurative stromal keratitis.32,39 a large case series suggests that c. parapsilosis accounts for approximately 10% of all causes of yeast keratitis in southern florida.32 recently , c. parapsilosis has been reported as a cause of chronic postoperative endophthalmitis.28,40 furthermore , c. parapsilosis has emerged as an important nosocomial pathogen with many systemic clinical manifestations in addition to the ocular manifestation of endophthalmitis.41 overall , c. parapsilosis has accounted for 3%27% of cases of fungemia in large hospital - based studies.41 candida and other yeasts are more frequently opportunistic than are filamentous fungi.42 infections caused by these agents are usually seen in compromised corneas with multiple predisposing alterations in host defense .
comparison by pcr of the dna isolated from the current case with various cultured strains of candida and with previously confirmed c. parapsilosis - infected corneal tissue was consistent with the infecting etiologic agent being c. parapsilosis . the sensitivity and specificity of any molecular diagnostic approach are important parameters for interpretation of assay results .
establishment of the sensitivity and specificity of the current assay has been previously detailed.17 the panfungal primers used bracket a region of the fungal genome encoding the highly conserved 5.8s ribosomal rna ( rrna ) , internal transcribed sequence-2 ( its-2 ) , and 28s rrna . the sequence specificity and assay stringency conditions allow for sensitive amplification of dna from a wide variety of fungal strains without amplification of human , bovine , murine , bacterial , or viral dna . while highly conserved , genomic sequence heterogeneity results in variation of the pcr amplicon / product size depending on the specific fungal strain used as a template source .
candida strains produce pcr amplicons ranging in size from 242 bp to 379 bp ( table 1 ) with c. parapsilosis producing a 311 bp pcr product .
this is the first reported case in the peer - reviewed literature of c. parapsilosis keratitis following insertion of intrastromal corneal ring segments .
the origin of the candida , whether from the normal ocular flora or introduced from an exogenous source , is not entirely clear and could not be determined from the current study .
while the source is unknown , exposure of the stroma by insertion of intacs may have allowed ingress of the fungus .
lipid accumulation on the external side of intrastromal ring segments is common and may complicate the clinical picture of an infectious process . during evaluation of any cornea with intrastromal ring segments , it is important to distinguish between the white color of lipid accumulation and that of an infectious infiltrate .
awareness and consideration of an infectious process being potentially responsible for any white banking is important and may play a critical role in correct diagnosis and subsequent treatment .
patients should be informed of the risk factors and warning signs of infectious keratitis and should be advised to seek medical attention immediately should they develop signs of symptoms of keratitis .
unlike bacterial keratitis , which can be controlled by potent antibiotics , fungal keratitis is difficult to manage because of the lack of effective antifungal agents , low drug bioavailability , ocular toxicity , decreased solubility , and late presentation with large infiltrates .
long - term postoperative observation following intrastromal corneal ring segments insertion is advocated , especially given that the onset of microbial keratitis post implant can vary from days to months.8,9,1114,16,43,44 a high degree of suspicion along with appropriate and complete microbiologic testing , coupled with prompt and appropriate treatment , may result in better visual recovery .
this case report adds to the growing and diverse list of organisms and presentations of infectious keratitis following insertion of intrastromal ring segments . | this case report describes the clinical and histopathologic features , including molecular confirmation , of fungal keratitis after intrastromal corneal ring segments placement for keratoconus .
a 52-year - old woman underwent insertion of intacs corneal implants for treatment of keratoconus .
extrusion of the implants was noted 5 months post insertion and replaced .
three months later , monocular infiltrates and an epithelial defect were observed .
the intacs were removed and the infiltrates were treated with ofloxacin and prednisolone acetate .
microbial cultures and stains were negative .
the patient demonstrated flares and exacerbation one month later .
mycoplasma and/or fungus were suspected and treated without improvement .
therapeutic keratoplasty was performed 10 months following initial placement of the corneal ring implants .
the keratectomy specimen was analyzed by light microscopy and a panfungal polymerase chain reaction assay .
a histopathologic diagnosis of candida parapsilosis keratitis was made and confirmed by polymerase chain reaction .
one year postoperatively , a systemic workup of the patient was done with no signs of recurrence .
this rare report of fungal keratitis following intacs insertion is the first reported case of c. parapsilosis complicating intacs implantation . |
apoptosis is a type of genetically regulated programmed cell death that controls the development of multicellular organisms and tissues by eliminating physiologically redundant , physical damaged , and abnormal cells .
studies focusing on the genes and signals regulating apoptosis have played an important role in basic oncology research .
radiotherapy and chemotherapy are essential for the treatment of malignant tumors in pre- and post - operative cancer patients .
the efficacy of anticancer drugs is measured by their ability to detect cancer cells and selectively promote their apoptosis .
the tumor drug sensitivity test ( dst ) is a method to identify the most effective drug to treat tumors based on their sensitivity .
the genotype and pathogenesis of tumors vary , and malignant tumors are made up of a polymorphous , heterogeneous , and multi - differentiated cell population .
tumors can be resistant to one or more drugs , or exhibit sensitivity towards many drugs . by reducing drug resistance and increasing the efficiency of dsts ,
multiple methods to detect apoptosis have been developed that detect changes in cell morphology and surface markers associated with apoptosis .
this can obscure the result of dsts , affecting the accuracy and validity of the drugs they select .
dual acridine orange / ethidium bromide ( ao / eb ) fluorescent staining , visualized under a fluorescent microscope , can be used to identify apoptosis - associated changes of cell membranes during the process of apoptosis .
dual ao / eb staining was used to examine apoptosis in a human osteosarcoma cell line treated with the tumor inhibitor kappa - selenocarrageenan .
we qualitatively and quantitatively analyzed the effects of kappa - selenocarrageenan using the ao / eb staining method .
flow cytometry can be used to measure the fluorescence intensity of intracellular ca(2 ) from bone marrow mesenchymal stem cells ( mscs ) .
cell - cycle distribution can be determined by flow cytometric analysis using propidium iodide ( pi ) staining and cellular apoptosis can be evaluated by flow cytometry and terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ) assay . by comparing our technique with flow cytometry analysis technology , we concluded that dual ao / eb staining is a simple and accurate method that can be used in tumor dsts .
after obtaining irb approval , a human osteosarcoma cell line ( os-732 cells , purchased from beijing jishuitan hospital , beijing , china ) in the logarithmic growth phase were digested with 0.25% trypsin ( hyclone , logan , ut ) .
rpmi1604 culture medium ( hyclone , logan , ut ) containing 10% fetal calf serum ( fcs , hyclone , logan , ut ) was deposited in each well of a 96-well plate ( 100 l / well ) .
cells were added to a final concentration of 210/ml , and the plates were incubated .
cells were left untreated or treated with 30 , 60 , or 120 g / ml of kappa - selenocarrageenan ( shanghai tiancifu biological engineering co. ltd . , shanghai , china ) .
the samples in a 96-well plate were divided into 4 groups , with 24 well samples in each group corresponding to different reagent concentrations . after being cultured for 24 h , 48 h , and
when cells had sloughed off , suspensions ( 25 l ) were transferred to glass slides .
dual fluorescent staining solution ( 1 l ) containing 100 g / ml ao and 100 g / ml eb ( ao / eb , sigma , st . louis , mo ) was added to each suspension and then covered with a coverslip .
the morphology of apoptotic cells was examined and 500 cells were counted within 20 min using a fluorescent microscope ( olympus , japan ) .
dual acridine orange / ethidium bromide ( ao / eb ) staining method was repeated 3 times at least .
cells were trypsinized ( 0.25% trypsin ) 24 h after kappa - selenocarrageenan was applied to the sample .
single - cell suspensions ( 210 cells ) were extracted and washed using phosphate - buffered saline .
0.1% triton x-100 ph 7.8 ) was added to the samples and was incubated at room temperature for 45 min .
next , the cells were digested with 50 g / ml rnase for 10 min .
cells were stained with pi ( 50 g / ml ) for 30 min ( pi , sigma , st .
the samples were analyzed using a flow cytometer ( bd , franklin lakes , nj ) .
the cells with dna content less than that of cells at the g1 phase were identified as apoptotic cells . also , flow cytometry analysis was repeated at least 3 times .
all statistical analyses were performed using the spss 20.0 software ( spss inc . , chicago , il , usa ) for windows .
after obtaining irb approval , a human osteosarcoma cell line ( os-732 cells , purchased from beijing jishuitan hospital , beijing , china ) in the logarithmic growth phase were digested with 0.25% trypsin ( hyclone , logan , ut ) .
rpmi1604 culture medium ( hyclone , logan , ut ) containing 10% fetal calf serum ( fcs , hyclone , logan , ut ) was deposited in each well of a 96-well plate ( 100 l / well ) .
cells were added to a final concentration of 210/ml , and the plates were incubated .
cells were left untreated or treated with 30 , 60 , or 120 g / ml of kappa - selenocarrageenan ( shanghai tiancifu biological engineering co. ltd . , shanghai , china ) .
the samples in a 96-well plate were divided into 4 groups , with 24 well samples in each group corresponding to different reagent concentrations . after being cultured for 24 h , 48 h , and
when cells had sloughed off , suspensions ( 25 l ) were transferred to glass slides .
dual fluorescent staining solution ( 1 l ) containing 100 g / ml ao and 100 g / ml eb ( ao / eb , sigma , st . louis , mo ) was added to each suspension and then covered with a coverslip .
the morphology of apoptotic cells was examined and 500 cells were counted within 20 min using a fluorescent microscope ( olympus , japan ) .
dual acridine orange / ethidium bromide ( ao / eb ) staining method was repeated 3 times at least .
cells were trypsinized ( 0.25% trypsin ) 24 h after kappa - selenocarrageenan was applied to the sample .
single - cell suspensions ( 210 cells ) were extracted and washed using phosphate - buffered saline .
0.1% triton x-100 ph 7.8 ) was added to the samples and was incubated at room temperature for 45 min .
next , the cells were digested with 50 g / ml rnase for 10 min .
cells were stained with pi ( 50 g / ml ) for 30 min ( pi , sigma , st .
the samples were analyzed using a flow cytometer ( bd , franklin lakes , nj ) .
the cells with dna content less than that of cells at the g1 phase were identified as apoptotic cells . also , flow cytometry analysis was repeated at least 3 times .
all statistical analyses were performed using the spss 20.0 software ( spss inc . , chicago , il , usa ) for windows .
osteosarcoma cells were labeled by ao / eb 24 h after kappa - selenocarrageenan was applied .
early - stage apoptotic cells , marked by crescent - shaped or granular yellow - green ao nuclear staining , were detected in the experimental group ( figure 1b ) .
staining was localized asymmetrically within the cells . with increasing concentrations and treatment lengths , the number of early - stage apoptotic cells increased .
late - stage apoptotic cells , with concentrated and asymmetrically localized orange nuclear eb staining , were also detected ( figure 1c ) .
necrotic cells increased in volume and showed uneven orange - red fluorescence at their periphery .
the hypodiploid peak appearing to the left of the g1 peak in the cell cycle histogram formed as a result of apoptosis .
no distinctive apoptotic peak was observed for the negative control group cultured for 24 h ( figure 2a ) .
the apoptotic peak to the left of the g1 peak immediately appeared 24 h after 30 g / ml of kappa - selenocarrageenan was applied to the osteosarcoma cells ( figure 2b ) .
a student s t - test showed no significant difference between the 2 methods ( table 1 ) .
osteosarcoma cells were labeled by ao / eb 24 h after kappa - selenocarrageenan was applied .
early - stage apoptotic cells , marked by crescent - shaped or granular yellow - green ao nuclear staining , were detected in the experimental group ( figure 1b ) .
staining was localized asymmetrically within the cells . with increasing concentrations and treatment lengths , the number of early - stage apoptotic cells increased .
late - stage apoptotic cells , with concentrated and asymmetrically localized orange nuclear eb staining , were also detected ( figure 1c ) .
necrotic cells increased in volume and showed uneven orange - red fluorescence at their periphery .
the hypodiploid peak appearing to the left of the g1 peak in the cell cycle histogram formed as a result of apoptosis .
no distinctive apoptotic peak was observed for the negative control group cultured for 24 h ( figure 2a ) .
the apoptotic peak to the left of the g1 peak immediately appeared 24 h after 30 g / ml of kappa - selenocarrageenan was applied to the osteosarcoma cells ( figure 2b ) .
a student s t - test showed no significant difference between the 2 methods ( table 1 ) .
greater than 80% of patients have systemic metastatic cancer when they enter the hospital , with the lungs being the most common site of osteosarcoma metastasis .
the long - term survival rate of the patients receiving surgical interventions is only 2030% . over the past 30 years
, rosen has been a strong proponent and has advocated for high - dose systemic chemotherapy , namely neoadjuvant chemotherapy , prior to surgical intervention , which has been a milestone in the history of osteosarcoma treatment .
this approach has greatly improved the 5-year survival rate of patients with osteosarcoma ; however , nearly half of patients die from pulmonary metastasis because cancers become resistant to chemotherapy drugs within 23 years of treatment .
because tumors are composed of a polymorphous , heterogeneous , and multi - differentiated cell population , their sensitivity to various chemotherapeutic drugs differs depending on the individual .
the action and metabolism of drugs in vivo is a complex process , and chemotherapy is time - consuming and cost - ineffective .
individualized treatment strategies have been proposed . in individualized treatment , dsts are carried out in vitro to identify and select the most effective chemotherapeutics , and to determine its effective dose . therefore , individualized medication could improve the targeting and efficacy of chemotherapeutic agents and reduce adverse reactions and drug resistance
multiple methods , such as pi , in situ nick translation , terminal deoxynucleotidyl , acidic denaturation , and thermal denaturation assays , have been developed to detect apoptosis by monitoring changes in cell morphology and surface markers .
these methods involve multi - step procedures , including diversion , washing , and transfer of samples , using both time and materials .
mtt assays have been used as dsts in vitro , but this method has some weaknesses .
for example , this assay can not distinguish between apoptotic and necrotic cells , and therefore can not detect the toxic effects of drugs on the body .
while tumor cells undergo apoptosis in the presence of anticancer drugs , normal cells become necrotic if the drug is toxic .
therefore , detection of tumor cell apoptosis is more valuable than generally assessing tumor cell viability .
in addition , it allows for the distinction between normal cells , early and late apoptotic cells , and necrotic cells .
therefore , ao / eb staining is a qualitative and quantitative method to detect apoptosis .
we speculate that ao penetrated normal and early apoptotic cells with intact membranes , fluorescing green when bound to dna .
eb only entered cells with damaged membranes , such as late apoptotic and dead cells , emitting orange - red fluorescence when bound to concentrated dna fragments or apoptotic bodies . furthermore , dual ao / eb staining is able to detect mild dna injuries . therefore , to distinguish normal , early apoptotic , late apoptotic cells , and dead cells , nuclear morphology must be assessed .
fluorescent staining using ao alone has been used in the past ; however , detection of cell apoptosis using ao / eb is a relatively new approach , and few papers have reported its use . in comparison to ao staining , the ao / eb method improves the detection of apoptosis and can distinguish between late apoptotic and dead cells .
flow cytometry is a classical method to detect cell apoptosis with high sensitivity and can be used for simultaneous cell - cycle analysis . however
, a general laboratory may not be equipped with a flow cytometer , and they are costly to run .
no significant difference was observed between the ability of flow cytometry and ao / eb staining to detect apoptosis .
therefore , fluorescent staining can feasibly be applied to evaluate apoptosis in dsts of osteosarcomas or other malignant tumors . | backgroundthe aim of this study was to evaluate the ability of dual acridine orange / ethidium bromide ( ao / eb ) staining to detect tumor cell apoptosis . according to apoptosis - associated changes of cell membranes during the process of apoptosis , a clear distinction is made between normal cells , early and late apoptotic cells , and necrotic cells.material/methodwe cultured human osteosarcoma cells with 30 , 60 , and 120 g / ml kappa - selenocarrageenan . to assess the rates of cell proliferation and apoptosis ,
cells were fluorescently stained with acridine orange / ethidium bromide ( ao / eb ) or stained with propidium iodide ( pi ) and analyzed by flow cytometry . all experiments were repeated at least 3 times.resultnormal tumor cells , early and late apoptotic cells , and necrotic cells were examined using fluorescent microscopy .
early - stage apoptotic cells were marked by crescent - shaped or granular yellow - green acridine orange nuclear staining .
late - stage apoptotic cells were marked with concentrated and asymmetrically localized orange nuclear ethidium bromide staining .
necrotic cells increased in volume and showed uneven orange - red fluorescence at their periphery .
cells appeared to be in the process of disintegrating .
the percentage of apoptotic osteosarcoma cells detected by dual acridine orange / ethidium bromide ( ao / eb ) staining was not significantly different from that detected using flow cytometry ( p>0.05).conclusionsour results suggest that dual acridine orange / ethidium bromide staining is an economic and convenient method to detect apoptosis in tumor cells and to test tumor chemosensitivity compared with flow cytometry . |
the spect acquisition in lung perfusion scan provides higher sensitivity and specificity to detect pulmonary thromboemboli ( pte ) compared to planar imaging ( 1 , 2 ) . because of higher specificity , there are hopes that the lung perfusion spect could be interpreted without a ventilation study .
the value of higher specificity is more highlighted in complicated cases with vascular abnormality and shunts . in this report , a case with tetralogy of fallot ( tf ) and multiple cardiovascular abnormalities and corrective shunts are presented underscoring the importance of spect imaging in complicated patients .
a 22-year - old female patient was admitted for surgery to revise her waterston central aortopulmonary shunt .
she had the history of tf , pulmonary valve stenosis , ventricular septal defect and major aortopulmonary collateral artery .
her shunt was placed at the age of 5 years then complicated with stenosis for which a stent was placed and re - dilated 1 year ago .
after echocardiography reported minimal aorto - pulmonary shunt flow , a decision was made to operate the patient for revision of the central shunt .
midsternal incision was done and a polytetrafluoroethylene ( ptfe ) tube was inserted between the ascending aorta and right pulmonary branch , where the previous gortex was located .
patient recovery and rehabilitation after surgery was optimal until the patient experienced abrupt left - sided pleuritic chest pain and dyspnea .
the patient was referred to the department of nuclear medicine for perfusion and ventilation lung scanning .
perfusion lung scan was done with 4 mci 99 m technetium labeled macroaggregated albumin ( total particle count 120,000 ) in planar anterior , posterior , lateral and multiple oblique projections ( figure 1 ) .
additionally , the brain , renal parenchyma and the thyroid demonstrated uptake due to the right to left shunt ( figure 2 ) .
ventilation scan was not performed due to logistic problems ; the available ventilation tracer in iran is 81kr , which is distributed only on wednesdays .
there was a large wedge - shaped peripheral defect in the apicoposterior segment of the left upper lobe .
the finding was interpreted as either acute / chronic pte or secondary to developmental / iatrogenic vascular abnormality .
meanwhile , the patient underwent ct angiography reporting postoperative atelectatic changes in the bilateral lower lobes without evidence in support of pte .
the iv heparin was changed to warfarin after 5 days when the symptoms improved gradually and the arterial o2 saturation increased to 92% in room atmosphere .
tetralogy of fallot is a congenital cyanotic heart disease in about 3.5% of infants with congenital heart diseases .
there are a wide spectrum of anatomic defects with varying severity including ventricular septal defect , overriding aorta , right ventricular outflow tract obstruction , and right ventricular hypertrophy ( 3 ) .
diagnosis of pulmonary emboli is a great diagnostic challenge and it should be suspected in any patient with unexplained dyspnea , tachypnea , or chest pain ( 4 , 5 ) .
lung perfusion and ventilation spect is superior to planar scan in detecting pulmonary embolism with a high sensitivity and specificity ( 6 ) .
perfusion lung scan without ventilation scan has also been shown to have a high specificity of 92% and sensitivity of 87% ( 7 ) .
the advantages of lung perfusion spect imaging include higher contrast for better detection of the shape and location of defects and higher sensitivity ( 6 ) . in complicated cases similar to the patient described here ,
this is of foremost importance . because pulmonary angiography was not performed and ct angiography was negative , pte was not confirmed in this case .
ct angiography is a sensitive and optimal technique for diagnosing large pulmonary emboli , but it has a relatively low sensitivity ( below 80% ) for sub - segmental ptes ( 4 ) . | a 22-year - old woman presented with acute left - sided pleuritic chest pain and dyspnea 6 days after surgery for revision of the stenotic central aortopulmonary shunt .
she had a history of tetralogy of fallot ( tof ) , pulmonary valve stenosis , ventricular septal defect and major aortopulmonary collateral artery .
her waterston shunt was placed when she was 5 years old and stented and re - dilated after stenosis .
acute pulmonary thromboemboli ( pte ) was suspected and pulmonary perfusion scan was performed with 4 mci 99 m technetium labeled macroaggregated albumin .
the left lung was globally hypoperfused with evident uptake in the brain , renal parenchyma and thyroid .
spect images revealed a segmental wedge - shaped peripheral defect in the posterior segment of the left upper lobe .
the scan was interpreted as acute / chronic pte or vascular abnormality .
ct angiography excluded pte ; nevertheless the patient was treated with a therapeutic dose of heparin changed to warfarin and was discharged with improvement of the symptoms .
pulmonary artery angiography was not performed . |
the primary complaint of most patients with this tumor is testicular swelling , which is usually painless .
it is not uncommon for this tumor to be of an advanced stage at the time of the diagnosis , and the prognosis is dismal .
although scrotal swelling is the most common manifestation , the clinical presentation is often nonspecific and may not suggest a neoplasm , such that the diagnosis can be delayed . to our knowledge , this is the first report in the published literature documenting incidental adenocarcinoma of the rete testis with preceding detection of unilateral hydronephrosis .
the patient was a 54-year - old man with right flank pain that had started 1 month previously .
the kidney - ureter - bladder and urinalysis findings were normal , but ultrasonography showed mild right hydronephrosis .
therefore , abdominopelvic computed tomography ( ct ) was performed to determine the cause of the hydronephrosis .
we found multiple enhanced lesions around the right obturator muscle that were compressing the right distal ureter and causing the hydronephrosis .
additionally , a heterogeneous , low - density lesion with an irregular margin was found with hydrocele in the right testis ( fig .
according to the patient 's medical history , the painless swelling of the right scrotum had started about 1 year previously with no other significant medical history . on the physical examination , a relatively movable
iu / l ) and carcinoembryonic antigen ( cea , 40.8 ng / ml ) levels were higher than normal , but the alpha - fetoprotein and beta - human chorionic gonadotropin ( beta - hcg ) levels were normal .
we decided to perform cystoscopy and retrograde pyelography with radical orchiectomy to rule out any metastatic lesions in the right ureter .
a right ureteral stricture was found and a d - j ureteral catheter was inserted .
microscopic examination showed conventional adenocarcinoma , which shows a glandular pattern of growth with atypical stratified cuboidal to columnar epithelial tumor cells ( fig .
significant immunohistochemical reactivities for cea , muc1 , cytokeratin 20 , and cdx2 were seen ( fig .
there was no reactivity for prostate - specific antigen ( psa ) , carbohydrate antigen 19 - 9 , thyroid transcription factor-1 , or s-100 protein .
positron emission tomography - ct ( pet - ct ) , gastroduodenoscopy , colonoscopy , and transrectal ultrasound were performed in addition to measuring the serum psa level to rule out metastatic adenocarcinoma from another site .
, we finally diagnosed a right testicular lesion as the primary adenocarcinoma in the rete testis .
after the radical orchiectomy , the patient received combined chemotherapy 6 times , but the progression of the disease was unstoppable . during the course of the chemotherapy , pet - ct was done , which revealed newly developed lung metastasis .
up to one quarter of these tumors have been reported in association with scrotal hydrocele , as in our case .
the diagnosis is often difficult and made incidentally and is often delayed , because of the nonspecific clinical presentation , which can suggest only an inflammatory process .
the time from the initial problem to presentation averages approximately 24 months . in an extensive review of reported cases ,
54% of patients had metastases at diagnosis with lymphatic spread to the lumboaortic and iliac nodes and metastases to the bones , lungs , and skin .
the common sites of early metastases are the lymph nodes , lungs , liver , and bones .
the diagnostic criteria of nochomovitz and orenstein for adenocarcinoma of the rete testis include 1 ) tumor origin around the hilum , 2 ) no involvement of the tunica , 3 ) demonstration of transition from normal to tumor epithelium , and 4 ) no evidence of teratoma or any other primary tumor elsewhere in the body .
our case fit 3 of these 4 diagnostic criteria : 1 ) , 2 ) , and 4 ) .
a few cases have been examined immunohistochemically and are positive for cytokeratins and negative for beta - hcg , psa , and alpha - fetoprotein .
positivity for cea may be seen , and it is helpful in excluding the diagnosis of mesothelioma . in our case ,
however , reliable markers that can define the origin of the adenocarcinoma as the rete testis have never been indicated .
in advanced stages , adenocarcinoma of the rete testis is highly resistant to adjuvant radiotherapy and any known chemotherapy .
the natural history of this tumor appears to be highly malignant , with 3- and 5-year disease - free survival rates of 49% and 13% , respectively . | primary adenocarcinoma of the rete testis is a rare malignant testicular tumor with a poor prognosis . here
we report the case of a 54-year - old man with right hydronephrosis that was detected before the diagnosis of the primary testicular lesion . during the evaluation of the right hydronephrosis by use of abdominopelvic computed tomography , a painless , hard
, solid lesion was found on the right testis .
the patient underwent radical orchiectomy , and the pathologic examination revealed an adenocarcinoma of the rete testis .
multiple metastases were present at the time of diagnosis .
the patient received combined chemotherapy after the surgery but lived only 8 months after the initial diagnosis . |
functional fitness not only accounts for the traditional physical fitness parameters such as muscle strength , cardiorespiratory endurance , and flexibility , but also includes balance . however , it is well known that , with age , functional fitness declines , resulting in a reduced ability to perform activities such as rising from a chair or climbing stairs , which can be viewed as functional components of activities of daily living ( adl ) performance , and may eventually compromise the ability to perform adl [ 35 ] .
in addition , approximately a third of community - dwelling older adults fall at least once a year , and over 30% of fallers suffer injuries requiring medical attention .
strategies to prevent or attenuate the decline in physical function and balance are important for promoting independence in the elderly and thereby enhancing quality of life .
research over the past two decades clearly shows that regular physical exercise is effective for maintaining and promoting health , physical fitness , and functional independence in older adults , especially in terms of endurance , muscular strength , flexibility , and balance .
physical training programs can be undertaken in the clinic , commercial fitness centers or gymnasiums , in the community or at home .
in contrast to clinic and gym - based exercise , community- and home - based exercise does not require special facilities or expensive equipment .
moreover , community- and home - based exercise is capable of reaching a broad audience and , importantly , the community setting provides an opportunity for social contact and support which may have an array of benefits , especially for those living in regional and rural settings . in order to maximize compliance to community- and home - based programs
thus , community- and home - based exercises have become increasingly popular as an alternative to gym - based resistance exercise .
providing feedback and prompting have been shown to be effective in increasing and maintaining physical activity as well as other positive health behaviors .
moreover , feedback may also enhance the exercise response which may prove valuable in enhancing recovery of functional abilities after injury or illness [ 11 , 12 ] .
however , a paucity of information is currently available regarding the effects of feedback on muscular performance and functional mobility improvement in older adults undertaking resistance training in the community- and home - based setting .
hasegawa and tomiyama have previously reported , that in middle - aged and older persons participating in a community- and home - based setting , periodic feedback enhanced muscle performance compared to those not receiving feedback .
consequently , the purpose of this study was to confirm and extend these findings by examining the effects of periodic task - specific test feedback on not only muscle performance but also functional mobility in older adults undertaking resistance training with elastic bands in the community- and home - based setting .
in response to a public relations magazine advertisement , 80 older adults from tokai city , aichi prefecture , japan , volunteered to participate in the study .
inclusion criteria were 65 years of age , community residing , functionally independent , and able to perform physical exercise .
participants were excluded if they had been advised by their physician to refrain from exercise . prior to acceptance into the study , a brief health examination was performed by an occupational therapist and questionnaires regarding medical history were completed .
current presence of disease was determined using self - reported physician - diagnosed disease information .
three others were excluded from the study because they had scheduling conflicts , transportation issues , or no further interest in joining the study .
the remaining 75 participants ( 65 to 83 years ) were included in this study .
they were divided into a muscular performance feedback group ( mpg ) or a functional mobility feedback group ( fmg ) by their residential area ( the two groups were from two separate communities within the same city ) .
both groups participated in an identical 9-week community- and home - based resistance exercise program .
all participants were asked to not alter their diet or physical activity patterns for the duration of the study .
twenty - three participants did not complete follow - up measures due to travel - related reasons ( n = 18 ) and medically - related problems ( low back pain , n = 2 ; dizziness , n = 1 ; upper respiratory tract infection , n = 2 ) .
regarding baseline characteristics , there was no difference between those who dropped out and those who completed the study within each group for age , weight or any functional fitness measure , with the only difference being in the mpg with more women than men dropping out ( due to travel ) and hence height of those who dropped out was lower than those who completed .
the ethics committee of seijoh university approved the study and all participants provided written informed consent .
instruction and progression of the exercise routine occurred at a community center once per week and participants were asked to exercise an additional two times each week at home .
the community - based exercise sessions consisted of 15 min of warmup , 60 min of resistance exercise , and 15 min of cooldown .
pictorial guidebooks were provided to all participants in order to assist them to perform the exercises correctly .
the home - based exercise program also consisted of the same resistance and stretching exercises .
participants were asked to record exercises they performed and submit a diary every week while attending the exercise classes . in order to train all major muscle groups ,
resistance exercises were prescribed as a combination of 3 upper body exercises , 6 lower body exercises , and 2 trunk exercises performed using an elastic resistance band ( thera - band , hygenic , usa ) .
exertion was rated using borg 's rate of perceived exertion ( rpe ) scale [ 15 , 16 ] .
participants were instructed to start resistance exercises at an intensity level of 13 on the rpe scale and then to progressively increase resistance to a level of 15 to 17 .
participants were instructed to progressively increase resistance every two to four weeks by advancing to the next color of elastic band ( lower to higher resistance of bands in order : red , green , and blue ) or shortening the initial length of the band for increased resistance ( figure 2 ) . stretching exercises consisted of eight upper body exercises and seven lower body exercises .
the participants were asked to stretch to the point where they felt moderate tension without feeling pain in joints or muscles .
fortnightly assessments were performed by testers for three performance tests : arm curls , chair stands in 30 seconds , and the timed up and go ( tug ) test .
task specific feedback , which is extrinsic or augmented information provided to a performer in regard to a specific task with the goal to enhance future performance of that task , was provided by the testers ( not instructors ) who were blinded to the participant 's previous results .
mpg received immediate test feedback ( verbal and written ) only on the arm curls and chair stand test while fmg received immediate feedback only for the tug .
in addition to the participant 's results for the respective tests ( muscle performance or balance ) , verbal feedback from the tester included statements ( in front of other participants ) such as you have improved in your performance and it shows in your current score and keep up the exercise and you are going to improve your performance .
anthropometrics , physical symptoms by questionnaire , muscle performance , and functional mobility were evaluated at baseline and followup .
participant 's height and weight were assessed and body mass index ( bmi ) was calculated as body weight ( kg ) divided by the square of height ( m ) .
upper body muscle performance was assessed using the 30-second arm curl test ( arm curl ) [ 18 , 19 ] . on a signal , participants were instructed to flex and extend the elbow of the dominant hand , lifting a weight dumbbell ( men : 8-lbs [ 3.6 kg ] , women : 5-lbs [ 2.3 kg ] ) through the complete range of motion , as many times as possible in 30 seconds .
a practice trial of one or two repetitions was given , followed by two test trials .
the score was the number of repetitions completed with the best performance used for analysis .
lower body muscle performance was assessed using the 30-second chair stand test [ 18 , 19 ] .
the participant 's arms were crossed at the wrists and held against the chest . on a signal
, participants rose to a standing position from a chair and then returned to a seated position and continued to complete as many full stands as possible in 30 seconds .
a practice trial of one or two repetitions was given , followed by two test trials .
the score was the total number of stands executed correctly with the best performance used for analysis .
functional mobility was assessed using the 8-foot timed up and go ( tug ) test [ 1820 ] .
participants sat in a chair with their hands on their thighs and feet flat on the floor . on a signal
, participants stood from the chair without pushing off with the arms , walked as quickly as possible around a cone placed 8-feet ( 2.44 m ) ahead of the chair , and returned to a fully seated position in the chair .
participants walked through the test one time as a practice and then were given two test trials with the best performance time used for analysis .
comparisons between mpg and fmg at baseline were performed using an independent student 's t - test or chi - square test as appropriate .
the effect of the intervention was determined using a repeated measures analysis of covariance ( ancova ) adjusted for gender and within group changes by a paired t - test .
all tests were two tailed and a p value of less than 0.05 was considered statistically significant .
there were no differences between groups at baseline for age , height , weight , and prevalent disease ( table 1 ) or muscle performance and functional mobility ( table 2 ) .
the average adherence rates in the exercise class at the community center were 92 13% for mpg and 87 12% for fmg .
both groups performed home - based exercises in addition to community - based classes for a total of 2.7 1.3 days / week in mpg and 2.1 1.0 days / week in fmg .
no differences were observed between the two groups in adherence or home - based training frequency ( table 1 ) . there were no accidents or injuries during the exercise classes at the community center or at home .
there was a significant group time interaction for the arm curl ( f = 15.2 ) and chair stand test ( f = 5.2 ) with mpg improving more than fmg , while fmg improved more than mpg for the tug ( f = 4.1 ) ( table 2 ) . in the mpg , the muscle performance measures for the arm curl and chair stand test
increased by 31.4% ( p = 0.001 ) and 33.7% ( p < 0.001 ) , respectively , while tug performance time was reduced by 3.5% ( p < 0.001 ) . in the fmg ,
arm curl and chair stand test significantly increased by 15.9% ( p < 0.001 ) and 24.9% ( p
< 0.001 ) , respectively , while time to perform the tug was reduced by 9.7% ( p = 0.049 ) .
maintaining or enhancing muscle performance and functional mobility in older persons is critical for undertaking daily activities and for sustaining an appropriate quality of life . in the current study
, we found significant improvements in muscle performance and functional mobility following a 9-week community- and home - based exercise regimen undertaken with elastic bands .
importantly , gains were partly dependent on fortnightly test feedback suggesting that this is an important component of the training session in order to maximize gains resulting from the program .
the muscular performance results are in agreement with other studies that have used elastic bands in a community setting .
reported significant increases of 16% in arm curl and 18% in chair stand performance in older women who participated in a 12-week exercise program ( 3 d / wk ) using elastic bands .
similarly , a 23% and 19% increase in arm curl and chair stand performance , respectively , was reported by rogers et al . who enrolled older women in a 4-week ( 3 d / wk ) elastic band resistance exercise program .
however , gains resulting from home - based programs may not be of the same magnitude as that of supervised gym- or community - based programs with yamauchi et al .
reporting improvements of 18% in the arm curl test and only 6% in the chair stand test for older adults following 12 weeks of a home - based exercise program .
in the current study , the fmg had an average improvement for the arm curl test of 16% , which is comparable to other studies with a similar training period . however , the mpg improvement on this task was double that of the fmg with gains of 31% .
these results suggest that task - specific feedback of serial testing may be an effective strategy to enhance performance in older persons . for the chair stand test
, there was also a significant difference between the two feedback groups with the task - specific group enhancing their performance by ~34% while the fmg increased by ~25% .
moreover , the results in mpg were higher than previous studies with comparable training programs [ 9 , 21 , 22 ] .
the improvements in the chair stand test are particularly important given the role that lower body muscle performance , as does balance , plays in maintaining physical function .
similarly , for the timed up and go test , which was used to assess functional mobility in our cohort , improvement was greater for the fmg who decreased their performance time by ~10% whereas the mpg experienced a reduction of only 3.5% .
this suggests that improvements in functional mobility are also associated with the use of feedback in older persons .
these results indicate that the magnitude of improvement in muscular function and functional mobility are associated with the feedback provided and would be beneficial to older persons in order to maximize program - related gains .
the importance of data feedback has been previously described by mihalko et al . who reported that individual fitness feedback influences exercise attendance .
in addition , it is suggested that relatedness is intensified in group work and exercise programs in which the participant 's interaction is increased . moreover , bourbonnais et al . reported that treatment of the lower limb in stroke patients based on muscle force - feedback produced an improvement in gait velocity , while feedback has also been found to improve pelvic floor muscle training in those with urinary incontinence . in athletes , providing verbal feedback has been associated with a modest enhancement in muscle performance and tuck - jump performance , although not in time trial cycling .
feedback can be classified as either intrinsic or extrinsic with task - specific feedback categorized as knowledge of results .
indeed , the observations of leventthal have documented the importance of providing relevant information in order to encourage an action .
it may well be that awareness of muscular performance or functional mobility improvement and the encouragement provided by the tester ( in delivering the feedback in a face - to - face situation ) enhanced the participant 's self - efficacy for these respective tasks and their intrinsic motivation [ 33 , 34 ] and this contributed to task - specific performance differences in the mpg and fmg .
in addition , the feedback may have reinforced or enhanced the participant 's goal - setting which in turn enhanced their intrinsic motivation .
the finding that feedback of performance may have increased motivation is consistent with previous literature and cognitive evaluation theory which proposes that , when feelings of competence are enhanced , motivation increases .
motivating older adults to perform exercises on a regular basis is an important factor in maintaining the effects of exercise . in the current study
, we held community - based exercise classes once a week in addition to the home - based exercise program 2 days per week .
the adherence to the community - based classes was over 85% and the frequency of the community- and home - based exercise was over 2 days per week .
most participants mentioned that they were glad to make new friends and enjoyed exercising as a group in the community setting .
this supports previous findings that community - based exercise classes have positive psychological effects as well as physical effects on community - dwelling older adults .
after completion of this 9-week program , participants continued to exercise and currently attend exercise classes twice a month .
enhanced self - efficacy resulting from participation may be one factor that contributed to the participation rates during the study period as well as with the participation postintervention .
given the homogeneity of participants in the study ( age , socioeconomic status , and geographical location ) , caution should be taken when extrapolating our results to all older community - dwelling persons .
moreover , the participants were not randomized to the treatment conditions as it was not possible to blind participants to the exercise program , leaving them vulnerable to a variety of tester and subject effects that may influence test results and introduce bias in our findings .
our program was also of a relatively short - term nature and it is unclear if differences based on feedback would exist with a longer program , such as 6 or 12 months .
nevertheless , our results show , even within a relatively short training period , that task - specific test feedback appears to have a beneficial effect in enhancing physical performance , and , regardless of the underlying mechanism for the improvement , this is an important practical outcome for those involved in the exercise training of older persons .
a future direction would be in determining if task - specific feedback is a valuable strategy to enhance performance in those disabled or recovering from illness / disease or injury .
in addition , a more extensive test battery could be employed to measure additional aspects of functional and ambulatory ability .
lastly , it would be of interest to determine if performance was enhanced by motivation , whether it was motivation to train harder or to perform the tests or to the combination .
this study was designed to determine the efficacy of test performance feedback to improve muscle performance and functional mobility in older adults . following the 9-week community- and home - based intervention ,
a significant interaction was noted for the muscle performance and functional mobility tests suggesting that periodic test feedback during resistance training may enhance task - specific physical performance in older persons . providing regular feedback on test performance in the community and ,
if possible , the home exercise setting may facilitate gains in muscle and balance performance resulting in enhanced physical function and a greater safety margin for functional thresholds . | the purpose of this study was to determine the effects of periodic task - specific test feedback on performance improvement in older adults undertaking community- and home - based resistance exercises ( chbre ) .
fifty - two older adults ( 6583 years ) were assigned to a muscular perfsormance feedback group ( mpg , n = 32 ) or a functional mobility feedback group ( fmg , n = 20 ) .
both groups received exactly the same 9-week chbre program comprising one community - based and two home - based sessions per week .
muscle performance included arm curls and chair stands in 30 seconds , while functional mobility was determined by the timed up and go ( tug ) test .
mpg received fortnightly test feedback only on muscle performance and fmg received feedback only on the tug .
following training , there was a significant ( p < 0.05 ) interaction for all performance tests with mpg improving more for the arm curls ( mpg 31.4% , fmg 15.9% ) and chair stands ( mpg 33.7% , fmg 24.9% ) while fmg improved more for the tug ( mpg-3.5% , fmg-9.7% ) .
results from this nonrandomized study suggest that periodic test feedback during resistance training may enhance task - specific physical performance in older persons , thereby augmenting reserve capacity or potentially reducing the time required to recover functional abilities . |
participants were chinese women with a hong kong permanent identity card who were :
aged 1865 years;able to read chinese or english;not pregnant . able to read chinese or english ;
the study was approved by the institutional review board of the university of hong kong .
a total of 379 women were recruited in a private medical center in hong kong .
those who consented to take part were given a set of questionnaires to be completed while waiting to see the doctors , and these completed questionnaires were returned to the researcher directly .
a validated scale was used to assess women s health perception , knowledge about cervical screening and the risk factors of cervical cancer .
the original questionnaire was developed by seow and associates in singapore.18 the scale was translated into chinese and used in studies in hong kong.13,19,20 minor modifications were made from a telephone survey to a self - administered questionnaire for this study .
the questionnaire consisted of the following sections :
health perception and health behavior;general knowledge on cervical screening , cervical cancer and the risk factors;socio - demographic characteristics . health perception and health behavior ; general knowledge on cervical screening , cervical cancer and the risk factors ; socio - demographic characteristics .
section a consisted of 11 questions including how the respondents perceived their own health , perceived control over health , their concerns about family / relatives health and the influence of family / relatives health .
section b consisted of 9 items to test the participants on their general knowledge of cervical screening and cervical cancer , and 9 items on the risk factors .
cronbach s alphas were 0.62 , 0.60 and 0.63 for general knowledge , knowledge on risk factors and total knowledge scales , respectively .
section c consisted of demographics data including age , education level , monthly household income , marital status , number of children , and regular sources of health care .
pearson s correlation was examined for the relationships between cervical screening and age , education , income , knowledge , and health perception .
the test was used to examine the relationships between screening and non - parametric variables .
differences between screening and non - screening for knowledge were demonstrated by independent t - test .
logistic regression was used to indicate health perception , knowledge , and risk of cervical cancer as well as demographic factors associated with cervical cancer screening .
factors included in the logistic model were age , education , income , perceived health , perceived controlled over health , concerns about family / relatives health , and health behaviors influenced by their health , general knowledge on cervical screening and cancer , and knowledge on risk factors .
the hosmer and lemeshow test of goodness - of - fit was used to test the overall fit of the logistic model .
participants were chinese women with a hong kong permanent identity card who were :
aged 1865 years;able to read chinese or english;not pregnant . able to read chinese or english ;
the study was approved by the institutional review board of the university of hong kong .
a total of 379 women were recruited in a private medical center in hong kong .
those who consented to take part were given a set of questionnaires to be completed while waiting to see the doctors , and these completed questionnaires were returned to the researcher directly .
a validated scale was used to assess women s health perception , knowledge about cervical screening and the risk factors of cervical cancer .
the original questionnaire was developed by seow and associates in singapore.18 the scale was translated into chinese and used in studies in hong kong.13,19,20 minor modifications were made from a telephone survey to a self - administered questionnaire for this study .
the questionnaire consisted of the following sections :
health perception and health behavior;general knowledge on cervical screening , cervical cancer and the risk factors;socio - demographic characteristics . health perception and health behavior ; general knowledge on cervical screening , cervical cancer and the risk factors ; socio - demographic characteristics .
section a consisted of 11 questions including how the respondents perceived their own health , perceived control over health , their concerns about family / relatives health and the influence of family / relatives health .
section b consisted of 9 items to test the participants on their general knowledge of cervical screening and cervical cancer , and 9 items on the risk factors .
cronbach s alphas were 0.62 , 0.60 and 0.63 for general knowledge , knowledge on risk factors and total knowledge scales , respectively .
section c consisted of demographics data including age , education level , monthly household income , marital status , number of children , and regular sources of health care .
pearson s correlation was examined for the relationships between cervical screening and age , education , income , knowledge , and health perception .
the test was used to examine the relationships between screening and non - parametric variables .
differences between screening and non - screening for knowledge were demonstrated by independent t - test .
logistic regression was used to indicate health perception , knowledge , and risk of cervical cancer as well as demographic factors associated with cervical cancer screening .
factors included in the logistic model were age , education , income , perceived health , perceived controlled over health , concerns about family / relatives health , and health behaviors influenced by their health , general knowledge on cervical screening and cancer , and knowledge on risk factors .
the hosmer and lemeshow test of goodness - of - fit was used to test the overall fit of the logistic model .
385 were eligible and agreed to participate giving a response rate of 92% and 35 found to be ineligible and/or unwilling to participate .
more than half of the participants ( n = 194 , 51.2% ) were educated to tertiary or higher level , almost half of them ( n = 183 , 48.3% ) had received secondary education and only 2 ( 0.5% ) had received no formal education .
54.4% ( n = 206 ) of the participants were married and 41.4% ( n = 157 ) were single .
sixty - nine ( 18.2% ) of the participants had never attended screening and 33 ( 8.7% ) said that they never thought of having such a test .
table 1 shows the frequencies of health perception on self and families , age and education , and their correlations with cervical screening .
screening was also significantly correlated with knowledge on cervical screening , cervical cancer and risk factors ( table 1 ) .
the mean total knowledge score was higher for screening than non - screening ( t = 6.47 , p < 0.001 ) .
do nt know answers to each item on general knowledge and knowledge of risk factors .
only about one - fifth of the women ( 19.3% ) received a full score on general knowledge and only 0.3% had a full score on the risk factors .
high percentages of women reported they did not know whether some items were risk factors ( 17.9% to 41.2% for 5 out of the 9 risk factors ) .
more than half of the participants ( 66.2% ) considered themselves healthy and only 5.5% reported themselves as not healthy .
less than one - third of the participants ( 28.5% ) believed that they did not have control of their health . very high percentages ( 95.3% ) were concerned about family or relatives health and half of the participants reported that their health behaviors were influenced by family or relatives health .
cervical screening was significantly correlated with health perception except when influenced by family / relatives health ( table 1 ) .
hosmer and lemeshow goodness - of - fit test indicated a good fit of the logistic model ( = 4.734 , df = 8 , p = 0.786 ) .
nagelkertke s r was 0.608 which was an index of approximation to r from ols regression .
the significant factors identified were age , education , control over their own health , and knowledge on risk factors , after controlling for all other factors in the model .
younger participants ( 37 years ) , had attained tertiary education , perceived having control over their own health , and had better knowledge of risk factors were more likely to attend cervical screening .
385 were eligible and agreed to participate giving a response rate of 92% and 35 found to be ineligible and/or unwilling to participate .
more than half of the participants ( n = 194 , 51.2% ) were educated to tertiary or higher level , almost half of them ( n = 183 , 48.3% ) had received secondary education and only 2 ( 0.5% ) had received no formal education .
54.4% ( n = 206 ) of the participants were married and 41.4% ( n = 157 ) were single .
sixty - nine ( 18.2% ) of the participants had never attended screening and 33 ( 8.7% ) said that they never thought of having such a test .
table 1 shows the frequencies of health perception on self and families , age and education , and their correlations with cervical screening .
screening was also significantly correlated with knowledge on cervical screening , cervical cancer and risk factors ( table 1 ) .
the mean total knowledge score was higher for screening than non - screening ( t = 6.47 , p < 0.001 ) .
do nt know answers to each item on general knowledge and knowledge of risk factors .
only about one - fifth of the women ( 19.3% ) received a full score on general knowledge and only 0.3% had a full score on the risk factors .
high percentages of women reported they did not know whether some items were risk factors ( 17.9% to 41.2% for 5 out of the 9 risk factors ) .
more than half of the participants ( 66.2% ) considered themselves healthy and only 5.5% reported themselves as not healthy .
less than one - third of the participants ( 28.5% ) believed that they did not have control of their health . very high percentages ( 95.3% ) were concerned about family or relatives health and half of the participants reported that their health behaviors were influenced by family or relatives health .
cervical screening was significantly correlated with health perception except when influenced by family / relatives health ( table 1 ) .
table 3 shows odds ratios of the predictors on cervical screening with logistic regression . hosmer and lemeshow
goodness - of - fit test indicated a good fit of the logistic model ( = 4.734 , df = 8 , p = 0.786 ) .
nagelkertke s r was 0.608 which was an index of approximation to r from ols regression .
the significant factors identified were age , education , control over their own health , and knowledge on risk factors , after controlling for all other factors in the model .
younger participants ( 37 years ) , had attained tertiary education , perceived having control over their own health , and had better knowledge of risk factors were more likely to attend cervical screening .
this study reports the determinants of cervical screening among demographic variables , knowledge on cervical screening and cancer and health perception . in this study , females aged 37 years or younger were more likely to attend cervical screening .
however , statistics in hong kong showed that the average age - specific incidence rate of cervical cancer increased sharply after 55 years of age with a peak at aged 65 over the period of 2003 to 2007 , and the cervical cancer was the eighth most common cause of registered death in females in 2007.9 more active health promotion and education efforts should target females approaching 40 years and older .
the 3 questions most often answered correctly in the general knowledge section were :
it has to be done routinely to be effective;screening was still needed even if they had not had sex for years;getting old was not a reason to not attend cervical screening .
it has to be done routinely to be effective ; screening was still needed even if they had not had sex for years ; getting old was not a reason to not attend cervical screening .
however , many participants thought that once women reach menopause , cervical screening was not needed .
these apparent contradicting findings showed that participants might define menopause as an indicator of being old enough to be spared from cervical screening .
education on menopause and screening should be emphasized by doctors and nurses engaged in health - promoting activities .
our findings also showed that participants were aware of the importance of having regular screening and a healthy lifestyle .
however , quite a number of participants did not have accurate knowledge on risk factors and had a higher chance of failing to attend cervical screening .
such knowledge deficits probably explain why a significant number of women do not take advantage of cervical screening even though its efficacy is well known .
these results also lend support to a recent qualitative study among african americans which found that personal beliefs on screening , perception of vulnerability , and providing correct information might increase screening rates.21 the majority of participants correctly identified that having a previous abnormal cervical smear was one of the risk factors .
the next most commonly identified risk factors were : having sex at early age ; many different sexual partners ; and sexually transmitted disease .
this finding was somewhat different from reports in the united kingdom in which only 14% of the population sample were aware of a link between cervical cancer and sexual transmission.22 another qualitative study on women who took part in human papillomavirus ( hpv ) testing also found that many participants did not recognize the link between cervical cancer and sex or sexually transmitted disease.23 authors in those earlier studies conjectured that generally women were aware of the link between cervical cancer and sexual activity but did not clearly conceptualize the linkage between cervical cancer and hpv .
they thus emphasized the role of healthcare providers in providing clear and consistent information of the sexually transmitted nature of cervical cancer .
the findings in this current study agreed with kemm and close s concept of internal locus of control.24 most of the participants ( 71.5% ) perceived they have control of their health , which concurred with the high cervical screening .
this group of women is more likely to take the initiative to engage in health - promoting activities such as cervical screening .
women s knowledge on cervical cancer and risk factors affected their perception of the severity of illness and their own susceptibility to it .
health care providers need to take into account women s knowledge and health perception when developing a cervical screening program .
accurate information must be readily available to women to increase their knowledge , especially on the risk factors associated with cervical cancer , to guide them on the importance of regular screening .
health professionals also need to further explore women s perceived ability to their control health and the effect on changing their beliefs of clearer information on the treatability and significant reduction in mortality and morbidity with early detection .
women educated to higher level are more likely to have a better knowledge of cervical screening and cervical cancer , leading to their participation in cervical screening .
women with a higher level of education are more likely to gather new information by themselves or have a faster channel to reach up - to - date information .
more active promotion strategies ought to be channeled through media that are appropriate to reach women with less education .
publicity through different means should be considered , such as tv , radio commercials and newspapers .
health education through trained lay persons in community centers could also be considered as this has been reported to be an effective method in several studies.2527 this study has limitations on the design and sampling .
it was a cross - sectional study with some longitudinal data for identifying prediction of screening at follow - up .
there were a number of issues where this sampling methodology could be improved :
participants were recruited with convenience sampling and showed to have higher percentage with tertiary education compared to the general populations which raises some questions about generalizing these findings to the greater hong kong population.screening was self - reported data without comparison to any objective medical records and thus the identified factors may be different from those related to the actual cervical screening.the questionnaires did not assess participants knowledge on human papilloma virus which was important information relating to cervical screening .
participants were recruited with convenience sampling and showed to have higher percentage with tertiary education compared to the general populations which raises some questions about generalizing these findings to the greater hong kong population .
screening was self - reported data without comparison to any objective medical records and thus the identified factors may be different from those related to the actual cervical screening .
the questionnaires did not assess participants knowledge on human papilloma virus which was important information relating to cervical screening .
the study identifies that the lack of knowledge particularly on risk factors of cervical cancer and the importance of perceived control over health are significant factors influencing cervical cancer screening among chinese .
this study can contribute to the development of a multi - strategy health program that will take into account women s perceptions of cervical screening and cancer and emphasize a non - stigmatizing approach to encourage chinese women to attend cervical screening .
the role of nurses in cervical screening programs needs to be further explored as public health nurses are commonly the ones who take smears.28 women need to accept the correct concept at an earlier stage , thus normalizing the behavior of taking part in health - promoting activities such as cervical screening . | purpose : cervical cancer screening has been consistently shown to be effective in reducing the incidence rate and mortality from cervical cancer . however , cervical screening attendance rates are still far from satisfactory in many countries .
strategies , health promotion and education programs need to be developed with clear evidence of the causes and factors relating to the low attendance rate . the study aims to assess the prediction of cervical screening attendance rate by chinese women s knowledge about cervical cancer and cervical screening as well as their perception of health.patients and methods : a survey with self - reported questionnaires was conducted on 385 chinese women recruited from a community clinic in hong kong .
participants were chinese women , hong kong residents , aged 1865 years , able to read chinese or english , and were not pregnant.results:women aged 37 years or less , with at least tertiary education , who perceived having control over their own health and had better knowledge on risk factors , were more likely to attend cervical cancer screening .
many participants had adequate general knowledge but were unable to identify correct answers on the risk factors.conclusion:health promotion efforts need to focus on increasing women s knowledge on risk factors and enhancing their perceived health control by providing more information on the link between screening and early detection with lower incidence rates and mortality from cervical cancer . |
we report on the prevalence of different antimicrobial - drug resistance profiles among danish salmonella ser .
furthermore , we show results indicating that a dt104 outbreak strain lost resistance genes by shifting to the sgi1-b variant during the outbreak . consequently , the outbreak included isolates with both the acssut and the asu phenotype .
typhimurium dt104 and 16 dt104b were obtained from persons with clinical disease in denmark . several outbreaks and clusters of dt104
the 323 isolates were characterized by mlva ( 97% of isolates ) , pfge ( 78% ) , and susceptibility testing ( 100% ) .
susceptibility to a standard panel of antimicrobial agents ( www.danmap.org ) was determined by microbroth dilution and interpreted according to clinical and laboratory standards institute ( clsi ) guidelines ( 9 ) , except for ciprofloxacin ( > 0.125 g / ml was used as breakpoint ) .
( 10 ) and pfge , according to the pulsenet protocol ( 11 ) , were performed and analyzed as previously described ( 5 ) . to obtain a cluster - independent data set
, we reduced the 323 isolates to 146 unique strains on the basis of the mlva and resistance profile ( i.e. , with different mlva and/or resistance profile ) ( table 1 ) .
* one isolate per multilocus variable number of tandem repeats analysis ( mlva ) and antimicrobial resistance profile is represented in the data set of 146 strains with unique types . a , ampicillin ; c , chloramphenicol ; s , streptomycin ; su , sulfonamides ; t , tetracycline ; tm , trimethoprim .
genotype determined by the sgi1-related pcr assays described in the text ( 1 isolate per unique type ) ; see table 2 for selected details .
additional antimicrobial - drug resistances : nalidixic acid and ciprofloxacin ( 22 strains ) , amoxicillin / clavulanic acid ( 3 strains ) , gentamicin ( 1 strain ) .
only a small subset of 10 acssut strains was tested by pcr for the presence of sgi1 genes .
additional antimicrobial - drug resistances : nalidixic acid , ciprofloxacin , and gentamicin ( 1 strain ) .
strains with phenotypes lacking resistance to > 1 of the antimicrobial agents ( acssut ) typical for sgi1 were further investigated for the presence of sgi1 variants .
pcr was performed for detection of the sgi1 left and right junctions by using primers u7-l12 , lj - r1 , 104-rj , and c9-l2 ( 2 ) ; detection of the integron conserved segments ( 5-cs and 3-cs ) was done by using primers l1 and r1 ( 3 ) ; and detection of the antimicrobial resistance genes aada2 , qac / sul1 , flor , tet(g ) , and blap1 was performed by using the primers described by boyd et al .
most dt104 and dt104b isolates displayed the phenotype acssut ( table 1 ) , which is compatible with the possession of sgi1 ; 122 ( 84% ) of the 146 strains were resistant to acssut with or without additional antimicrobial drug resistance that is regarded as independent of the sgi1 ( e.g. , flouroquinolones ) .
three percent of the strains were additionally resistant to trimethoprim ( tm ) and therefore likely to possess the sgi1-a variant , and 6% of the strains were fully sensitive ( all of these were dt104 ) .
the remaining strains had a variety of resistance profiles , including profiles that comply with the descriptions for variants sgi1-b and sgi1-c ( table 1 ) .
this outbreak was shown to be caused by raw beef , served as carpaccio at a restaurant in a limited period of 4 weeks ( 6 ) . the isolates displayed variation with all applied typing methods : phage typing , mlva , pfge , and resistance profile ; 33 isolates were typical mdr dt104 ( acssut ) with 1 pfge and mlva profile .
one isolate differed in phage type ( nontypeable ) and had a variant of the pfge profile that differed by 1 band .
three isolates had different mlva types ( variation in 1 of the 5 loci ) ( 6 ) .
detection of sgi1-related genes and regions by pcr showed that the acssut isolates were positive in all pcrs as expected for sgi1 ( table 2 ) , whereas the 3 asu isolates were positive for the left / right junction , but only 1 integron and pse-1 and qac / sul1 , in accordance with the phenotype and the sgi1-b variant , were positive ( 3,12 ) .
the most likely mechanism for the creation of variants of sgi1 is considered to be homologue recombination between identical dna segments ( 12 ) .
recombination could have taken place in the intestine of the infected animal , during processing or storage of the beef , or during infection of the patients .
it is less likely that the outbreak was caused by multiple unrelated strains of dt104 because both the pfge and mlva types , including the variants , were distinctive and unique to this outbreak . *
mlva , multilocus variable number of tandem repeats analysis ; pfge , pulsed - field gel electrophoresis .
in addition to the 3 isolates related to the carpaccio outbreak , 21 human salmonella dt104 isolates in 20032006 had the phenotype asu .
all of these had the same pfge profile ( worldwide , the typical dt104 pfge profile ) ( 13,14 ) but 3 different mlva profiles ( represented by 18 isolates , 2 isolates , and 1 isolate each ) .
the cluster of 18 isolates had the phenotype asu and the mlva profile 133 ( table 2 ) , and most of these isolates were likely part of an outbreak related to pork from denmark ( december 2003march 2004 ) .
another cluster of 3 isolates was received in the laboratory within a 3-day period in june 2003 from patients living in the same region .
the phenotype of 2 of these isolates was asu , and 1 was acssut ( table 2 ) .
no outbreak investigation was carried out at that time , and we can only speculate that this is a similar occurrence of shift in antimicrobial - drug resistance during an outbreak .
a cluster of 9 isolates with mlva 443 occurred late in 2005 and in january 2006 ; most patients lived in a specific geographic area .
eight of these isolates had the phenotype ssu ( equivalent to sgi1-c ) , and 1 isolate , which appeared late in the cluster , was assuttm .
pcr confirmed the presence in all 9 mlva 443 isolates of the left and right junction , the typical sgi1 genes conferring resistance to ssu , and 1 cassette of class 1 integrons ( lacking the pse1-associated cassette ) ( table 2 ) .
, it seems likely that the variation in phenotype was caused not by variation in sgi1 but rather by acquisition of antimicrobial - drug resistance genes independent of sgi1 ( e.g. , on plasmids ) .
most of the salmonella dt104 isolates from patients in denmark had the typical acssut phenotype ; however , a small fraction ( 6% ) of dt104 isolates were fully sensitive and correspondingly negative for all sgi1-related genes and regions tested for by pcr .
approximately 10% of the isolates had a different phenotype , and most of these had an antimicrobial - drug resistance profile corresponding to sgi1-a , -b , or c , which could be verified by pcr assays mapping parts of sgi1 .
a number of clusters and outbreaks occurred in the study period , and shifts in antimicrobial - drug resistance phenotype and genotype seem to have taken place in several outbreaks .
these shifts were most clearly seen in the carpaccio outbreak ( 6 ) , in which all evidence pointed at a single source outbreak , though the applied typing methods showed some variation among isolates , including a shift from sgi1 to sgi1-b in 3 of 40 isolates .
we therefore conclude that variability of both genotypic and phenotypic characteristics can be expected during a foodborne outbreak ; this variability should be taken into consideration when defining outbreak - related cases . | the prevalence of different antimicrobial resistance profiles and variants of the salmonella genomic island 1 ( sgi1 ) was reported for salmonella enterica serovar typhimurium dt104 strains isolated from patients in denmark .
variation in antimicrobial resistance and corresponding changes of sgi1 were shown among isolates from a foodborne outbreak . |
the connection may occur as a result of iatrogenic causes during root canal treatment ( at the level of the floor of the cameral cavity or at different levels of the root ) or during prosthetic treatment for postcanal penetration .
prognoses for perforated roots depend on the time lapsed before the perforation is sealed , the localization and size of the perforation , and the quality of the sealing material used .
mineral trioxide aggregate ( mta ) has been used in a variety of surgical and nonsurgical endodontic applications .
several studies have demonstrated that mta offers good - quality sealing of dentine [ 27 ] . unlike other commonly used materials such as glass ionomer and reinforced zinc oxide - eugenol cement ( super - eba ; harry j ; bosworth company , skokie , il , usa )
mta is commonly accepted as the best choice for root perforation treatment . on the other hand , raldi et al
. demonstrated that the surface of dentin irradiated by an er : yag laser has better cell adhesion than mta surfaces and unlased dentinal surfaces .
furthermore , baraba et al . showed that an er : yag laser , used under specific irradiation conditions , is more efficient than mechanical drills for enamel and dentin ablation .
. demonstrated that the use of acid etching is mandatory to obtain good adhesion and retention with resin composites .
the purpose of this study was to evaluate the capacity of the er : yag laser to improve the quality of mta sealing and to compare the microleakage of roots sealed using mta versus those sealed by mta assisted by an er : yag laser .
forty - two recently extracted human mono - roots were used ( mainly canines and second premolars ) , stored in distilled water at 4c .
two cavities were prepared on the opposite sides of the cervical part of each root , using a standard diamond bur of 1.7 mm diameter and 2 mm depth with a stopper ( meissinger 828g017 , hager - meisinger gmbh , neuss germany ) ( figure 1 ) .
the laser apparatus was an er : yag laser ( wavelength : 2940 nm ; fidelis plus ii , fotona , slovania ) .
laser settings were 200 mj , 10 hz , ssp mode ( 50 sec ) , energy density per pulse : 44,1 j / cm , water spray : 10 ml / min , air : 20 ml / min , noncontact hand piece ( model r 02-c ) , beam diameter of 0.7 mm , and distance to target : 79 mm . on each root , one of the cavities
a total number of 39 teeth were used for sealing measurements , and three teeth were used for sem views ( group ml ) .
one commercial brand of gray mta ( proroot ; dentsply maillefer , ballaigues , switzerland ) was used .
the surface was burnished with a b-3 condenser / ball burnisher ( analytic , synbron endo , orange , usa ) in order to improve the marginal sealing .
the roots were stored in moist conditions at 37c for one week . before mta sealing of the cavities ,
the dentine of the drilled and unlased cavities was left without any complementary treatment ( group m ) .
one week after mta sealing of the cavities and before thermocycling , the root surfaces were polished by means of sof - lex pop - on discs ( medium , 3 m , espe , usa ) under cold water , with the aim of avoiding an eventual excess of mta overhanging the border of the cavities .
next , the roots were thermocycled for 1000 cycles , for 24 h , from 5c to 55c ( willytec thermocycler v 2.9 , westerham , germany )
. two coats of an acid - resistant marine varnish ( alkydurethan varnish , trimetal , belgium ) were applied , leaving a 0.5 mm border around the edge of each cavity in order to protect the rest of the tooth from the dye solution .
the roots were immersed in 2% methylene blue solution for 12 h. great care was taken to keep the apices of the roots out of the dye solution ( the coronal parts of the teeth were held in wax to prevent the root apices from being immersed ) .
penetration of the dye was measured in millimeters using visilog 5.3 analysis software ( noesis vision , st laurent , pq , canada ) . for each cavity ,
the deepest penetration of the methylene blue dye was recorded ( d value ) after agreement of the examiners .
the measurements were calibrated by reference to the total length of the diamond bur ( 2 mm ) .
the percentage of dye penetration was calculated by dividing the deepest penetration of the dye by the total depth of the cavity ( 2 mm ) ( d/2 mm 100 = % of dye penetration ) .
percentages of dye penetration in all cavities were used for statistical analysis ( t - student ) . a normality test , using the kolmogorov and smirnov method , was performed and followed by a test for the qualitative paired data .
the comparison of means and sd of microleakage was expressed in percentage of dye penetration .
three more monoroots were used for sem analysis ( hitachi s-4700-ii fesem , tokyo , japan ) .
the aspect of the drilled dentine without any complementary treatment was compared to the aspect of the drilled and er : yag - conditioned dentine .
the mean and standard deviation value of the percentage of dye penetration in the unlased cavities filled with mta ( group m ) was 25.17 17.53% ( minimum value = 10 ; maximum value = 73 ) .
the mean value and standard deviation of the percentage of dye penetration in the group of cavities filled with mta following er : yag laser irradiation ( group ml ) was 23.91 14.63% ( minimum value = 10 ; maximum value = 58 ) ( figure 2 ) .
the mean values of microleakage and dye penetration resulting from the use of an er : yag laser to precondition dentine were the lower of the two groups ( figure 2 ) . all groups passed normality tests using the kolmogorov and smirnov method .
the difference between the percentage of dye penetration in cavities sealed with mta ( group m ) that in those sealed with mta following er : yag laser irradiation ( group ml ) was not statistically significant ( paired and two - tailed t - test ; p = 0.2519 ; t = 1.235 with 8 degrees of freedom ; 95% confidence interval ) .
the dentine drilled without any complementary treatment exhibited a smear layer covering the tubules and the total surface of the exposed dentine ( figure 3 ) . on the other hand , on the dentine conditioned by er : yag laser ,
the er : yag laser had removed the smear layer ( figure 4 ) . the tubules were totally opened .
the er : yag laser produced a selective and preferential ablation of intertubular dentine , while the peritubular dentine ( higher mineralization ) was less ablated ( figure 4 ) .
however , methylene blue is not the best choice for the measurement of the quality of sealing of root canal fillings . in our study
we used this dye because of its common use in several articles for the evaluation of microleakage of dentinal fillings [ 1517 ] .
several studies showed that the use of an er : yag laser for cavity preparation has many advantages .
er : yag laser ( 2940 nm ) conditioning of perforated dentine under an air - water spray is noncontact and decontaminating .
furthermore , according to our results and other studies , an er : yag laser beam is able to remove the dentinal smear layer and open the dentinal tubules [ 19 , 20 ] . on the other hand , the conditioning of dentin by er : yag laser irradiation can favor cells adhesion . for these reasons , we selected the er : yag laser for the dentinal conditioning of root perforation treatment .
garthner and dorn proposed the ideal characteristics of mta : simple manipulation , radio - opacity , stability in three dimensions and in a moist environment for a long period , nonresorbability , quality adhesion to dentin , and biocompatibility with desmodontal cells .
fournier and bouter recommended the consideration of three properties : cytotoxicity of filling material , long - term sealing strength , and simplicity of manipulation .
mta contains a hydrophilic powder of tricalcic silicate , iron , tricalcic aluminate , tricalcic oxide , silice oxide particles , and a bismuth oxide for radio - opacity 9 .
several studies explained that the biocompatibility of the mta may be due to the release of calcium hydroxide in water [ 3 , 10 , 26 , 27 ] .
other studies reported that mta is well tolerated by periodontal tissues [ 28 , 29 ] .
because of these qualities of tightness , mta is mainly indicated for retroapical fillings , apexification , and root perforations [ 1 , 57 , 33 ] .
the prognosis for a perforated root depends on the time elapsed before the sealing of the perforation , the localization and size of the perforation , and the sealing quality of the material used . in accordance with the previous articles ,
our results demonstrate a higher sealing ( in mean ) of mta to er : yag laser - conditioned dentine [ 35 , 36 ] versus unlased mta - sealed dentine .
our sem study shows that er : yag laser conditioning of dentine removes the dentinal smear layer and has a less ablative effect on peritubular dentine , which has higher mineral content .
the more effective sealing of mta to er : yag - lased dentine may be due to the interaction between mta components and the exposed mineral content of er : yag - lased dentine ( chemical effects )
. a second explanation may be the increase of microretention in lased dentin and dentinal opened tubules ( physical effect ) ( figure 4 ) .
an intimate contact between mta and the dentine is required , but in the literature , the necessity to remove or not the smear layer prior to mta application to exposed dentine is not clear .
there is a necessity to have a standard clinical procedure mentioning the necessity or not to remove the smear layer .
for this reason , we did not remove the dentinal smear layer from the exposed dentin prior to the application of mta .
the use of an er : yag laser beam for dentinal conditioning prior to mta filling of perforated roots or localized root crack treatments did not decrease significantly the microleakage of mta sealing when compared to the conventional use of mta filling . | the aim of this in vitro study is to compare the microleakage of a root perforation sealed with mta ( mineral trioxide aggregate ) ( group m ) to that sealed with mta following er : yag laser irradiation ( group ml ) .
forty - two recently extracted human monoroot teeth were used .
two cavities were prepared on each root surface . randomly , on each root , the exposed dentine of one cavity was irradiated prior to mta filling using an er : yag laser with the following settings : 200 mj / pulses under an air water spray , 10 hz , pulse duration of 50 sec , and 0.7 mm beam diameter .
all cavities were then sealed with mta . submitted to thermocycling and immersed in 2% methylene blue dye solution for 12 h. the penetration of methylene blue in the microleakage of cavity was observed and recorded . the mean value dye penetration in cavities sealed with mta following er : yag laser irradiation ( 23.91 14.63% ) was lower than that of unlased cavities sealed only with mta ( 25.17 17.53% ) .
no significant difference was noted .
the use of an er : yag laser beam for dentinal conditioning prior to mta filling of perforated roots did not decrease significantly the microleakage of mta sealing when compared to the conventional use of mta filling . |
in teeth with extensive coronal tissue loss , a post - and - core foundation is usually needed to retain a definitive restoration.in the past decades , a commonly employed system included a prefabricated fiber post and adhesive cement followed by core build - up .
advantages of using prefabricated adhesive posts include improved aesthetics and retrievability , as well as the need for only one treatment / visit . due to the lower elastic modulus of fiber posts which are closer to that of dentin , better stress distribution is expected .
clinical studies ; however , show that failure can occur with all restorative systems , including post - and - core systems . debonding and retention loss are common failure modes with fiber posts .
the retention properties of posts depend on their design , length , surface treatment , fitting accuracy and the resistance of luting agents to dislodging forces generated during function .
of the prefabricated posts , the retentive value of parallel posts is higher than that of tapered posts .
however , since the root canal anatomy may be neither circular in cross section nor cylindrical in length , shaping the canal using the drill supplied with the post kit frequently sacrifices the remaining dentin , especially in the apical third . therefore , tapered posts are preferred to avoid weakening the teeth that already have lost a part of their hard tissues due to caries or root canal therapy
. however , passive tapered posts are less retentive than other prefabricated post types . to improve the retentive ability of passive tapered posts ,
it is important to maintain both close adaptation of the post to the post space and a homogeneous thin cement layer .
however , due to the individual curvature and cross - sectional shape of root canals , differences between post space preparation and the root canal wall have been reported even when the corresponding preparation burs of a post system was used .with sub - optimally adapted posts , cement can fill the excess space between the post and root dentin , allowing excessive and hazardous stress to concentrate within the cement layer during functional loading .
the mechanical properties of the luting agent must therefore be considered when determining the localized stress .
voids and imperfections along the bonded interface can result in gap formation and enhance microleakage , leading to coronal and/or apical failure .
these have revealed that shrinkage stress in the resin cement layer and external loading can cause irregularities , crack formation and post debonding [ 1820 ] , which are related to increased microleakage . however , few studies have directly addressed the microleakage .
less microleakage was found in overflared canals restored by individually shaped posts using a polyethylene woven fiber ribbon ( ribbond ) compared to those restored by a preshaped glass fiber or quartz fiber post .
however , another study revealed similar microleakage values when a custom ribbond post or glass fiber post was used . to our knowledge , it remains unknown how the close adaptation of a fiber post may affect the microleakage of such systems under fatigue loading .
therefore , the aim of the present study was to compare the microleakage of teeth restored with a custom adapted fiber post and those restored with an indirect resin composite , both with and without cyclic loading .
the null hypothesis was that there was no difference between the microleakages of a prefabricated fiber post , a custom adapted fiber post with a highly - filled resin composite and a custom casting post .
sixty - five extracted single - rooted human teeth were used , none of which displayed cervical lesions , cracks , craze lines or resorption of the immature apices .
the teeth were almost straight in the 10-mm coronal portion of the root length . to ensure similarity in teeth dimensions , the buccolingual and mesiodistal dimensions and the root length
were measured using a digital caliper ( series 500 caliper , mitutoyo , tokyo , japan ) with an accuracy of 0.01 mm .
teeth within 0.5 mm of an average 7-mm buccolingual dimension and 5-mm mesiodistal dimension and within 1 mm of a 15-mm rolength were selected .
experimental group teeth were decoronated from the most coronal portion of the cementoenamel junction ( cej ) using a diamond bur in a high - speed hand piece under water spray .
each tooth was endodontically treated with a working length 1-mm shorter than the length at which a # 10 k file tip ( mani inc .
, kiohara industrial park , tochigi , japan ) passed the apical foramen , using the flat surface of a coronal section as a reference point .
canals were prepared to a master file size of 40 using the step - back technique and were flared up at the middle third and coronal third using gate glidden # 2 and # 3 ( maillefer , dentsply , ballaigues , switzerland ) , respectively .
the access cavity was filled with a non - eugenole provisional material ( cavit - g , 3 m espe , st paul , mn ) .
the apical fourth was sealed externally with a glass ionomer cement ( ionofill , lot 002033 , voco , 3 m espe ) and stored in normal saline for 72 h before post space preparation .
gutta - percha was removed with # 2 specified reamers of a dt light post ( rtd , st .
specimens were randomly allocated into three experimental groups ; namely , cast post ( cp ) , custom adapted fiber post ( cf ) and prefabricated post ( pf ) groups . a groove ( 1 mm deep , 2 mm long ) was prepared as an antirotation using a diamond bur ( # 856 - 010 , d - z co. , bern , switzerland ) to prevent post rotation during cementation .
passively fitting polycarbonate posts ( pin - jet , angelus dental solution , lonrina , brazil ) were adapted to the post space with auto - polymerizing acrylic resin ( pattern resin , lot 0505061 , gc america inc . ,
the occlusal surface was formed with two cusps of 15. a silicone index ( putty , speedex , coltene / whaledent inc .
patterns were cast in a base metal alloy ( verabond , fairfield , ca ) .
canals were cleaned with water and dried . a self - etching primer ( ed primer , lot 51169 , kuraray co. ltd . ,
osaka , japan ) was applied to each canal with a microbrush tip ( microbrush international , waterford , ireland ) and was air - thinned after 60 s. margins were light - polymerized ( coltolux 75 , coltene / whaledent ) for 60 s at 500 mw / cm and a distance of 1.0 mm .
a # 2 glass fiber post ( reforpost , angelus dental solution ) was placed in the canals and cut 3 mm above the tooth surface with a diamond disk ( edenta ag dentalprodukte , hauptstrausse , switzerland ) .
post spaces were coated with a thin layer of a separating agent ( gc gradia separator , gc corp . ,
airborne abrasion was used on the post surface with 50-m aluminum oxide particles ( shenzen co , shanghai , china ) under 3 kg / cm of pressure for 10 s at a distance of 5 cm .
post surfaces were conditioned with two layers of a saline solution ( monobond s , vivadent , schaan , liechtenstein ) .
one layer of a self - etching light - polymerizing bonding agent ( g bond , lot 1002051 , gc corp . ) was applied to the post surfaces , gently air dried and exposed to 500 mw / cm light ( coltolux 75 ) for 20 s at a distance of 1.0 mm . a microhybrid microfilled resin ( mfr ) composite ( shade da2 , lot 0909102 gradia indirect , gc corp .
) was applied to the post surfaces and in the canals using a microbrush ( microbrush international ) .
any voids or defects on the post surfaces were corrected by adding composite and light polymerizing for 60 s. the core was fabricated by indirect composite ( da2 gc gradia , gc corp . ) to a height of 5 mm .
each layer was initially light polymerized in the gradia step light unit for 10 s. a quartz fiber post ( dt light post , rtd ) was inserted in the prepared post space cut 12 mm from the apical end using a diamond disk ( edenta ag dentalprodukte ) .
the negative control group included 10 untreated teeth that were sectioned 4 mm above the most coronal portion of the cej . the teeth were sealed on both ends with cyanoacrylate ( super glue , razi chemicals , terhan , iran ) . in the positive control group ( n= 10 ) , the canals were obturated with gutta percha , but no sealer was used and the access cavity was not temporized .
for the experimental groups , a double layer of nail varnish ( my , kahl & co , ghazvin , iran ) was painted on the root surfaces at 1-mm apical to the core interface . for the control groups , all surfaces except ( positive control ) or including ( negative control ) the access cavity were coated .
all specimens were immersed for 24 h in 50 cc of a thallium 201 chloride solution ( mds nordion , fleurus , belgium ) with the specific activity of 1 mci .
the specimens were washed with a liquid detergent material ( prill , henkel , saveh , iran ) on a swab tip for 60 s. specimens were inserted in a specific tube , with care being taken to position them identically and were placed in a gamma counter device ( kontron gammamatic , neufahrn , germany ) .
the radiation was counted with a thallium 201 photon pick of 77 kev and an energy window of 15% for 60 s. to eliminate the radioactive physical contamination , the specimens were then kept in a lead shield container for 12 day . to simulate the oral condition ,
all specimens were subjected to thermal cycling ( tc ) between 5 and 55 for 60 s each and a dwelling time of 12 s. the periodontal ligament was simulated on the roots of teeth in the cyclic loading ( cl ) test groups using a silicone index .
specimens were subjected to 100,000 cycles of 50 n load at a frequency of 1.5 hz to simulate 6 months of service .
the microleakage was measured after immersing the specimens in thallium 201 solution , as described above .
the net microleakage in experimental groups was calculated by subtracting the mean microleakage value of negative controls known as surface contamination from the measured value of each specimen .
one - way anova , tukey hsd post - hoc and dunnett t3 tests were used to reveal any difference between the mean microleakage values of the tested groups , either before or after tc / cl . to compare the microleakage values of each group before and after tc / cl ,
the significance level was set to = 0.05 and the statistical software spss ver .
each tooth was endodontically treated with a working length 1-mm shorter than the length at which a # 10 k file tip ( mani inc .
, kiohara industrial park , tochigi , japan ) passed the apical foramen , using the flat surface of a coronal section as a reference point .
canals were prepared to a master file size of 40 using the step - back technique and were flared up at the middle third and coronal third using gate glidden # 2 and # 3 ( maillefer , dentsply , ballaigues , switzerland ) , respectively .
files were replaced after use in four canals . the access cavity was filled with a non - eugenole provisional material ( cavit - g , 3 m espe , st paul , mn ) .
the apical fourth was sealed externally with a glass ionomer cement ( ionofill , lot 002033 , voco , 3 m espe ) and stored in normal saline for 72 h before post space preparation .
gutta - percha was removed with # 2 specified reamers of a dt light post ( rtd , st .
specimens were randomly allocated into three experimental groups ; namely , cast post ( cp ) , custom adapted fiber post ( cf ) and prefabricated post ( pf ) groups .
a groove ( 1 mm deep , 2 mm long ) was prepared as an antirotation using a diamond bur ( # 856 - 010 , d - z co. , bern , switzerland ) to prevent post rotation during cementation . passively fitting polycarbonate posts ( pin - jet , angelus dental solution , lonrina , brazil )
were adapted to the post space with auto - polymerizing acrylic resin ( pattern resin , lot 0505061 , gc america inc . ,
the occlusal surface was formed with two cusps of 15. a silicone index ( putty , speedex , coltene / whaledent inc .
patterns were cast in a base metal alloy ( verabond , fairfield , ca ) .
canals were cleaned with water and dried . a self - etching primer ( ed primer , lot 51169 , kuraray co. ltd . ,
osaka , japan ) was applied to each canal with a microbrush tip ( microbrush international , waterford , ireland ) and was air - thinned after 60 s. margins were light - polymerized ( coltolux 75 , coltene / whaledent ) for 60 s at 500 mw / cm and a distance of 1.0 mm .
a # 2 glass fiber post ( reforpost , angelus dental solution ) was placed in the canals and cut 3 mm above the tooth surface with a diamond disk ( edenta ag dentalprodukte , hauptstrausse , switzerland ) .
post spaces were coated with a thin layer of a separating agent ( gc gradia separator , gc corp . ,
airborne abrasion was used on the post surface with 50-m aluminum oxide particles ( shenzen co , shanghai , china ) under 3 kg / cm of pressure for 10 s at a distance of 5 cm .
post surfaces were conditioned with two layers of a saline solution ( monobond s , vivadent , schaan , liechtenstein ) .
one layer of a self - etching light - polymerizing bonding agent ( g bond , lot 1002051 , gc corp . ) was applied to the post surfaces , gently air dried and exposed to 500 mw / cm light ( coltolux 75 ) for 20 s at a distance of 1.0 mm . a microhybrid microfilled resin ( mfr ) composite ( shade da2 , lot 0909102 gradia indirect , gc corp .
) was applied to the post surfaces and in the canals using a microbrush ( microbrush international ) .
any voids or defects on the post surfaces were corrected by adding composite and light polymerizing for 60 s. the core was fabricated by indirect composite ( da2 gc gradia , gc corp . ) to a height of 5 mm .
a quartz fiber post ( dt light post , rtd ) was inserted in the prepared post space cut 12 mm from the apical end using a diamond disk ( edenta ag dentalprodukte ) .
the negative control group included 10 untreated teeth that were sectioned 4 mm above the most coronal portion of the cej . the teeth were sealed on both ends with cyanoacrylate ( super glue , razi chemicals , terhan , iran ) . in the positive control group ( n= 10 ) , the canals were obturated with gutta percha , but no sealer was used and the access cavity was not temporized .
for the experimental groups , a double layer of nail varnish ( my , kahl & co , ghazvin , iran ) was painted on the root surfaces at 1-mm apical to the core interface . for the control groups , all surfaces except ( positive control ) or including ( negative control ) the access cavity were coated .
all specimens were immersed for 24 h in 50 cc of a thallium 201 chloride solution ( mds nordion , fleurus , belgium ) with the specific activity of 1 mci .
the specimens were washed with a liquid detergent material ( prill , henkel , saveh , iran ) on a swab tip for 60 s. specimens were inserted in a specific tube , with care being taken to position them identically and were placed in a gamma counter device ( kontron gammamatic , neufahrn , germany ) .
the radiation was counted with a thallium 201 photon pick of 77 kev and an energy window of 15% for 60 s. to eliminate the radioactive physical contamination , the specimens were then kept in a lead shield container for 12 day . to simulate the oral condition ,
all specimens were subjected to thermal cycling ( tc ) between 5 and 55 for 60 s each and a dwelling time of 12 s. the periodontal ligament was simulated on the roots of teeth in the cyclic loading ( cl ) test groups using a silicone index .
specimens were subjected to 100,000 cycles of 50 n load at a frequency of 1.5 hz to simulate 6 months of service .
the microleakage was measured after immersing the specimens in thallium 201 solution , as described above .
the net microleakage in experimental groups was calculated by subtracting the mean microleakage value of negative controls known as surface contamination from the measured value of each specimen .
one - way anova , tukey hsd post - hoc and dunnett t3 tests were used to reveal any difference between the mean microleakage values of the tested groups , either before or after tc / cl . to compare the microleakage values of each group before and after tc / cl
the significance level was set to = 0.05 and the statistical software spss ver .
dislodgements that caused loss of contact with the loading head or fracture were considered failures .
greatest microleakage occurred in the positive control and pf groups before and after tc / cl . except for the negative control ,
the lowest microleakage was found in the cp and cf groups before and after tc / cl ( table 1 ) . before tc
/ cl , anova revealed significant differences in the mean microleakage values among post systems .
multiple comparison tests indicated that cp and cf had significantly lower microleakage values than pf and the positive control , but pf and the positive control were not significantly different ( table 2 ) .
no difference was found between the microleakage values of each test group before and after tc / cl .
the null hypothesis of the present study was rejected , because the custom adapted fiber post and the cast post had significantly lower microleakage values than the prefabricated fiber post .
however , tc / cl did not affect the microleakage values in the tested groups .
due to the use of different combinations of post systems , luting materials and methodologies , it is not possible to directly compare the results of previous studies to the present results .
however , previous studies have generally shown that a lower microleakage value corresponds to improved fiber post fitness . using systems without cores , usumez et al .
found significantly less microleakage using a direct custom fiber post with a polyethylene woven fiber post ribbon ( ribbond ) compared to systems using a prefabricated stainless steel ( para post ) or zirconia post .
no difference was seen compared to a prefabricated fiber post in contrast to the results of the present study .
our custom adaptation procedure improved post accuracy through a process of removing the post from the canal and directly repairing any voids with composite resin .
a third generation indirect composite ( gradia ) was used to closely adapt the custom frc post .
the mfr composite incorporates ceramic fillers with a mean particle size of less than 1 m , which is twice the content of organic matrix ( 66% inorganic fillers and 33% resin matrix ) .
the increased filler content improves fracture toughness ( resistance to crack propagation ) and decreases polymerization shrinkage .
surfaces bonded to gradia are more compliant ( i.e. , can relieve shrinkage strain ) than those directly bonded to the post surface ( such as in the pf group ) .
furthermore , the elastic modulus of gradia ( 50 mpa ) is 400 times lower than that of the dt light post ( 20 gpa ) , permitting the composite resin layer to undergo more plastic deformation and to compensate for the negative effect of stress production under loading and during polymerization .
the poor correlation among different microleakage evaluation methods is related to differences in the mechanisms of action .
the fluid filtration method assesses the liquid forced through the voids and gaps between dentin , cement , post and/or sealer and the filtration law is applied to any liquid movement .
the results of the present study were consistent with those of a study conducted by erkut et al .
, in which individually formed fiber posts performed better than pre - shaped glass or quartz fiber posts in term of microleakage measured by dye penetration .
a dual polymerizing adhesive cement ( rely x arc ) was used to compensate for the remaining space in the coronal portion of the overflared canals , which appear to be weaker .
this finding may have contributed to the higher microleakage values of the two prefabricated post types in their study .
thermal cycling and mechanical loading did not influence the microleakage values of posts in the present study . repeatedly applying a low magnitude force to restorative materials in the oral environment
can introduce failure over time , by propagating flaws and microcracks along the interface between the post - and - core and tooth structure .
it is recommended to simulate the masticatory cyclic loading when investigating the clinical application of a restorative material .
application of an insufficient number of loading cycles may help explain our obtained results , because sufficient time is required for the cracks to become macroscopically detectable .
mechanical loading protocols that simulate years of clinical service may be considered as accelerated aging protocols . in reality
, it takes years for a cement to disintegrate due to microleakage subsequent to a gap .
in addition , simultaneous tc / cl was not possible because of the design limitations of the machine used .
more cycles and simultaneous tc are recommended for future investigations .several attempts were made to standardize the specimens in the present study .
the dimensions of included teeth were measured to be similar and standardized rotary instruments were used to finish the root canals and prepare post spaces . however , no crown or ferrule ( a minimum residual 1.52 mm vertical length of coronal dentin ) was considered .
the reinforcement effect of these factors could obscure the effect of the post and cement types . in vitro microleakage investigations
are increasingly criticized for their limited clinical application . nevertheless , quantified methods ( e.g. , fluid filtration ) are preferred over non - quantified methods ( e.g. , dye penetration ) .
the method adopted in the present study , namely gamma counting , may be considered a quantified method .
radioisotopes are routinely used in visualizing physiologic body images , and thallium 201 is a cyclotron - generated radionuclide that degrades to mercury ( emitting x - rays ) and thallium ( emitting gamma rays ) .
a specific gamma counter or high resolution camera can produce the gamma profile absorbed by a specimen immersed in the appropriate solution for several hours .
the solution concentration used in the present study was determined by trial and error , since similar studies were unavailable from the literature .
similar to fluid filtration , the gamma counting method is repeatable and non - destructive to the specimens , allowing other tests to be performed simultaneously .
the size of the hydrated thallium 201 ion is similar to that of the hydrated potassium ion , which can enter any gap along in a water molecule .
thallium tracing may reveal the amount of water contaminating a test specimen and is therefore relevant to the physiological oral condition . in the future
, a specified camera could be used to obtain more information on the pattern of microleakage .
within the limitations of the present study , we conclude that the microleakages of the custom - adapted fiber post - and - core and cast post - and - core systems were significantly less than that of the prefabricated fiber post system .
the microleakage values of the test groups showed no significant difference after short - term tc / cl . | objective : the effects of closely adapting a prefabricated fiber to the post space remain unknown .
the purpose of this study was to quantify the microleakages of a custom adapted fiber - reinforced post , a prefabricated quartz fiber post and a cast post using nondestructive methods.materials and methods : sixty - five extracted human premolars were endodontically treated and randomly divided into three groups ( n=15 ) , which were restored using a cast post - and - core , a custom adapted fiber post ( refropost ) with a microhybrid microfiller resin composite ( gradia ) , or a prefabricated quartz fiber post ( dt light post ) and two groups of control ( n=10 ) .
all groups were cemented using a dual polymerizing resin cement ( panavia f2.0 ) .
a composite core ( z100 ) was used for the fiber posts .
the microleakage was calculated for the experimental and control groups before and after thermal cycling and cycling loading using a radiotracer solution ( thallium 201 chloride ) and a gamma counter device .
data were subjected to statistical analysis of anova and tukey hsd at significant level of p < 0.05.results:significantly lower microleakage values were found for the cast post - and - core ( mean value = 16.04 104 ) and custom adapted fiber post groups ( mean value=14.36104 ) .
thermal cycling and cyclic loading had no significant effect on the microleakage value of any tested group.conclusion:post systems with improved adaptation showed similar microleakage to casting posts . |
diseases that belong to spondyloarthritides ( spa ) are frequently manifested as asymmetric peripheral arthritis of the large joints with knee involvement .
tumour necrosis factor ( tnf ) inhibitors are highly effective for the treatment of peripheral arthritis in patients with ankylosing spondylitis ( as ) [ 1 , 2 ] , psoriatic arthritis ( psa ) , undifferentiated spa ( unspa ) , or spa as a whole independently of the phenotypic disease , even in the case of arthritis resistant to disease - modifying antirheumatic drugs ( dmards ) .
however , data on the effect of anti - tnf therapy specifically on knee synovitis are limited in peripheral spa .
the aim of this retrospective study was to evaluate the effectiveness of anti - tnf agents as adjunctive therapy for knee synovitis that did not respond to dmards and other standard treatments in patients with peripheral spa .
we retrospectively studied patients with spa according to the european spa study group criteria and peripheral arthritis involving the knee joint , who were monitored every 24 months at the rheumatology outpatient clinic of the 424 general military hospital ( thessaloniki , greece ) between january 2005 and january 2012 .
inclusion criterion was the addition of an anti - tnf agent for active peripheral arthritis with knee synovitis unresponsive to dmards and standard treatment with low - dose oral corticosteroids ( prednisone 7.5 mg / day ) and/or nonsteroidal anti - inflammatory drugs ( nsaids ) and intra - articular ( ia ) corticosteroids .
exclusion criteria were ( i ) the use of ia corticosteroids and ( ii ) the increase of the dmards ' dose or the addition of a new dmard during anti - tnf therapy .
knee synovitis was defined as the presence of at least 2 of the following 3 clinical criteria : swelling , tenderness , or decreased range of movement . as response of knee
synovitis the absence of the aforementioned three clinical joint signs after 4 months of anti - tnf therapy were considered .
the association of knee synovitis response with gender , age , disease subtype , and pattern of arthritis at anti - tnf initiation excluding interphalangeal joint involvement was also investigated .
knee synovitis responders and nonresponders were compared using the mann - whitney u test for continuous variables and the test or fischer 's exact test for categorical variables .
statistical analysis was performed by using spss software for windows , version 13 ( spss inc . ,
twenty - one patients were on dmard monotherapy ( 9 on methotrexate ( mtx ) , eight on leflunomide ( lef ) , and four on sulfasalazine ( ssz ) ) while the remaining 6 patients were on dmards combination treatment ( 2 patients on mtx + lef , two on mtx + ssz , one on mtx + cyclosporine a ( cysa ) , and one on mtx + cysa + lef ) .
thirteen patients received adalimumab , 8 infliximab , 4 etanercept , and 2 golimumab . in all but three of the patients there was response of knee synovitis to anti - tnf therapy , which sustained for an average observation period of 31.9 months .
furthermore , all knee synovitis responders achieved at least low disease activity ( lda ) according to the disease activity score in 28 joints ( das28 ) . among the 3 patients with unresponsive knee synovitis , tenderness and decreased range of movement were the remaining signs in the two of them , who had psoriatic arthritis ( psa ) with knee monoarthritis and achieved lda .
knee joints of these two patients presented osteoarthritis grade 2 according to the kellgren - lawrence radiologic scoring system on x - ray , leading us not to consider treatment modification as appropriate approach , because the residual clinical signs of their knees were attributed to osteoarthritis secondary to joint destruction . in the third patient , who had oligoarticular psa with remaining knee monoarthritis and achieved moderate disease activity ( das28 = 3.29 ) , both tenderness and swelling of the knee joint persisted . in this patient ,
whose knee x - ray was normal , a switch of adalimumab to infliximab was ineffective ; finally , radiation synovectomy led to the resolution of knee synovitis .
there were no differences in gender ( p = 0.53 ) , age ( p = 0.88 ) , disease subtype ( p = 0.22 ) , and pattern of arthritis ( p = 0.20 ) between knee synovitis responders and nonresponders .
fourteen patients ( 51.9% ) managed to stop dmard therapy , while the rest 13 patients ( 48.1% ) remained on combination of anti - tnf agent and dmard in order to maintain both knee synovitis response and disease activity on remission or lda ; however , in 6 of these patients , all initially on dmards combination , the number of dmards was decreased to one .
there is only one study in the literature evaluating the efficacy of anti - tnf agents focusing on knee synovitis . in this prospective study , including 20 patients with peripheral spa and knee synovitis who received etanercept as monotherapy , a statistically significant decrease in the physician 's assessment of the knee joint ( median visual analog score from 53.5 at baseline to < 10 at week 12 and thereafter ) was observed .
accordingly , clinical synovitis of the knee joint was present in all patients at baseline and decreased to a frequency of 12 ( 60% ) of the 20 knee joints with synovitis at week 12 and 6 ( 30% ) at week 52 . in comparison ,
knee synovitis persisted in approximately 11% of our spa patients at month 4 following anti - tnf addition .
the combination of anti - tnf agent with dmards could explain the more favorable results of our study .
indeed , at least in regard to psa , the superiority of anti - tnf agent plus mtx to mtx alone is well documented in mtx - nave patients and , according to the danish registry , concomitant use of dmard ( mtx ) is a predictor of clinical response in patients treated with an anti - tnf agent .
furthermore , it has been reported that in psa patients , early treatment before joint damage resulted in better outcome and polyarticular disease had poorer prognosis than oligoarticular disease [ 10 , 11 ] .
thus , the addition of an anti - tnf agent shortly after dmard failure ( mean time interval between initiation of dmards and anti - tnf agent was 11.4 months , table 1 ) and the fact that none of our patients with psa had polyarticular disease could be additional reasons for the better response of knee synovitis in our study .
notably , the two psa patients with radiographic damage of the affected knee joint at baseline were nonresponders to anti - tnf therapy . in conclusion , the high percentage of our spa patients with knee synovitis responsive to anti - tnf agent may be explained by the combination of anti - tnf therapy with dmards and the presence of good response predictors in most of them , although in the literature this could be supported only for the psa patients , who were the majority of the studied patients .
it is a retrospective study with limited number of patients , who had heterogeneity on pattern of dmard therapy and were treated with different anti - tnf agents and dmards .
furthermore , we did not include a control group . however , this is the first study to attempt to evaluate the efficacy of anti - tnf agents as adjunctive therapy for knee synovitis refractory to dmards in patients with peripheral spa . in all patients ,
knee synovitis had already failed to respond to dmards and other standard treatments before the addition of an anti - tnf agent .
thus , it is reasonable to attribute knee synovitis response to anti - tnf therapy itself .
the absence of swelling , tenderness , and decreased range of movement on clinical examination , which has been used as outcome in randomized controlled studies [ 12 , 13 ] , was considered as response of knee synovitis in our study .
we used the term response over remission in order to exclude the possibility of mild knee joint inflammation not detectable with clinical examination .
our results imply a sustained beneficial effect of adjunctive anti - tnf therapy on knee synovitis unresponsive to dmards and other standard treatments in patients with peripheral spa . because of both knee synovitis response and remission or lda , approximately half of the patients stopped dmards and all patients on dmards combination decreased the number of them to one | our aim was to evaluate the effectiveness of tumour necrosis factor ( tnf ) inhibitors as add - on therapy for knee synovitis that did not respond to disease - modifying antirheumatic drugs ( dmards ) and other standard treatments in patients with peripheral spondyloarthritis ( spa ) .
we retrospectively studied 27 spa patients , in whom an anti - tnf agent was added for active peripheral arthritis with knee synovitis refractory to dmards and treatment with low - dose oral corticosteroids and/or nonsteroidal anti - inflammatory drugs ( nsaids ) and intra - articular ( ia ) corticosteroids . as response of knee synovitis , were considered the absence of swelling , tenderness , and decreased range of movement in the clinical examination , after 4 months of anti - tnf therapy . in twenty - four ( 88.9% ) of the patients there was response of knee synovitis .
no statistical differences in gender ( p = 0.53 ) , age ( p = 0.88 ) , disease subtype ( p = 0.22 ) , and pattern of arthritis ( p = 0.20 ) between knee synovitis responders and nonresponders were found .
fourteen patients managed to stop dmard therapy and six , all of whom were initially on dmards combination , to decrease the number of dmards to one , maintaining simultaneously the response of knee synovitis .
our results imply a beneficial effect of adjunctive anti - tnf therapy on knee synovitis not responding to dmards and other standard treatments in patients with peripheral spa . |
type 1 diabetes is an autoimmune disease resulting from destruction of pancreatic - cells by infiltrating immune cells .
the nod mouse is an important model of spontaneous autoimmune diabetes that has been used to understand type 1 diabetes pathogenesis and develop therapeutics to prevent - cell destruction .
similar to humans who have genetic susceptibility to type 1 diabetes and islet autoimmunity , not all nod mice progress to overt diabetes , and progression to diabetes occurs at a variable time point after the start of autoimmunity [ 2 , 3 ] .
nod mice have alterations in leukocyte populations as compared to other mouse strains [ 4 , 5 ] .
it is therefore possible that the extent of change may associate with the likelihood and rate of diabetes development in individual mice and that this may reveal markers that may help stratify the rate of progression to diabetes in man .
we , therefore , quantified leukocyte populations in peripheral blood from nod and nor mice in biweekly intervals from 4 weeks to 36 weeks of age or until diabetes onset .
we compared trajectories of t cell and b cell subpopulations and of nk cells , granulocytes , and monocytes between nod and nor mice and within nod mice that progressed and did not progress to diabetes .
nod ( n = 71 ) and nor ( n = 40 ) mice were longitudinally followed up ( suppl .
figure a.1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4208156 ) . in the experimental groups ( nod , n = 58 mice ; nor , n = 22 mice ) , follow - up included biweekly venous blood sampling ( heparin coated tubes ) for facs phenotyping , determination of insulin autoantibody ( iaa ) levels , and blood glucose concentration and for assessment of weight .
in addition , a control group of 13 nod and 18 nor mice was naturally followed up for growth and diabetes development without blood sampling to assess whether frequent blood sampling would affect diabetes development .
all mice were kept under the same specific pathogen - free ( spf ) conditions at the institute of diabetes research , helmholtz zentrum mnchen .
diabetes development was monitored by weekly measurement of urine glucose ( diastix , bayer , leverkusen , germany ) and confirmed by a venous blood sugar level > 13.9
mouse breeding and experiments were carried out in accordance with german legal guidelines and were approved by the local animal welfare authority ( license number tva 70/07 ) .
cells were resuspended and incubated with fc - blocking solution and subsequently with surface antibodies for 15 minutes on ice ( suppl .
dead cells were excluded using propidium iodide - staining and leukocytes were gated via cd45 .
data were acquired with an lsr ii - hts cytometer ( franklin lakes , nj , usa ) .
the resulting data were analyzed with flowjo ( flowjo enterprise , oregon , usa ) using boolean gating ( suppl .
kaplan - meier survival analyses and log - rank statistics were used to estimate diabetes progression in nod mice .
longitudinal trajectories of iaa , t , b , and other cell subpopulations were modeled using first- , second- , and third - order polynomial growth models for each measure and model selection performed according to the akaike information criterion .
trajectories were compared between ( a ) nod and nor mice , ( b ) nod mice that developed overt diabetes and nod mice that did not develop diabetes and between nod mice that progressed rapidly ( by age of 21 weeks which was the median age of diabetes development in nod mice that developed diabetes ) and nondiabetic nod mice . for comparisons ,
trajectories were analyzed until age of 30 weeks except for comparisons with the nod mice that developed diabetes rapidly , which were analyzed until age of 21 weeks .
the overall level of significance was set to a two - tailed p value of 0.05 . for the group comparisons of the 14 cell subpopulations we accounted for multiple testing resulting in the bonferroni corrected significance level of 0.004 .
longitudinal trajectories were visualized by time pointwise means and confidence intervals ( ci ) and smoothed locally via loess .
in the experimental groups , 33 of the 58 female nod mice developed diabetes at a median age of 22 weeks ( iqr 18 to 27 weeks ) and a cumulative risk at age of 36 ( end of the study ) of 32% ( ci 21%49% ) . none of the 22 nor mice developed diabetes during follow - up .
the incidence of diabetes in nod mice was stable from age of 16 weeks ( 9% per 2 follow - up weeks from age of 10 weeks to age of 30 weeks , figure 1(a ) ) .
no difference in diabetes development was observed between the nod mice that were bled weekly as part of the study ( 57% by age of 36 weeks ) and nod mice that were not bled ( 62% , p = 0.89 ) , indicating that frequent blood sampling did not affect diabetes development ( figure 1(b ) ) .
weights were comparable between the nor and nor mice and between the nod mice that developed diabetes and nod mice that remained diabetes - free ( suppl .
trajectories of iaa were higher in nod mice than in nor mice ( figure 2(a ) , p = 0.001 ) and higher in nod mice that developed diabetes as compared to nod mice that did not develop diabetes ( figure 2(b ) , p = 0.002 ) .
trajectories of total t cells ( cd3 ) , t helper cells ( cd4cd3 ) , and cytotoxic t cells ( cd8cd3 ) were higher in nod mice compared to nor mice ( p < 0.001 , figure 3(a ) ) . despite this , cd4cd25 regulatory t cells and the cd44 subpopulation of cd8 t cells were lower in nod mice compared to nor mice ( p < 0.001 , figure 3 ) .
no differences were found for any of the t cell populations between nod mice progressing to diabetes and nod mice that remained diabetes - free ( figure 3(b ) ) , nor between the nod mice that developed diabetes early as compared to mice that remained diabetes - free ( suppl .
trajectories of total b cell frequencies ( cd19 and cd19b220igd ) and b cell subpopulations were significantly different between nod and nor mice with nod mice presenting higher frequencies of later stage b cells ( p < 0.001 , figure 4 ) . within the b cell compartment , nor mice showed a higher percentage of nave ( igdigm ) and activated ( igdigm ) b cells , whilst nod mice had higher percentages of igd igm cells ( figure 4 ) .
the trajectories of b cells and b cell subpopulations were similar between nod mice that progressed to diabetes and nod mice that remained diabetes - free ( figure 4 ) .
the trajectories of granulocyte and monocyte frequencies were lower in nod mice compared to nor mice and the trajectory of nk cell frequencies was higher in the nod mice ( figure 5 ) .
no differences were found within nod mice relative to diabetes progression ( figure 5 ) .
here we used the nod mouse model to portray longitudinal alterations of multiple leukocyte populations from birth to age of 36 weeks with the intention to identify potential biomarkers that may associate with the likelihood and rate of autoimmune diabetes development .
we found profound differences in the circulating b cell compartment of nod compared to nor and additionally significant but less pronounced differences in t cells , granulocytes , and monocytes .
however , none of these differences were able to predict which nod mice would develop diabetes or when the mice would develop diabetes .
the only assessed immune marker which distinguished progressor from nonprogressor nod mice was the autoantibody response to insulin as previously described .
the study is unique because it included prospective frequent phenotyping and utilized advanced statistical modeling of longitudinal data for the analysis .
limitations include the low blood volume that restricts how many cell populations were examined as well as the ability to precisely measure rare cell phenotypes .
since we did not examine cells in lymphoid organs and in the pancreas , we do not know if any of the observed alterations are generalized to the immune and hematopoietic systems .
some of the findings in the nod mice were similar to what is observed in man .
the relationship between increased iaa and more diabetes development is reflected by the faster rate of progression in antibody children if they have an earlier seroconversion or have higher islet autoantibody titers [ 13 , 14 ] . similar to nod mice , although much less pronounced , girls with islet autoantibodies progress to diabetes faster than boys .
finally , it appears that both nod mice and multiple islet autoantibody positive children progress to diabetes at a constant rate . in mice ,
this is from around 10 weeks of age in our colony , while in children it is from the time of seroconversion .
such a constant rate raises the possibility that progression is stochastic rather than induced by single events .
nod mice are reported to have alterations in immune cell numbers and function [ 16 , 17 ] .
we confirm this and show that , as compared to genetically similar nor mice , they have more circulating cd4 and cd8 t cells but lower circulating cd4cd25 t cells .
most striking were the novel differences observed in the b cell population with a more general evidence of b cell maturation from early age in the nod mice .
fiorina and colleagues describe that depletion of mature ( cd22-positive ) b cells reverses autoimmune diabetes in nod mice , which argues for a role of b cells in diabetes development in nod mice .
more subtle differences in circulating b cell subpopulations have also been observed in patients with type 1 diabetes .
we confirmed that nor mice have insulin autoantibodies , but to a lower extent than nod mice as previously demonstrated . circulating monocyte and
some of the observed differences in blood cell populations may provide susceptibility to developing autoimmunity .
disappointingly , however , none of the differences were in any way related to whether the nod mice progressed to diabetes , suggesting that they neither contribute to progression nor reflect other changes that occur during progression .
longitudinal trajectories of leukocyte subpopulations were not found to be valuable biomarkers to predict disease progression in nod mice . | immune phenotyping provides insight into disease pathogenesis and prognostic markers .
trajectories from age of 4 to 36 weeks were modeled for insulin autoantibodies and for leukocyte subpopulations in peripheral blood from female nod ( n = 58 ) and nor ( n = 22 ) mice .
nod mice had higher trajectories of insulin autoantibodies , cd4 + and cd8 + t lymphocytes , b lymphocytes , igd+igm b lymphocytes , and nk cells and lower trajectories of cd4+cd25 + t lymphocytes , igm+ b lymphocytes , granulocytes , and monocytes than nor mice ( all p < 0.001 ) . of these , only the increased iaa trajectory was observed in nod mice that developed diabetes as
compared to nod mice that remained diabetes - free .
therefore , the profound differences in peripheral blood leukocyte proportions observed between the diabetes - prone nod mice and the diabetes - resistant mice do not explain the variation in diabetes development within nod mice and do not provide markers for diabetes prediction in this model . |
lagophthalmos is defined as the inability to close the eyelids completely.1,2 orbicularis oculi muscle that closes eyelids is innervated by facial nerve ( cnvii ) .
paresis of the orbicularis oculi muscle leads to diminished blink , lagophthalmos , and impairment of the nasolacrimal pumping system .
lagophthalmos may be the result of the residual effect of seventh cranial nerve damage secondary from congenital ( moebius syndrome ) , acquired ( bell s palsy , vascular lesions ) , tumors , iatrogenic ( during surgery ) , trauma , infections , or degenerative diseases.3,4 the blink reflex and lid closing are critical to maintain the ocular health .
each blink spreads the tear film over the ocular surface and allows a continuous layer of moisture .
the inability to close the eyelid may lead to corneal problems such as epithelial defects , stromal thinning , exposure keratitis , bacterial infection , perforation , and blindness.59 the prior treatment of lagophthalmos is conservative and symptomatic such as ointments , eye drops , taping , and moisture chambers .
surgical intervention may be required in patients who have failed medical therapy or in whom the facial paralysis is not expected to improve.5 upper lid loading in the treatment of paralytic lagophthalmos brings out the highest patient satisfaction in lid closing during the day and during sleep as well as in the aesthetic appearance of the lid .
when implanted into lid margin , the weight will essentially allow earth s gravity to gently pull the eye to the closed position when the muscles relax . in this procedure ,
a gold weight is inserted in the upper eyelid to allow closure by the force of gravity .
the presence of the weights seems to contribute to the mimics and blinking of the eyelids which provides a better aesthetic appearance .
gold is the preferred material due to its color , specific gravity , and tissue compatibility.2,912 gold weight eyelid implants can elicit a gradually progressive inflammatory response . in at least some cases ,
local corticosteroid injection may suppress the inflammation and permit retention of the implant.13 common complications are related to the selection of wrong weight , and insufficient or excessive correction.14 the eyelid implant method of treatment has proven to be very successful , easily accomplished , and one that may involve the dental profession.6 in this case report the treatment method of the paralyzed eyelid of a patient by a gold weight implant was explained .
a 35-year - old caucasian woman , applied to our department for consultation due to the development of lagophthalmos at her left eyelid 6 months ago caused by acoustic neuroma ( figure 1 ) .
gold weight implantation to left upper lid was planned for solution of the problem . to determine the weight of the implant , a container was tied to a string and was attached to the eyelid ( figure 2).container was made of a plastic amalgam capsule ( permite , sdi , australia ) .
when the patient s head was at upright position , mercury ( gulsa , atlas - enta , izmir , turkey ) was dropped step by step into the container and the patient was instructed to close her eyes .
mercury was preferred due to its specific gravity and easy manuplation . the container including mercury and string and sticking plaster weighted by a jewellery balance ( radwag wpx 250 , radom , poland ) .
the load at which the patient was able to close her eyelid was weighted to be 1.1 g ; 0.2 g was added to this amount and the final weight of the implant was calculated as 1.3 g. irreversible hydrocolloid impression material ( kromopan 100 , lascod , s.p.a . ,
florence , italy ) was used to make the impression and a plaster model was obtained on this model , the implant design was made and the plaster was trimmed arbitrarily ( figure 3 ) .
the patient s affected eyelid was protected with a sticky band - aid , during the impression procedure . commercially available
2.5 g square shaped pure gold ( 99.99% ) , ( ziraat bank , turkey ) , was wrought by a rolling machine , ( lion gold smith production , model no : mrmc 001 , gujarat , india ) than trimmed to a desired shape . wrought and trimming procedures gradually performed until the defined shape and weight is attained .
this gold piece was placed on the plaster model and the margins were trimmed and burnished thus the form of the curvature was given .
two holes were drilled on the gold plate to fix the suture during the surgery . after polishing , final weight of gold plate
gold implant was tried on the patient by the help of a both side sticky tape .
after ultrasonic cleaning with ethanol solution , the gold implant was delivered to the surgeon for sterilization and insertion . the gold weight implant was inserted and fixed by plastic surgeons to the tarsal plate ( figures 4 and 5 ) .
patient s complete eyelid closure and restoration of blink reflex were obtained at postoperative examination .
corneal irritation symptoms were not observed after the procedure . after the healing phase of the edema on the eyelid ( figure 6 ) , the patient was instructed to exercise to close her eyelid manually .
the patient was able to close her eye and pleased with the cosmetic and esthetic results after three months and 2 years control trial .
gold weight implantation is the most commonly used static procedure and method for surgical correction of paralytic lagophthalmos.7,15 other mechanical techniques for reanimating lid closure are palpebral springs , encircling the upper and lower eyelids with silicone or fascia lata , and temporalis muscle transfer.15 although other materials are also used , gold weights are frequently preferred because of gold s high specific gravity , inertness to the body and malleability.4 especially platinum weights should be used in patients with suspected gold allergy.11 the use of gold weight eyelid implants is certainly a desirable option for treatment of patients suffering from lagophthalmos , but it is not always successful or may not give the desired aesthetic result due to the thickness of the prefabricated implants and the anatomical structures of the eye .
there are distinct anatomic differences between the caucasian and asian eyelids , which dictate the overlying aesthetic differences.5 commercially manufactured gold implants are available in several weights and are usually used but may create a
custom - made weights that produce a much more aesthetic result can be fabricated by the dental professionals.6 the gold weight trial procedure was considered a success when postoperative lagophthalmos met the predefined preoperative success criteria ( minimum 50% reduction in lagophthalmos without induction of > 2 mm ptosis ) and was considered a failure when the reduction in the lagophthalmos did not reach 50% of the preoperative value.16 adequate preoperative evaluation is necessary to determine optimum size , weight , and position of the gold implant.11 the most common complications result from inappropriate gold weights . the insufficient gold weight is not effective for closing the eyelid ; contrary excessive estimation leads to unaesthetic appearance and ptosis of the eyelid .
however , it is also frequently associated with certain complications like ulceration , migration , extrusion , etc.17 at the present case , care was taken to find the appropriate weight before the operation . as a result ,
the selected weight and curvature of the gold implant was unique to patient , and there were no complications during the follow - up period of at least 2 years .
kelly and sharpe suggested that the implants must be fabricated in perforated design to allow tissue penetration and to prevent migration.18 it was also reported that gold implants which do not adapt the slope of cornea cause astigmatism.8,19 our patient did not complain of any visual discomfort . because of the aforementioned complications due to fabricated weighs , we preferred a custom - made gold weigh implant .
the estimated weight of the implant from the trials on the skin of the upper lid fails to obtain the expected eye closure outcomes after surgical implantation on the tarsal plate.20 the results show that an addition of 0.2 g to the gold weight estimated in the trial is required to achieve a similar closure of the eye by means of the gold implant on the tarsal plate.20 smellie suggested 0.75 to 1.0 g of lid loading for most effective eyelid closure , whereas gilbard and daspit concluded that a much heavier weight ( 1.2 to 2.6 g ) is required to achieve complete lid closure.21,22 according to aggarwa et al the median predicted ideal gold weight was 1.2 g ( range , 1.0 to 2.0 g).16 in this case the gold weight was 1.3 g. usually to fabricate the gold implant at the determined weight , the density of the gold and the weight of the wax pattern is helpful for calculation.23 in this case casting procedures and related failures were easily eliminated by using a rolling machine .
a custom - made gold weigh eyelid implant can be fabricated by the means of a rolling machine . by this way casting procedures and related failures
fabrication of gold weight implants with the aid of a rolling machine is an effective and easy way and should be considered in management of lagophthalmos . in the present case , management of lagophthalmos was easily accomplished with a custom - made gold implant . | lagophthalmos is defined as the inability to close the eyelids completely .
this situation may lead to corneal problems such as epithelial defects , stromal thinning , exposure keratitis , bacterial infection , perforation , and blindness .
a patient with lagophthalmos underwent insertion of a custom - made gold weight implant .
after implantation , the patient was able to close her eye and expressed high cosmetic and aesthetic results .
fabrication of gold weight implant by the means of a rolling machine is an effective and easy way and should be considered in the management of lagophthalmos . |
superior mesenteric artery syndrome ( smas ) is a rare condition causing acute or chronic compression of the third part of the duodenum , due to a reduction in the aortomesenteric angle .
laparoscopic duodenojejunostomy is a minimally invasive option that has been reported in up to 10 cases .
we describe our operative technique in one case and review the literature on this condition .
a previously well 66-year - old man presented with acute gastric dilatation . an abdominal computerized tomography ( ct )
scan and oral gastrografin meal revealed a dilated stomach and proximal duodenum due to compression of the third part of the duodenum between the superior mesenteric artery ( sma ) and aorta .
gastrografin administration on the fifth day showed no leak , with free flow of contrast into the jejunum .
it gives the same results as open surgery with all the advantages of minimally invasive surgery .
superior mesenteric artery syndrome ( smas ) , also known as wilkie 's syndrome , is a very rare cause of proximal small - bowel obstruction that was first described by rokitansky in 1861 .
although well documented in the english literature , some surgeons doubt the existence of smas as a real entity .
smas is characterized by extrinsic compression of the third part of the duodenum between the aorta and the superior mesenteric artery ( sma ) , due to narrowing of the aortomesenteric angle , and results in acute or chronic intermittent duodenal obstruction .
a 66-year - old man , with no previous medical history , presented with severe upper abdominal pain and distension that had been present for a few days .
he had no history of nausea , vomiting , recent weight loss , altered bowel habits , or previous abdominal surgery . on examination , he was alert , afebrile , with a pulse rate of 107/min and blood pressure of 155/95 mm hg .
an urgent computerized tomography ( ct ) scan revealed marked distension of the stomach and proximal duodenum with narrowing of the third part of the duodenum between the superior mesenteric artery ( sma ) and aorta ( figure 1 ) .
the sagittal - ct showed a marked narrowing of the aortomesenteric angle ( white arrow ) to 8.6 degrees compatible with a diagnosis of superior mesenteric artery syndrome ( smas ) ( figure 2 ) .
the patient 's symptoms improved with nasogastric decompression , and he got himself discharged against medical advice the next day .
he presented again in the emergency department 2 days later with similar symptoms , bilious vomiting and severe dehydration .
the first and second parts of the duodenum were dilated with a collapsed lumen in the third part . the scope , however , could pass through the third part .
gastrografin administration showed hold up of contrast in the third part of the duodenum with proximal dilatation and slow passage into the distal small bowel ( figure 3 ) .
conservative management with nasojejunal feeds was not successful because the patient was non - compliant and refused tube feeding .
axial computed tomographic scan showing markedly dilated stomach and proximal duodenum with compression of the third part of the duodenum between the sma and aorta .
preoperative gastrografin meal showing a dilated proximal duodenum with hold up of contrast in the third part and delayed passage distally .
the patient , while under general anesthesia , was positioned supine with both legs abducted to allow the surgeon to stand in between .
four ports were inserted - 1st port ( 10-mm camera ) : supraumbilical ; 2nd port ( 5 mm ) : left midclavicular line midway between the umbilicus and costal margin ; 3rd port ( 12 mm ) : right midclavicular line midway between the umbilicus and costal margin ; 4th port ( 5 mm ) : epigastrium ( figure 4 ) .
the small bowel was traced from the duodenojejunal ( dj ) flexure to the cecum and found to be normal .
the hepatic flexure , transverse mesocolon , and gastrocolic omentum were mobilized to visualize the duodenum and the head of the pancreas , which were elevated by the underlying aortic aneurysm .
an on - table egd helped in visualization and kocherization of the dilated duodenum with harmonic shears .
a proximal loop of jejunum about 25 cm from the dj - flexure was brought below the dilated duodenum and fixed with it by using interrupted seromuscular 2 0 vicryl sutures .
the duodenum and jejunal lumen was then opened by using hook diathermy , and an endogia stapler ( ethicon endosurgery , somerville , nj ) was inserted and fired ( figure 5 ) . after confirming hemostasis of the staple line , the openings in the duodenum and jejunum were sutured together with continuous full - thickness 2 0 vicryl sutures .
the anastomosis was tested for a leak , by instillation of methylene blue solution via the nasogastric tube .
a tube drain was placed adjacent to the anastomosis , and the 10- and 12-mm port sites closed .
a gastrografin meal study on the fifth postoperative day showed the anastomosis to be functioning well with no contrast leak ( figure 6 ) .
the delay in discharge was due to social reasons and transfer to a step - down facility .
the patient remained well and asymptomatic during follow - up at 1 and 3 months .
position of trocar sites on the patient . stapled side - to - side laparoscopic duodenojejunostomy .
postoperative day 5 gastrografin meal showing no leak with free flow into jejunum across the anastomosis .
the patient , while under general anesthesia , was positioned supine with both legs abducted to allow the surgeon to stand in between .
four ports were inserted - 1st port ( 10-mm camera ) : supraumbilical ; 2nd port ( 5 mm ) : left midclavicular line midway between the umbilicus and costal margin ; 3rd port ( 12 mm ) : right midclavicular line midway between the umbilicus and costal margin ; 4th port ( 5 mm ) : epigastrium ( figure 4 ) .
the small bowel was traced from the duodenojejunal ( dj ) flexure to the cecum and found to be normal .
the hepatic flexure , transverse mesocolon , and gastrocolic omentum were mobilized to visualize the duodenum and the head of the pancreas , which were elevated by the underlying aortic aneurysm .
an on - table egd helped in visualization and kocherization of the dilated duodenum with harmonic shears .
a proximal loop of jejunum about 25 cm from the dj - flexure was brought below the dilated duodenum and fixed with it by using interrupted seromuscular 2 0 vicryl sutures .
the duodenum and jejunal lumen was then opened by using hook diathermy , and an endogia stapler ( ethicon endosurgery , somerville , nj ) was inserted and fired ( figure 5 ) . after confirming hemostasis of the staple line , the openings in the duodenum and jejunum were sutured together with continuous full - thickness 2 0 vicryl sutures .
the anastomosis was tested for a leak , by instillation of methylene blue solution via the nasogastric tube .
a tube drain was placed adjacent to the anastomosis , and the 10- and 12-mm port sites closed .
a gastrografin meal study on the fifth postoperative day showed the anastomosis to be functioning well with no contrast leak ( figure 6 ) .
the delay in discharge was due to social reasons and transfer to a step - down facility .
the patient remained well and asymptomatic during follow - up at 1 and 3 months .
position of trocar sites on the patient . stapled side - to - side laparoscopic duodenojejunostomy .
postoperative day 5 gastrografin meal showing no leak with free flow into jejunum across the anastomosis .
about 400 cases have been described in the english literature with a slight female preponderance .
the availability of dynamic ct - scans has made the diagnosis of this rare and controversial condition much easier .
normally , the sma leaves the aorta at an angle of 45 degrees , and it is through this aortomesenteric vascular angle that the third part of the duodenum passes through . a mass of fat and lymphatic tissue around the sma normally provides an adequate cushion to prevent extrinsic duodenal compression .
any condition that narrows the aortomesenteric angle to approximately 6 degrees to 16 degrees can lead to smas . in our patient ,
he also had an accompanying 4-cm infrarenal aortic aneurysm that may have contributed to this narrowed angle .
ahmed and taylor stressed the importance of differentiating smas from other conditions causing a similar clinical picture , such as scleroderma , systemic sclerosis , diabetes , myotonic dystrophy , and myxoedema .
gondos suggested that the compression in these cases is brought on by dilatation and loss of muscle tone of the duodenum with secondary imprinting of the duodenum by the sma .
, patients may present with features of acute gastric dilatation or proximal small bowel obstruction depending on whether the vomitus is bile - stained .
the majority of cases are however chronic and present many times over several years for investigation of episodic upper abdominal pain with vomiting , anorexia , and early satiety .
typically , the pain is relieved by postural changes that increase the angle between sma and the aorta .
a high index of suspicion , in the presence of known risk factors , is the key to early diagnosis and avoiding complications like massive gastric dilatation , necrosis , or even perforation .
investigations like abdominal x - rays usually reveal a dilated stomach and proximal duodenum but are nonspecific .
endoscopy does not indicate the diagnosis but is important to rule out more common intraluminal causes of obstruction .
barium or gastrografin contrast findings suggestive of smas are dilatation of the stomach , first and second parts of the duodenum , abrupt vertical or linear cutoff of contrast in the third part of the duodenum with normal mucosal folds , delay of 4 hours to 6 hours in gastroduodenal transit and reverse peristalsis in the proximal dilated duodenum . with the advent of modern ct scanners ,
the dynamic ct - scan is a safe , rapid , noninvasive diagnostic tool , which can clearly demonstrate the compression of the duodenum between the sma and aorta and the narrowed aortomesenteric angle .
conservative treatment of smas involves nasogastric decompression , prokinetic agents to improve gastrointestinal transit , and reversing or removing the precipitating factor .
nutrition may be either in the form of small , frequent feeds in the left lateral or knee - chest posture , or nasojejunal feeding , or total parenteral nutrition .
the aim is to provide a high - calorie diet to increase mesenteric fat and expand the aortomesenteric angle .
conservative management was attempted , but the patient was noncompliant and refused prolonged tube feeding . we , therefore , decided to perform a surgical bypass for a definitive cure .
risk factors that predispose to superior mesenteric artery syndrome several surgical procedures have been described in the literature .
strong 's technique of mobilizing the duodenum by dividing the ligament of treitz has the benefit of avoiding an anastomosis but an unacceptably high failure rate .
gastrojejunostomy , due to inadequate decompression of the duodenum and the high risk of stomal ulceration , is controversial .
duvie has described transposition of the third part of the duodenum anterior to the superior mesenteric vessels to permanently circumvent the obstruction .
duodenojejunostomy , first described by staveley , remains the most frequently performed and favored operation over the years , with excellent results .
subsequent successful results with acceptable operating time , faster recovery , and shorter hospitalization have been reported by other authors . in our case ,
laparoscopic duodenojejunostomy showed excellent results with rapid recovery and relief of symptoms until the final follow - up at 6 months .
it gives the same results as open surgery with all the advantages of a minimally invasive procedure and should be the treatment of choice whenever surgical correction of smas is indicated . | background : superior mesenteric artery syndrome ( smas ) is a rare condition causing acute or chronic compression of the third part of the duodenum , due to a reduction in the aortomesenteric angle .
traditionally , an open duodenojejunostomy is recommended when conservative management fails .
laparoscopic duodenojejunostomy is a minimally invasive option that has been reported in up to 10 cases .
we describe our operative technique in one case and review the literature on this condition.methods:a previously well 66-year - old man presented with acute gastric dilatation . an abdominal computerized tomography ( ct ) scan and oral gastrografin meal revealed a dilated stomach and proximal duodenum due to compression of the third part of the duodenum between the superior mesenteric artery ( sma ) and aorta.results:esophagogastroduodenoscopy ( egd ) ruled out intraluminal causes .
a laparoscopic duodenojejunostomy was performed when conservative management failed .
postoperative recovery was quick and uneventful .
gastrografin administration on the fifth day showed no leak , with free flow of contrast into the jejunum .
the patient resumed a normal diet and remained asymptomatic at 6-month follow-up.conclusion:laparoscopic duodenojejunostomy is feasible , safe , and effective .
it gives the same results as open surgery with all the advantages of minimally invasive surgery . |
bipolar disorder ( bd ) is a clinically severe psychiatric disorder , characterized by recurrent alternating episodes of mania and depression , with a lifetime prevalence of 0.7%6% [ 13 ]
. given its high morbidity , disability , and premature mortality , bd is a major health problem with severe socioeconomic implications .
the economic burden of bd has been estimated at $ 45 billion per year in the united states in 1991 , with a total of $ 7 billion of direct costs consisting of expenditures for inpatient and outpatient care [ 5 , 6 ] . a very recent report analyzing the inpatient care costs for bipolar i disorder in europe demonstrated that huge resources and frequent hospitalizations are required , especially in managing manic episodes .
moreover , it has been shown that bd has a significant negative effect on work relationships , attendance , and functioning , with an overall increase in costs deriving from lost productivity .
family , twin and adoption studies have demonstrated that bd is characterized by a high heritability , suggesting that genes exert a high - magnitude effect on disease development [ 9 , 10 ] . in light of this evidence
, great effort has been made to identify the genetic factors involved in the pathogenesis of bd , although fulfilling results have not yet been obtained .
investigations carried out to date on a genome - wide level using case - control study designs have thrown light on two genes , the ankirin g ( ank3 ) and the alpha 1c subunit of the l - type voltage - gated calcium channel ( cacna1c ) .
consequently , a number of studies have further investigated the role of these genes in bd . in regard to ank3
, its association has been displayed ex novo or replicated in several other studies , although with different effect size and association magnitude for the polymorphisms analyzed [ 1216 ] .
following the first report of its association with bd , cacna1c has been further investigated in some studies . in 2008 , using the wellcome trust case - control consortium dataset as comparison , a genome - wide association study ( gwas ) of bd identified concordant association signals in the cacna1c gene , although the genome - wide significance was not reached . since then and upholding the report by ferreira et al .
, the possible causative role of cacna1c in bd has been confirmed by gene - wide analysis , although further evidence is needed to assess its effects on the disease .
very recently , a meta - analysis of genome wide association data on major mood disorders , including bd , has identified a risk locus on 3p21.1 with the most significant association signal located in the protein polybromo-1 ( pbrm1 ) and guanine nucleotide binding protein - like 3 ( gnl3 ) genes , whose encoded products are , respectively , involved in chromatin remodelling and the proliferation of stem cells . among the genes possibly contributing to bd susceptibiliy the diacylglycerol kinase eta ( dgkh ) , encoding for an enzyme that phosphorylates the second messenger diacylglycerol ,
was first found to be associated with bd in the gwas by baum et al . ,
although replication was not uniformly displayed by subsequent studies [ 2224 ] . on the whole , this evidence seems to indicate that the biological underpinnings of bd appear to be highly heterogeneous on the genetic and molecular level .
moreover , it is unlikely that common variants of large effect - size are causative of bd , if , for instance , meta - analysis of gwas data in bd displayed relatively low odds ratios ( or ) of 1.45 and 1.18 and minor allele frequencies ( maf ) in cases of 0.07 and 0.38 for ank3 and cacna1a genes , respectively .
this highly heterogeneous genetic framework characterizing bd reflects on the broad variability of its phenotypic expression , with clinical symptoms presenting different patterns within subjects ( longitudinal variability ) and between subjects [ 2527 ] .
the genetic and phenotypic heterogeneity of bd clearly plays a pivotal role in pacing the way to the discovery of biological determinants of the disease .
specifically , indisputable associations between the genetic variants identified to date and the phenotypic changes manifested in bd are still lacking . in this context ,
an appropriate phenotype definition represents a powerful tool in the search for susceptibility genes in bd [ 27 , 28 ] . to this end
, several subphenotypes have been studied to refine the phenotype and consequently increase the possibility of detecting genetic determinants [ 27 , 29 ] .
the response to long - term treatment with mood stabilizers has been suggested to be a clinical trait suitable for identifying distinct subgroups of bd with greater homogeneity and for the mapping of genes involved in the biological basis of treatment response and in the disorder itself [ 27 , 30 , 31 ] . on the one hand , the reduction of heterogeneity might depend on the identification of common phenotypic patterns in the clinical presentation of the disease . on the other hand ,
the narrower are the criteria for the definition of treatment response , the greater the probability of reducing genetic heterogeneity and consequently of detecting causative determinants of differences in response to medications . phenotypic assessment is the harbinger of more complex research strategies such as pharmacogenomics , which aim to assemble the molecular and phenotypic heterogeneity jigsaw puzzle in the study of treatment response .
indeed , pharmacogenomics is the genome - wide analysis of the role of genetic determinants in modulating an individual 's response to a drug treatment or the onset of adverse drug reactions ( adrs ) . along with its use as a strategy for reducing heterogeneity and consequently for shedding light on the biological underpinnings of the disease , the integration of pharmacogenomic data with specific individual phenotypic information , represents a powerful instrument for the development of personalized therapies .
although the data collected by numerous studies have not yet reached a sufficient magnitude to significantly impact the clinical field , pharmacogenomics in bd is a rapid - evolving research field .
specifically , most of the pharmacogenomic studies performed in bd have focused on candidate genes selected according to the biological rationale supporting their involvement in the drugs ' mechanism of action , in the biological basis of the disease and in the metabolic pathways implicated in the onset of adrs . in this paper , we present the current state of the pharmacogenomic research on long - term treatments in bd , specifically focusing on mood stabilizers . in the introductory section ,
we discuss the issues pertaining to the currently - available mood stabilizing treatments for bd , the appropriate definition of the treatment response phenotype and its implication for pharmacogenomics studies
. then we selectively review existing evidence on genetic predictors of maintenance treatment response in bd , firstly presenting an overview of the molecular targets involved in mood stabilizers mechanism of action and then focusing on presently available reports on the genetic determinants of treatment response .
it is widely accepted that the term mood stabilizers , although not yet uniformly validated , may be applied to several classes of drugs , such as anticonvulsants , antipsychotics ( particularly the second generation antipsychotics or atypical ) , and lithium which , although different in terms of mechanism of action and chemical structure , commonly act as prophylactic agents able to prevent illness recurrences . ideally , a mood stabilizer should be efficacious in : ( 1 ) treatment of acute manic symptoms , ( 2 ) treatment of acute depressive symptoms , ( 3 ) prevention of manic symptoms , and ( 4 ) prevention of depressive symptoms . among the mood stabilizing agents , lithium , more than the others , seems to meet these criteria , further strengthening the rationale for its use as first - line treatment in bd [ 3235 ] . in this regard
, numerous controlled and open trials have clearly shown that lithium is able to reduce the frequency of episode recurrences by at least 30% [ 33 , 35 ] .
a randomised open - label trial recently demonstrated that both combination therapy with lithium plus valproate ( vpa ) and lithium monotherapy are more likely to prevent relapse than is valproate monotherapy .
therefore , it is not surprising that since its first introduction in the clinical use in 1949 by cade , lithium has remained one of the mainstays in the treatment of bd , a practice further corroborated by its high efficacy in reducing suicidality in patients with recurrent major affective disorders [ 38 , 39 ] . along with lithium , anticonvulsants and antipsychotics ,
[ vpa , lamotrigine ( ltg ) and carbamazepine ( cbz ) ] have strong evidence - based support for use in clinical states of bd [ 40 , 41 ] .
specifically , vpa , ltg and cbz have data supporting their roles as potential long - term treatments to prevent relapse [ 40 , 41 ] .
ltg has a greater effect on the prevention of depression while has lacked efficacy in mania or in acute bipolar depression . on the other hand
, vpa is effective in the treatment of mania , somewhat more effective in certain patient subgroups ( mixed mania , mania with prominent irritability ) than other treatments ; recent proof - of - concept indicates benefits in bipolar depression , while there is clear evidence that cbz is effective in treating mania [ 41 , 42 ] . among the other anticonvulsants , gabapentin ( gbp ) and topiramate ( tpm )
have not been shown to be superior to placebo in randomized trials , although gbp might be useful in controlling anxiety in bd .
antipsychotics , and specifically the class of atypical , appear to be effective in the treatment of mania , although their potential in the long - term management of bd is still subject of debate [ 43 , 44 ] . a systematic review of randomized trials for treatment of the maintenance phase in bd supported evidence that , along with lithium , ltg and vpa , olanzapine is effective in long - term use significantly reducing manic relapses . although this evidence describes a background of multiple therapeutic tools available for the treatment of bd , a relatively high percentage of patients still present lack of response even after consecutive trials of different drugs . in this context
, it appears crucial to develop theoretical frameworks useful in identifying clinical predictors of response by categorizing bd patients according to different diagnostic spectra . in this regard , a three - spectrum model of bd
has been proposed , describing different subtypes of bd ( classical , psychotic and characterological ) characterized by specific clinical features and more importantly by specific patterns of drug treatment response .
this model supports the importance of the careful diagnostic assessment of bd at the phenomenological level , as some specific clinical features , such as family history , clinical course and comorbidity are strictly linked to the treatment response phenotype .
definition of the treatment response phenotype is a crucial process propaedeutic to any subsequent pharmacogenetic / genomic analysis the value and reliability of the latter based on an accurate assessment of the former .
diagnoses are expected to be heterogeneous in bd , and similarly , quantification of the treatment response in clinical terms , a complex trait in itself , may vary . in detail ,
evaluation of treatment response outcomes has for the most part been performed considering it a binary - trait ( 0 = unaffected / nonresponders , 1 = affected / responders ) .
however , it has been suggested that , since treatment response is a quantitative trait , this approach might not totally represent the complex nature of this phenotype .
indeed , deeming treatment response a quantitative trait can facilitate the identification of genetic variants involved in intermediate phenotypes , as for instance partial response to a drug .
it is important to note that the binary - trait approach does not take into account the degree of variation in the treatment response phenotype , which can be represented as a gradient lying within the two extremes of complete nonresponse and complete response . on the other hand ,
comparing and contrasting the genetic differences at the two extreme points of the gradient ( extreme discordant phenotype ( edp ) ) can increase the statistical power and consequently the probability of detecting the causative variants for the response or lack of response to a drug [ 46 , 47 ] . in bd , efforts thus far to develop a stringent definition of treatment response , especially in long - term therapies , have applied analytical tools such as the affective morbidity index ( ami ) [ 48 , 49 ] and the illness severity index .
briefly , these approaches take into consideration the severity and the duration of episodes before and after the introduction of the mood stabilizing therapy , allowing quantification of the degree of improvement under a specific treatment
. however , these approaches do not take into account the presence of possible confounders ( such as additional medication or lack of compliance ) that may interfere in the establishment of a causal relationship between the clinical improvement and the treatment . in this regard , in 2002 grof et al .
developed a rating scale that measures the degree of improvement in the course of treatment and weighs clinical factors considered relevant for determining whether or not the observed improvement is due to the treatment .
this scale allows enucleating the full response phenotype , meaning the absence of abnormal mood episodes during treatment .
moreover , since the scale consists of eleven items ( 010 ) , it also permits identification of intermediate response phenotypes ( i.e. , partial response ) and the edp approach , both aspects supporting its validity for the implementation in pharmacogenomic studies of treatment response .
in general , the assessment of treatment response phenotype in bd shows quite similar rates of reduction of illness recurrence among the different mood stabilizing therapies . in regard to lithium ,
clinical data show that ~80% of chronically - treated subjects are at least partial responders and that ~30% of these patients are excellent responders with a complete remission of symptoms . in a review of 20
randomized , double - blind controlled studies of monotherapy or combination therapy for the treatment of acute mania , the pooled response rates of 15 monotherapy studies were around 50% for lithium , vpa , cbz or atypical antipsychotics ( 21% greater than placebo ) , while the remaining 5 combination studies showed an incremental benefit of adding a second agent equal to 21% .
however , treatment refractoriness in bd still remains a challenge , further encouraging the search for response predictors that might allow identification of the most effective treatment for carriers of specific genetic - variation patterns . in light of the complexity of the drug response phenotype
, the pharmacogenomic approach might benefit from analysis of convergent findings on prediction of treatment response on the phenotypic and genetic level .
it has been suggested that pharmacogenomic studies should consider nongenetic factors that can interact in influencing the phenotype .
genetic work can be informed and guided by appropriate clinical details and can allow investigation of possible genotype - phenotype information . in bd , this joint analytical approach has led to intriguing findings on significant interactions between lithium treatment response , gene variation and specific clinical features . since clinical markers of treatment response
can have predictive power that may allow the targeting of therapies to subpopulation of patients characterized by specific phenotypic traits , it is important to establish their correlation with treatment response outcome . in this context
, a large amount of evidence has been gathered over the years on outcome predictors for long - term treatments with cbz , vpa , ltg and lithium [ 5759 ] .
treatment response to cbz seems to be predicted by clinical features such as mood - incongruent psychosis , lack of response or intolerance to lithium , while contrasting findings are present on the possible predictive value of rapid cycling , severe mania and dysphoric mania [ 25 , 57 , 60 ] .
responders to vpa are characterized by the presence of pure , mixed or dysphoric mania . moreover , early age at onset , rapid - cycling , concurrent substance abuse and lack of response or intolerance to lithium are clinical markers of positive outcome with vpa .
response to ltg seems to be predicted by earlier onset of symptoms , non episodic course of illness , rapid cycling , comorbidity with panic or substance use disorder , fewer hospitalizations , fewer prior medication trials and male gender [ 61 , 62 ] .
responders to lithium are a group of patients with distinct clinical features corresponding to the bipolar disorder core phenotype [ 33 , 63 ]
. an episodic pattern of mania - depression interval , low rates of comorbid conditions , absence of rapid cycling and high age at illness onset have been identified as potentially protective against a recurrence under lithium [ 59 , 6466 ] .
in addition , family history might also provide important insights on the prediction of treatment outcome .
for instance , relatives of ltg responders present a greater prevalence of schizoaffective disorders , major depression and panic attacks , while lithium responders had a higher risk of bd . in this regard ,
it is important to note that since the first report by mendlewicz et al . , which pointed out a significant correlation between response to lithium and the presence of bd in the proband 's first degree relatives , this finding has been confirmed over a period of more than thirty years [ 50 , 62 , 6874 ] .
moreover , grof et al . showed that lithium response in itself clusters in families .
[ 75 , 76 ] suggested that a recessive model with sex - specific penetrance could explain the mode of inheritance in bd responsive to lithium . on the whole
, these findings clearly show that treatment response is a heritable clinical feature , evidence that corroborates the rationale for pharmacogenomic approaches in bd .
moreover , they underscore the importance of an accurate collection of clinical data which , if analyzed jointly with genetic information , could lead to precise prediction of treatment outcome in bd patients .
considerable evidence has shown that bd is characterized by neurotrophic alterations resulting in impairment of signalling and neuroplasticity with a significant overall reduction in central nervous system ( cns ) volume ( for a review , see ) .
neuroimaging studies have demonstrated that bd and major depressive disorder ( mdd ) patients have several brain volumetric alterations such as enlargement of ventricles , reduced grey matter volumes in the orbital and medial prefrontal cortex ( pfc ) and reduced volumes in the frontal lobe and hippocampus [ 7882 ] .
post - mortem studies have also shown that the reduced volumes of pfc and orbitofrontal cortices are associated with decreased neuron numbers and/or size [ 83 , 84 ] .
other studies reported reductions in the number of interneurons in the anterior cingulated cortex of bd patients as compared to controls as well as reductions in nonpyramidal neurons in the hippocampus [ 85 , 86 ] .
it has been shown that the clinical efficacy of lithium correlates with measurable changes in the cns of treated subjects , who displayed increased volumes in specific brain areas after chronic treatment .
recently , a longitudinal study conducted from 1997 until 2004 evaluated the effect of lithium on grey matter volumes of different brain areas of 28 bipolar subjects with different response to lithium medication , using morphometric magnetic resonance imaging .
a significant increase in total brain grey matter volume was identified after chronic lithium administration .
interestingly , the regional analysis showed that the grey matter volume of pfc was significantly increased only in lithium responsive bd subjects .
these data show that the effect of lithium on the cns has clinical consequences and corroborate the evidence on its neurotrophic effect .
nevertheless , the mechanism by which lithium increases grey matter volumes is still under investigation .
a large body of evidence shows that lithium interacts with many elements of second messenger systems thus modulating processes both up- and downstream of its targets .
lithium competes with magnesium for a large number of enzymes requiring it as a cofactor [ 88 , 89 ] .
early reports show that lithium directly inhibits two enzymes of the inositol pathway , the inositol monophosphate phosphatase ( impase ) and the inositol polyphosphate 1 phosphatase ( ippase ) , at therapeutic serum concentration ( 0.61.2 mm ) .
this evidence led to the inositol depletion hypothesis of lithium 's mechanism of action , which would exert its mood stabilizing effects by decreasing inositol levels , thus ultimately influencing a number of processes relying on this pathway , including neurotrophin and g protein mediated signalling .
this hypothesis is also supported by findings from animal studies [ 9294 ] . like lithium , vpa has been shown to have neuroprotective effects .
it has been reported that vpa interacts with the inositol pathway , but through a different mechanism compared to lithium .
vpa does not directly inhibit impase , and the inositol depletion is mediated by a decrease in inositol phosphates and inositol levels [ 95 , 96 ] .
a number of findings have also shown that lithium and vpa interact with the activator protein 1 ( ap-1 ) , a family of transcription factors composed of heterodimeric complexes of c - jun and c - fos proteins . lithium has been shown to increase ap-1 binding activity and transcription in cultured cells [ 97 , 98 ] .
other studies have reported that vpa increases ap-1 dna binding and activation in cultured cells [ 99 , 100 ] . however , while lithium regulates ap-1 through the inhibition of glycogen synthase kinase 3 ( gsk-3 ) , vpa induces the expression of c - jun and c - fos by means of histone deacetylase ( hdac ) .
gsk-3 is a highly - conserved protein involved in the regulation of apoptosis , circadian rhythm and a wide range of neuronal functions and pathways involved in cell and tissue development ( for a review , see ) .
a large number of processes and pathways converge on gsk-3 including the wingless - int ( wnt ) signalling , several neurotransmitter pathways , and akt , with the latter shown to inhibit gsk-3 in response to multiple hormones and neurotrophic factors , including the brain derived neurotrophic factor ( bdnf ) .
lithium , like vpa , inhibits gsk-3 activity in mouse and rat brain at therapeutically relevant doses [ 103105 ] .
it has been reported that lithium inhibition of gsk-3 is mediated through competition for magnesium , an essential cofactor for gsk-3 , while vpa inhibition is indirect [ 89 , 106 , 107 ] .
the inhibitory effect of lithium and vpa on gsk-3 may represent one of the key mechanisms through which they regulate neuronal plasticity and neurogenesis . in this regard
, it has been shown that alternative inhibitors of gsk-3 affect neurogenesis in embryonic stem cells [ 108110 ] and stabilize growth cones in cultured sensory neurons .
along with lithium , vpa and cbz were shown to inhibit the collapse of sensory neuron growth cones and to increase growth cone area .
it has been shown that the serotonergic pathway is a downstream target of gsk-3 .
this evidence led to the hypothesis of an involvement of gsk-3 in depression and might explain the effectiveness of lithium as an add - on in the treatment of antidepressant - refractory depression [ 114 , 115 ] .
in this regard , a recent finding showed that lithium decrease serotonin release in primary serotonergic neurons from rat raphe nuclei . in the same study
, the authors reported that short - term lithium treatment ( 8 days ) resulted in a 45% decrease in tryptophan hydroxylase 2 ( tph2 ) expression and a 31% reduction in tph2 protein levels , completely compensated after long - term treatment ( 14 days ) .
as we mentioned earlier , the wnt signalling pathway converges on gsk-3. the activation of this pathway leads to the stabilization and then accumulation of -catenin and is involved in gene expression regulation .
lithium was shown to stabilize -catenin through inhibition of gsk-3 , while vpa interacts with the wnt pathway by increasing -catenin transcription . among the elements interacting with gsk-3 and involved in neurogenesis ,
the family of camp response element binding proteins ( creb ) is one of the most widely investigated .
lithium antagonizes phosphorylated creb loss and upregulates both creb and bdnf in several brain regions .
moreover , bdnf levels in the rat brain [ 119121 ] are increased after treatment with either lithium or vpa . a recent finding from a gene expression study performed on cultured rat cortical neurons showed that the treatment with lithium or vpa induced activation of the promoter iv of bdnf and that this activation was mediated by the inhibition of gsk-3 and hdac , respectively .
hdac is a key enzyme responsible for the hypoacetylation of histone proteins , resulting in gene silencing [ 123 , 124 ] , thus modulating transcription of about 2% of transcribed genes . as a whole
, these data show that vpa can potentially modulate a large number of long term processes through the epigenetic regulation of gene expression .
an effect of lithium on bdnf was also shown by tseng and coworkers in lymphoblasts from bd patients responsive to lithium .
the authors reported that lithium treatment in vitro decreased bdnf levels more significantly in bd patients than in controls .
another key element in neuroprotection is the b - cell cll / lymphoma 2 ( bcl-2 ) , a protein involved in regulating cell survival by blocking apoptotic death .
it has been shown that administration of both lithium and vpa induces a two - fold increase in bcl-2 levels in the frontal cortex of chronically - treated rodents and in the neuronal cell lines sh - sy5y . as discussed in section 2 , newer anticonvulsants , such as ltg , tpm and gbp ,
are effective in the maintenance treatment of bd ( ltg ) or as add - ons to first line mood stabilizers ( tpm , gbp ) .
in contrast to lithium and vpa , less is known about the mechanism of action of these drugs .
a number of studies have shown that ltg inhibits glutamate release through sodium and calcium channel blocking [ 129131 ] . moreover , ltg significantly enhanced the surface expression of glur1/2 ampa receptor in a time- and dose - dependent manner in cultured hippocampal neurons .
ltg was also shown to affect gaba - a receptor - regulated functions in the cns .
a gene expression study performed on primary cultured rat hippocampus cells treated with ltg 0.1 mm showed that the expression of the gaba - a receptor beta3 subunit was increased by the treatment .
ltg was also shown to inhibit gsk-3 in human neuroblastoma cells , a mechanism that is shared with vpa and lithium .
in addition to this function , tpm was shown to inhibit glutamate release in epileptic rats .
several studies have shown that tpm increased brain gaba levels in healthy humans and enhanced gaba - stimulated cl influx into cerebellar granule neurons and cerebral cortical neurons .
numerous findings also suggest that , like other mood stabilizers , ltg and tpm play a key role in neuroprotection .
ltg has been shown to have neuroprotective effects in animal models of ischemia [ 138141 ] and in the neuronal damage induced by excitotoxins , mitochondrial toxins and axotomy [ 142144 ] .
as regards gbp , the mechanism of its psychotropic action is still unknown , but some findings show that it has neuroprotective effects in ischemic animal models . moreover , some evidence suggests that gbp could negatively regulate glutamate neurotransmission . the first report from a genome wide gene expression ( gwge ) study on human derived cell lines
the transcriptome analysis showed that a total of 671 genes were differentially regulated after lithium treatment .
altered transcripts included genes of the apoptotic system and the phosphoinositide metabolism . in another study , plant and colleagues performed a transcriptome analysis on human neuroblastoma cells ( sh - sy5y ) treated with either lithium or valproic for 6 h or 72 h. findings showed that the treatment with both drugs determined an altered expression of 936 genes with the homeodomain protein six1 being the most significantly upregulated .
moreover , the evaluation of the antiapoptotic action of lithium and valproate showed that six1 over - expression protected the cells from staurosporine - induced apoptosis via the blockade of caspase-3 activation .
another gwge study was carried out on multiple prostate human cancer cell lines that were administered with therapeutic levels of lithium .
lithium significantly inhibited cell proliferation , which was associated with reduced dna replication and s - phase cell cycle arrest .
moreover , lithium significantly decreased the expression of multiple dna replication - related genes . during the 8th annual pharmacogenetics in psychiatry meeting ,
mclnnis reported on a gene expression study performed on lymphoblasts from bd subjects , incubated with 1 mm lithium chloride for 4 , 8 and 16 days .
results revealed that the chromosome 8 open reading frame 33 ( c8orf33 ) was significantly over - expressed after lithium treatment and that a total of 217 genes was significantly downregulated .
c8orf33 activity was found to be associated with g protein - coupled receptor protein signalling pathway , neuroactive ligand - receptor interaction , ca++ signalling pathway and the regulation of the actin cytoskeleton .
recently , sugawara and colleagues evaluated the effect of both lithium and valproate on gene expression levels in lymphoblasts derived from three healthy subjects .
the microarray analysis revealed that 44 and 416 genes were regulated by lithium and valproate , respectively . among the 18 genes
commonly altered , the strongest downregulation was reported for the vascular endothelial growth factor a ( vegfa ) gene .
as for lithium - specific effects , the most significant altered expressions were reported for the bcl-2 associated x protein ( bax ) and the platelet - activating factor acetylhydrolase isoform 1b , beta subunit ( pafah1b2 ) . in 2002 ,
bosetti and coworkers carried out a gwge to describe gene expression changes in brain of rats fed with lithium for 7 ( subacute ) and 42 ( chronic ) days respectively using an array of 4132 genes .
findings showed that lithium downregulated 25 genes after 7 days but did not upregulate any gene .
in contrast , after 42 days of lithium treatment , 50 genes were upregulated while no gene was downregulated .
interestingly , among the downregulated genes was the inositol polyphosphate-1-phosphatase gene ( inpp1 ) , whose expression was 2.7 times lower after 7 days and 2.6 times lower after 42 days of lithium treatment .
findings showed that lithium caused a significant change in the expression level of 121 genes . among these , lithium upregulated the period homolog 2 ( per2 ) , the secretogranin ii ( scg2 ) , the bdnf and the impa1 genes .
other interesting findings consisted of the upregulation mediated by lithium of the deiodinase ( dio2 ) and thyroid hormone receptor interactor 12 ( trip12 ) genes , encoding for elements that might be involved in the development of hypothyroidism , a long - term side effect caused by lithium treatment in clinical settings .
another study by chetcuti et al . identified a significantly - differential expression of a number of genes in brains of mice treated with lithium for 7 days as compared to a control sample .
these included genes involved in metal ion homeostasis and chemical / electrical gradients across membranes , and regulating rna polymerase ii , protein degradation and g - protein - coupled signal transduction . as regards to vpa , a recent study examined gene expression levels in rat cortical neurons treated with vpa using microarray technology and reported that 726 genes were upregulated while 577 were downregulated by the treatment .
interestingly , the expression of bdnf was upregulated by vpa while the expression of the 4 subunit of the gaba - a receptor gene ( gaba - ar4 ) and the k / cl cotransporter ( kcc2 ) , which are both involved in the development of gabaergic inhibitory neurons , were downregulated .
micrornas ( mirna ) are a class of endogenous post - transcriptional regulators binding to complementary sequences in the 3 utr of multiple target mrnas .
the mirna database ( mirbase : http://www.mirbase.org/ ) currently include more than 10000 entries but this number will presumably increase rapidly in the coming years .
a single mirna can regulate the expression of a large number of genes , usually resulting in their silencing . among their functions , mirnas have been shown to regulate neurite outgrowth , neurogenesis and synaptic plasticity , mainly through the regulation of mediators such as creb , methyl cpg binding protein 2 ( mecp2 ) , the fragile x mental retardation 1 protein ( fmrp ) and elements of the wnt pathway .
a recent study explored the role of mirnas in the mechanism of action of lithium in a sample of lymphoblasts from bd patients .
each cell line was divided into two lines : one line was treated with lithium 1 mm for 4 , 8 and 16 days while the other was grown in lithium - free medium .
results showed that lithium treatment determined expression changes in 7 mirnas after 4 days treatment and that 4 out of these 7 were significantly regulated after 16 days .
the most intriguing finding was reported for mir-221 and mir-34a , two mirnas previously shown to be regulated by lithium and vpa in rat hippocampus .
these mirnas are respectively responsible for regulating the expression of 29 and 10 genes , including some genes previously shown to be implicated in bd .
interestingly , the study by zhou and colleagues also showed that in primary culture of hippocampal neurons treated with lithium or vpa , levels of the protein glutamate receptor metabotropic 7 ( grm7 ) , one of the mir-34a targets , was increased thus showing that mir-34a might contribute to mediating the effect of lithium and vpa on grm7 .
as a result of its efficacy and wide clinical use , pharmacogenomic studies on bd have for the most part focused on response to lithium prophylaxis .
early linkage studies used pharmacogenomic approaches based on lithium response as a strategy for reducing heterogeneity in bd . in 1999 , ewald and colleagues carried out a haplotype sharing analysis on chromosome 18 in a sample of eight lithium responding bd subjects from the faroe islands who had common ancestors .
the study reported increased haplotype sharing on the distal part of chromosome 18 ( region 18q23 ) . in 1999 , turecki and coworkers reported a modest linkage with markers in the chromosome region 18q22.3 in unilateral families of bd patients responsive to lithium .
interestingly , the same region was previously shown to be associated with bipolar in a sample of narrowly defined bdi patients for which , however , the response to mood stabilizing treatment was not evaluated . another linkage study on
bd probands for which response to lithium treatment was considered as inclusion criteria , reported significant lod scores for the region of chromosome 12q23-q24 in a large pedigree from the homogeneous population from saguenay - lac - st - jean ( quebec ) .
more recently , turecki and colleagues performed a genome scan on 31 families of probands with excellent response to lithium using 378 markers .
however , when the response phenotype was considered in the analysis , the 15q14 region appeared to be more probably involved in bd phenotype and 7q11.2 in lithium response .
as shown by the large amount of evidence on the key role of the inositol pathway in the mechanism of action of lithium , numerous candidate gene studies on lithium response have been performed on inositol - related genes . however , while some authors reported intriguing findings , a strong evidence of high impact genes is still lacking .
the gene encoding for phospholipase c1 ( plcg1 ) , a key enzyme involved in g protein mediated signals and in the inositol pathway , has been investigated in several studies .
one paper reported association for a dinucleotide repeat in plcg1 in a sample of 136 bd patients excellent responders to lithium and 163 controls .
the same study reported a modest linkage with this polymorphism in a sample of unilineal families .
however , these findings were not replicated in a re - examination study performed in a sample of norwegian lithium - treated bipolar patients sub - classifed as lithium responders , nonresponders , or partial responders / unclassifed .
plcg1 was further investigated in the study by ftouhi - paquin and coworkers , in which the authors screened the gene for functional polymorphisms . while three single nucleotide polymorphism ( snps ) in the translated region of plcg1 were identified , none of the markers was found to be associated with bd in a sample of 133 excellent responders to lithium and 99 healthy controls .
other genes encoding for elements of the inositol pathway have been tested for association with lithium response .
the authors showed association for the silent variant c937a and response to lithium in a norwegian sample of 23 bd patients and 20 controls but not in a sample of 54 bd and 50 controls from israel .
nevertheless , a recent study failed to confirm the association for this polymorphism and lithium response in a sample of 134 bd patients comprised of 61 full responders , 49 nonresponders and 24 partial responders to lithium .
positive association with lithium response was also shown for two polymorphisms in the gene encoding the inositol monophosphatase 2 ( impa2 ) in the study by dimitrova and colleagues . however , the relatively small size and power of the bd sample characterized for lithium response did not allow the authors to draw definitive conclusions .
the gene encoding for the impa1 has been studied in two papers reporting no association with lithium response [ 56 , 165 ] .
several studies have investigated for association another gene encoding for a key element in lithium mechanism of action : the gsk-3 gene [ 56 , 184186 ] . however , among the four studies performed so far , only benedetti and coworkers reported association between the 50 t / c polymorphism and lithium response , thus providing no conclusive results .
rybakowski and colleagues reported positive association between snp rs6265 ( val66met ) and better response to lithium in a sample of 88 bd patients .
the val66met is a functional polymorphism with the met allele shown to be associated with lower depolarization - induced secretion of the protein in neurons .
the val/66met polymorphism was investigated by rybakowski and coworkers in a further study reporting an interactive effect between the val / val genotype and the s / s and l / s genotypes of the serotonin - transporter - linked promoter region ( 5-httlpr ) polymorphism in a sample of 107 bd subjects characterized for lithium response .
other association studies of 5-httlpr polymorphism in lithium response are discussed hereafter in this review .
another study by the same group recently investigated for association four snps in the bdnf gene and three snps in the gene encoding for the neurotrophic tyrosine kinase , receptor , type 2 ( ntrk2 ) in a sample of 108 bd patients .
findings showed association between the bdnf val66met polymorphism and degree of response to lithium , but no association was reported for ntrk2 polymorphisms .
in which significant association between lithium response and two snps within ntrk2 was shown in bd patients with suicidal ideation as well as in bd patients with posttraumatic stress disorder .
interestingly , in 2009 szczepankiewicz and coworkers have investigated the association between the src - family tyrosine kinases ( fyn ) gene and lithium response in a sample of 101 bd patients characterized for lithium response .
fyn is a member of the protein - tyrosine kinase oncogene family and belongs to the protein kinase family phosphorylating nmda receptor subunits , participating in the regulation of ion transmission and bdnf / trkb signal transduction pathway . in this
study the authors reported a trend for association for one of the polymorphisms with a worse response to lithium .
while the study does not strongly support the involvement of this gene in lithium response , findings are intriguing in the light of a previous paper by the same group in which an association for fyn polymorphisms and bd was reported . as a whole , these results corroborate the evidence indicating a key role of the bdnf / trk signaling pathway in lithium response and bd .
another interesting finding was reported in the paper by mamdani and coworkers , in which the authors investigated the association between creb1 , creb2 and creb3 genes and response to lithium in a sample comprised of 180 bd lithium responders , 69 non responders and 127 controls .
findings showed association between creb1 - 1h and 7h snps and response to lithium . as we mentioned earlier
recently investigated the three snps previously found to be associated with bd by baum et al .
, in a sample of 197 sardinian bd i patients , 91 of whom were characterized for lithium response .
while the authors failed to replicate the association for individual snps , a significant association with bd was reported for a haplotype of the three snps .
however , the study did not show statistically significant association with response to lithium .
moreover , this lack of association has been confirmed in an extended cohort of sardinian bd patients charachterized for lithium treatment . along with inositol pathway genes , a number of papers
three studies reported association between the short allele ( s ) and/or genotypes carrying the s allele and a worse response to lithium [ 176 , 178 , 179 ] .
another study investigated the interactive effect of 5-httlpr and bdnf val66met in a sample comprised of 31 patients classifed as excellent responders , 54 as partial responders and 26 as nonresponders to lithium prophylaxis .
findings showed that patients with 5-httlpr s / s and l / s genotypes having the bdnf val / val genotype were more frequent in non responders than in the other groups .
while to date the association with 5-httlpr has been replicated most frequently in lithium response studies , other authors failed to report positive findings for this polymorphism [ 186 , 194 ] .
another interesting finding for serotonin - related genes was reported in a paper by serretti et al .
in which the authors identified an association between variants in the tph gene and worse response to lithium in a sample of 90 bd and 18 major depressive patients .
a number of studies have also dealt with dopamine system genes , but to date only one paper showed positive association .
rybakowski and coworkers reported an association between the g allele and g / g genotype of the 48 a / g polymorphism in the drd1 gene and worse response to lithium .
a detailed list of pharmacogenetic studies in lithium response can be found in table 1 .
to date only one gwas has been published reporting findings on a sample of bipolar patients characterized for lithium response .
the results showed that a snp in the glutamate receptor ionotropic , ampa 2 ( glur2 ) gene was associated with time to recurrence .
however , the association did not reach the genome wide significance and the characterization of lithium response of the replication sample was performed using a different methodology .
some international joint efforts are being undertaken to identify the genetic variants involved in the modulation of lithium response .
the consortium on lithium genetics ( conligen , www.conligen.org ) , created in 2008 , has already collected a sample of 1,500 bd patients retrospectively assessed using narrow criteria for the phenotypic characterization of treatment response .
the gwas analysis is currently ongoing , and new genetic findings on lithium response and related adrs will soon be available to the scientific community .
only a small number of gene expression studies have been performed thus far using human tissues from patients characterized for the response to lithium . in 2004 ,
sun and coworkers published findings from a microarray gene expression study on lymphoblasts from bd patients excellent responders to lithium .
after chronic lithium treatment in - vitro , the authors found that the expression levels were altered for 7 genes , 5 of which were confirmed by northern blotting analysis .
these 5 genes codified for elements of pathways involved in neurotransmission and in processes that might be related to the lithium mechanism of action , such as the alpha i b - adrenoceptor ( ib - ar ) , acetylcholine receptor protein alpha chain precursor ( achr ) and camp - dependent 3 , 5-cyclic phosphodiesterase 4d ( pde4d ) .
a recent study measured the expression levels of the gene codifying for the pdz and lim domain 5 ( pdlim5 ) protein in a sample of bd patients characterized for lithium response and a sample of controls .
pdlim5 is an adaptor protein that selectively binds the protein kinase c epsilon ( pkc ) to its target , the n - type ca2 + channels , voltage - gated ca2 + channels specifcally expressed in axons .
the study failed to show significant differences in expression levels between lithium responders and partial / non responders or controls .
compared with lithium , much less evidence is available on the pharmacogenomics of other mood stabilizing treatments .
a recent study investigated the association between the -116c / g polymorphism in the x - box binding protein 1 ( xbp1 ) gene and reponse to vpa in a sample of 51 bipolar patients .
xbp1 is a an endoplasmic reticulum ( er ) stress - responsive transcription factor regulating major histocompatibility complex ( mhc ) class ii genes by binding to a promoter .
the study showed an association between the g allele ( that is associated with reduced transcription activity of xbp1 ) and a better response to vpa as compared to the c allele .
the xbp1 gene was also shown to be involved in the pathophogenesis of bd , thus supporting the notion that its product might play a role in both the disease and the reponse to mood stabilizers .
another candidate gene study examined the role of the val158met polymorphism in the catechol - o - methyl transferase ( comt ) gene in response to mood stabilizers in a sample of 144 bdi patients and 157 controls . during the treatment period ,
the results showed that the met / met genotype was more frequent in nonresponders than in responders , while no differences were detected between bd patients and controls .
however , since the sample was not sub - divided according to the different types of treatment , it is not possible to draw conclusions on the role of comt val66met in the response to specific mood stabilizers . to our knowledge , only one pharmacogenetic study based on response to ltg has been performed .
more precisely , perlis and coworkers examinated the association between 19 snps located in several genes and response to olanzapine / fluoxetine combination ( ofc ) or ltg in a sample of 88 ofc - treated and 85 ltg treated bipolar i depression patients . in regard to ltg treatment , findings showed that snps within the dopamine d2 receptor ( drd2 ) , dopamine -hydroxylase ( dbh ) , glucocorticoid receptor ( nr3c1 ) , histamine h1 receptor ( hrh1 ) and melanocortin 2 receptor ( mcr2 ) genes were associated with the treatment response .
table 2 lists most of the association studies carried out to date in response to other mood stabilizing medications . despite the constant increase in the use of atypical antipsychotics in the maintenance treatment of bd ,
still very few studies have investigated the genetic underpinnings of their therapeutic response . in 2006 , dvila et al . investigated the role of comt val158met polymorphism on psychotic features in 42 bdi patients , further testing the hypothesis of a possible influence of this variant on blood plasma concentration of metabolites of dopamine and noradrenaline and on the severity of the disorder and the response to olanzapine and lithium .
no significant findings emerged with regard to treatment response to olanzapine as well as for the other markers tested . aiming to evaluate common genetic variation for association with clinical improvement in bdi depression following treatment with ofc , perlis et al .
analyzed a cohort of 88 patients treated with ofc , showing significant association for dopamine d3 receptor ( drd3 ) and hrh1 genes and response to ofc .
the dearth of pharmacogenomic studies on antipsychotics in bd should encourage research efforts aiming to identify possible genetic predictors of response , especially in light of the ever - wider use of these agents as maintenance treatments .
pharmacogenomic approaches in bd have thus far concentrated on identifying genetic predictors of treatment response to mood stabilizers .
however , based on the evidence that lithium responsive bd patients share common clinical features , pharmacogenomics has also been used as strategy for enucleating subgroups of individuals characterized by lower phenotypic and presumably genetic heterogeneity , thus also possibly increasing the power of detecting causative variants of bd .
while linkage studies performed on bd patients responsive to lithium have highlighted some chromosomal regions ( 7q11.2 , 15q14 , 18q23 ) , further studies have reported no association for specific genes within these regions .
candidate - gene approaches have so far focused on genes codifying for elements of biological pathways shown to be target of lithium , such as proteins of the intracellular second messenger cascade mediated by inositol , wnt and neurotrophins pathways and the gsk-3 protein . along with findings from gene expression studies ,
these data have provided intriguing , although yet inconclusive , insights into the understanding of the genetic underpinnings of lithium response . as regards to the anticonvulsant vpa ,
the small number of pharmacogenetic studies performed so far failed to provide evidence for high impact genes . however , the associations reported for xbp1 and comt genes are still intriguing in light of their key role in the mechanism of action of vpa and in pathways putatively involved in modulating the pathophysiology of bd .
as for ltg , evidence for association with treatment response has thus far been reported for drd2 , -hydroxylase , glucocorticoid receptor , hrh1 and melanocortin 2 genes . while these findings come from the only candidate gene study performed to date , they shed light on genes encoding for elements shown to be involved in both the ltg mechanism of action and bd .
overall , genetic data on response to mood stabilizers are informative tools that along with data on phenotypic and environmental factors might be useful in predicting individual treatment response profiles thus paving the way to targeted therapies . to facilitate the implementation process from pharmacogenomics to personalized medicine in the long - term treatment of bd , various research areas still need to be developed .
for instance , findings are still substantially lacking as regards safety pharmacogenomics , an approach dedicated to avoiding adrs .
such events usually occur a short time after the patient experiences the drug , so the precise phenotypic response can be specifically , sensitively and accurately documented .
since it could accurately define individuals who might be at higher personal risk of an adverse event , safety pharmacogenomics acquires particular importance in mood stabilizing treatments in bd , given their long - term nature and their specific pharmacokinetic / pharmacodynamic properties .
for instance , lithium is characterized by a narrow therapeutic index , and its clinical management is greatly impacted by important side effects , above all the alteration of thyroid function and nephrogenic diabetes insipidus . moreover
, the other available therapeutic tools , anticonvulsants and antipsychotics , also present a wide range of side effects ranging from allergic reaction to haematological toxicity and weight gain . in clinical practice
, these side effects can dramatically affect the positive outcomes of long - term treatments , since patients having a high degree of response may be forced to stop effective maintenance treatments due to the onset of serious adrs . in conclusion ,
once pharmacogenomics produces clear data to reliably determine the extent of the genetic contribution to the treatment response phenotype and the predictive power of positive response outcome associated with them , the personalized therapy strategy can be quite easily integrated into clinical settings ultimately leading to the selection of drugs that will be both safe and effective for a patient .
the authors of this paper do not have any commercial associations that might pose a conflict of interest in connection with this paper . | bipolar disorder ( bd ) is a chronic and often severe psychiatric illness characterized by manic and depressive episodes . among the most effective treatments ,
mood stabilizers represent the keystone in acute mania , depression , and maintenance treatment of bd . however , treatment response is a highly heterogeneous trait , thus emphasizing the need for a structured informational framework of phenotypic and genetic predictors . in this paper
, we present the current state of pharmacogenomic research on long - term treatment in bd , specifically focusing on mood stabilizers .
while the results provided so far support the key role of genetic factors in modulating the response phenotype , strong evidence for genetic predictors is still lacking . in order to facilitate implementation of pharmacogenomics into clinical settings ( i.e. , the creation of personalized therapy ) ,
further research efforts are needed . |
the inhibition of 1 on cell - free melan - a tyrosinase
was demonstrated by a lineweaver burk plot .
the results shown
in figure 1 revealed both 1 and
resveratrol to be noncompetitive inhibitors of tyrosinase , with kojic
acid being a competitive inhibitor .
thus , the addition of 1 to the reaction mixture led to a reduction of the maximal velocity
( vmax = 4.7 10 ) and km value ( 2.4 mm l - dopa )
of tyrosinase activity . the vmax value
decreased in a dose - dependent manner from 3.1 10 to 2.4 10 and 2.0 10 in the presence of 60 , 120 , and 240 m of 1 ,
respectively .
the ki value also decreased
from 3.1 to 2.8 and 2.5 mm , respectively .
resveratrol , a stilbenoid
of the same oxidative level as 1 , exhibited comparable ki and vmax values
at 120 m ( table 1 ) .
these results are
in agreement with a previous observation that the tyrosinase inhibitory
activity of stilbenes varies with the extent of hydroxylation , having
more potent inhibitory activity with a higher degree of hydroxylation .
a pretreatment experiment was also performed
in which tyrosinase was mixed with 1 for 24 h prior to
kinetic analysis .
the ki and vmax values of pretreated samples ( data not shown ) were
similar to those without the pretreatment .
this finding suggests that
the binding between 1 and tyrosinase is likely reversible .
taken together , the available evidence indicated 1 to
be a reversible noncompetitive inhibitor of melan - a tyrosinase showing
a higher potency than kojic acid ( table 1 ) .
inhibitory
effects of 60 , 120 , and 240 m 1 ,
120 m kojic acid , or 120 m resveratrol on tyrosinase
activity in murine melan - a cells .
the vmax and km values in the absence ( control )
or presence of 1 with l - dopa as the substrate
are analyzed using lineweaver
the kinetic parameters are obtained
with l - dopa as a substrate using the lineweaver
km ( michaelis constant ) and ki ( inhibition constant ) values are represented
as molar concentration , and vmax ( maximum
velocity ) values are expressed as the change of absorbance at 490
nm per second .
forskolin
and 12-o - tetradecanoylphorbol-13-acetate
( tpa ) are inducers of melanogenesis via the pka and pkc pathways ,
respectively .
the present results showed
that 1 reduces tyrosinase activity in melan - a cells in
a dose - dependent manner in the presence or absence of these melanogenesis
inducers ( figure 2a ) . in the presence of forskolin ,
similarly , the enzyme activity was reduced from 100% to
65% in the presence of tpa .
these results suggest that 1 is a more potent blocker of the pka melanogenic pathway than the
pkc pathway .
possible inhibitory mechanisms of 1 include
the inhibition of melanocortin-1 receptor from forskolin binding ,
inhibition of camp synthesis , or inactivation of camp responding element ( cre ) binding protein .
further study
is warranted to elucidate the inhibitory mechanism of 1 on the pka pathway .
cells were incubated with or without 1 in the medium
containing 20 nm 12-o - tetradecanoylphorbol-13-acetate
( tpa ) , 1 m forskolin , or vehicle alone for 3 days .
tyrosinase
activity was measured by l - dopa oxidation ( a ) , and cell viability
was determined by the sulforhodamine b assay ( b ) .
the values are denoted
as mean sd of triplicate experiments . among stilbene compounds , resveratrol and oxyresveratrol
the number and location of the hydroxy group(s ) , as well as the trans - olefin structure of the stilbene skeleton , have been
shown to be associated with the inhibitory properties on murine tyrosinase .
on the other hand , glycosylation of the hydroxy
group did not show a significant effect . in our hands ,
1 was the least cytotoxic compound among
the hydroxystilbenes tested including resveratrol , oxyresveratrol ,
and picetanneol ( supporting information , figure s1 ) .
moreover , it did not exert any cytotoxic effect in
samples with or without forskolin ( figure 2 ) .
the nascent tyrosinase core is a polypeptide of 60 kda that is n - glycosylated to become a 70 kda folding intermediate and
further post - translationally modified to a mature form of 80 kda in
the golgi apparatus . in cases
where the
polypeptide is misfolded or not properly glycosylated , it will remain
bound to calnexin in the er until a quality glycoprotein intermediate
is formed .
if there is misfolding or aberration trafficking from the
late endosome to melanosome , the enzyme will likely be trapped in
the lysosome .
consistent with the observations
made by others , functional tyrosinase colocalizes
with trp-1 ( figure 3a and b ) , but not with
lamp-1 of the lysosome ( figure 3c and d ) or
calnexin of the er ( figure 3f and g ) , regardless
of the presence or absence of 1 .
these findings suggest
that no aberrant tyrosinase is trapped in the er and lysosome in 1-treated cells and tyrosinase is properly transported outside
to the melanosomes where it forms a complex with trp-1 . as a positive
control , calnexin and tyrosinase
clearly colocalize to the er in resveratrol - treated
cells ( figure 3h ) , because resveratrol is capable
of retarding the post - translational - modified tyronsinase in the er . colocalization of both tyrosinase and lamp-1
were also observed ( figure 3e ) in progesterone - treated
cells in which misfolded tyrosinase was induced and retarded in the
lysosomes .
confocal immunofluorescence
microscopy of melan - a melanocytes incubated
in the presence of 1 , progesterone , or resveratrol .
the
locations of trp-1 ( red , a , b ) , lamp-1 ( red , c e ) , or calnexin
( cnx , red , f
overlay
of the red and green images resulted in a yellow fluorescence , indicating
strong colocalization of the targeted proteins .
cells were treated
with vehicle alone ( a , c , f ) , or 1 ( 60 m , b , d ,
g ) for 3 days , progesterone ( 15 m ) for 4 days ( e ) , or resveratrol
( 85 m ) for 1 day ( h ) .
glycosylation analysis further confirmed the normal trafficking
of tyrosinase in both untreated and 1-treated cells ( figure 4 ) .
the immature oligosaccharide added to tyrosinase
in the er can be digested only with endoglycosidase h ( endo h ) , whereas
both mature and immature oligosaccharide added to tyrosinase in the
golgi apparatus can be removed by peptide - n - glycosidase
f ( pngase f ) .
results of western blotting
revealed two bands : an upper band at 80 kda for the mature and endo
h - resistant form of tyrosinase and a lower band at 60 kda for the
immature and endo h - sensitive form ( figure 4 ) .
no immature endo h - sensitive form of tyrosinase was present in
both the untreated control and 1-treated murine cells
after being digested with endo h. these findings agree with the immunofluorescence
results to indicate that 1 does not interfere with tyrosinase
maturation in the er , golgi apparatus , and protein trafficking to
the melanosome .
the deglycosylated tyrosinase digested by pngase f
( 60 kda ) represented the total amount of tyrosinase .
protein lysates
were obtained from 1-treated melan - a cells in forskolin - supplemented
medium .
the lysates
were incubated with either peptide - n - glycosidase
f ( pngase f ) or endoglycosidase h ( endo h ) following the manufacturer s
instruction . the western blot was analyzed for the amounts of glycosylated
or deglycosylated tyrosinase .
the upper band at 80 kda and the lower
band at 60 kda represent the mature endo h - resistant form and immature
endo h - sensitive form of tyrosinase , respectively .
stilbene derivatives such as resveratrol and 2,2-dihydroxy-5,5-dipropylbiphenyl
have been associated with the unfolded protein response that triggers
er stress and subsequent apoptosis , but 1 seems to induce neither er stress nor lysosomal storage
stress as suggested by the above results . despite the structural similarity
between 1 and resveratrol , they do not share the same
canonical maturation retardation and apoptotic properties .
being a weak acid as conferred from its phenolic
groups , and unlike basic compounds such as quinolines that would alter
the lysosomal ph and retard lysosomal function,1 appears not to alter the acidic lysosomal
ph , or otherwise it would have resulted in lysosomal storage stress .
melanogenesis is controlled by mitf , a key regulator of a number
of melanogenic proteins such as tyrosinase , trp-1 , and trp-2 . since
melanogenesis is significantly increased through the forskolin
pka
pathway ( figure 2 ) , the inhibitory action of 1 on these proteins was analyzed further in forskolin - supplemented
melan - a cells .
it was found that 1 suppressed the expression
of mitf and tyrosinase in a dose - dependent manner , but the reduction
of trp-1 expression was not prominent ( figure 5 ) .
such a down - regulation of mitf and tyrosinase protein could explain ,
at least partially , the reduction of l - dopa oxidation with
increasing doses observed in figure 2a .
expression
of melanogenesis - related proteins in melan - a melanocytes
after 1 treatment for 1 day in fsk - supplemented medium .
the expressions of mitf ( a ) , tyrosinase ( b ) ,
and trp-1 ( c ) were analyzed by western blotting , and the expression
of melanogenic proteins relative to actin is shown in panel ( d ) .
trp-1 influences tyrosinase activity
by stabilizing and forming
complexes with tyrosinase . without stabilization from trp-1 during
the processing and intracellular trafficking , tyrosinase will be prompted
to degrade in the proteasome , leading to a reduction of pigment formation .
this phenomenon has been demonstrated in mutant
brown melan - b melanocytes in which the trp-1 locus is mutated .
co - immunoprecipitation using antibodies against trp-1 resulted in
a reduction of immunoprecipitated tyrosinase after treatment with 1 ( figure 6b ) , whereas trp-2 was not
pulled down together with tyrosinase ( figure 6c ) .
these results are in agreement with the findings of hearing and
associates that trp-1 forms a complex with tyrosinase but not with
trp-2 .
therefore , reduction of tyrosinase
activity after treatment with 1 was not only caused by
the down - regulation of tyrosinase expression but also affected by
the reduction of tyrosinase / trp-1 complex formation .
association of tyrosinase
and trp-1 in melan - a melanocytes after
treatment with 1 for 3 days in forskolin - supplemented
medium .
co - immunoprecipitation with antibody against trp-1 proteins
showed that trp-1 ( a ) and tyrosinase ( b ) , but not trp-2 ( c ) , proteins
were co - immunoprecipitated . in conclusion , compound 1 displayed antimelanogenic
activity in murine melanocytes .
it noncompetitively inhibited the
enzyme activity of tyrosinase , down - regulated the expression of melanogenic
proteins , and reduced the tyrosinase / trp-1 complex formation .
studies
of the antimelanogenic properties of 1 and its analogues
in the future using human melanocytes are needed to confirm these
activities .
2,3,5,4-tetrahydroxystilbene-2-o--d - glucopyranoside ( 1 ) was
isolated from polygonum multiflorum , and a larger
quantity was obtained from the hong kong jockey club institute of
chinese medicine ; the purity was determined to be > 98% by hplc .
kojic
acid , forskolin , 12-o - tetradecanoylphorbol-13-acetate ,
progesterone , and resveratrol were purchased from sigma - aldrich co.
( st louis , mo , usa ) .
all antibodies were obtained from santa cruz
biotechnology ( dallas , tx , usa ) , unless otherwise specified . mouse melan - a melanocytes derived from
c57bl/6 mice were a gift from professor d. c. bennett ( st george s
hospital medical school , london , uk ) .
cells were cultured in rpmi-1640
medium supplemented with 5% fbs , 100 iu / ml penicillin ( sigma ) , 100
g / ml streptomycin sulfate ( sigma ) , and 200 nm tpa ( sigma ) for
cell attachment and proliferation . during treatment , cells were cultured
in the absence of tpa unless specified .
cells used in the experiments
were restricted to passages of < 30 as recommended by the donor .
the sulforhodamine
b ( srb , sigma ) colorimetric cytotoxicity assay was adopted in this
study .
briefly , melan - a melanocytes ( 20 000 cells/0.1 ml / well )
were treated with the test compounds in 96-well corning costar plates
for 72 h. the reaction was stopped and subjected to srb cytotoxicity
assays as described previously .
the 3,4-dihydroxy - l - phenylalanine ( l - dopa ) ( sigma ) conversion assay is
a colorimetric assay in which the oxidation rate of l - dopa
specifically reflects the tyrosinase activity .
tyrosinase inhibitory
property of 1 in the presence of various melanogenic
inducers was investigated under the same conditions .
after treatment ,
one volume cell lysate in 20 mm tris/0.1% triton x-100 ( ph 6 ) reacted
with one volume 1 g / ml l - dopa .
data represent the mean values and
standard deviations of triplicate assays in at least three separate
experiments .
the melan - a
cells were rinsed with phosphate buffer saline twice , then lysed with
20 mm tris/0.1% triton x-100 ( ph 7 ) .
the protein lysate ( 50 g )
was preincubated with the test compounds , i.e. , 1 , resveratrol ,
or kojic acid , for 10 min at room temperature before reacting with l - dopa ( ph 6 ) .
the rate of dopachrome formation
was measured at 490 nm per 20 s for a period of 10 min at 37 c
using a fluostar microplate reader ( bmg labtech , ortenberg , germany ) .
the tyrosinase activity was determined based on the rate of l - dopa oxidation ( absorbance at 490 nm per second ) ; michaelis
menten
constants ( km ) and maximal velocity ( vmax ) were calculated from the lineweaver
for colocalization of
tyrosinase and trp-1 , cells cultured on coverslips were fixed in 100%
methanol , permeablized with 0.2% triton x-100 , and blocked in 2% bovine
serum albumin before subjected to confocal immunofluorescence microscopy .
the procedures for costaining tyrosinase with organelle marker proteins ,
i.e. , lamp-1 and calnexin ( sigma ) for lysosome and endoplasmic reticulum ,
respectively , have been described in a previous paper .
cells cotreated
with 1 m
forskolin with or without 1 before total proteins were
extracted with phosphosafe extraction buffer ( novagen , darmstadt ,
germany ) .
the proteins were resolved on a 10% sds - page gel and transferred
onto a pvdf membrane using a semidry transfer cell ( bio - rad , hercules ,
ca , usa ) .
the blot was blocked with 3% bovine serum albumin before
incubation with primary antibodies against tyrosinase , trp-1 ( generous
gift from dr .
hearing , nih , bethesda , md , usa ) , trp-2 , mitf ( thermo
fisher scientific , pittsburgh , pa , usa ) , or -actin .
signals
were detected by incubating the blot for 1 h at room temperature with
secondary antibodies , horseradish peroxidase - conjugated goat anti - mouse ,
or donkey anti - goat , or goat anti - rabbit ( dako cytomation , glostrup ,
denmark ) , and visualized using an enhanced chemiluminescence system
( ecl , ge healthcare , lower chalfont , uk ) . for reprobing with other
primary antibodies ,
previous antibodies were stripped away using the
reblot plus western blot recycling kit ( millipore , temecula , ca , usa ) . protein lysates ( 10 g )
obtained in the immunoblotting analyses were digested with either
1000 units of endoglycosidase h ( new england biolabs , beverly , ma ,
usa ) or 1000 units of peptide - n - glycosidase f ( new
england biolabs ) according to the manufacturer s instruction .
in brief , the protein lysates were subjected to denaturation by treating
with glycoprotein denaturing buffer at 100 c for 10 min .
the
denatured lysates were incubated with endo h or pngase f at 37 c
for 1 h and then subjected to western blotting analysis using anti - tyr
antibody .
tyrosinase and trp-1 proteins
were co - immunoprecipitated using a dynabeads co - immunoprecipitation
kit with minor modifications ( invitrogen , grand island , ny , usa ) .
briefly , harvested cell pellets were lysed with immunoprecipitation
lysis buffer supplemented with 0.1 m nacl , 0.5 mm pmsf , and 10 g / ml
apotinin on ice for 30 min .
the extracted proteins of the same amount
from 1-treated or untreated samples ( 1 mg each ) were
reacted with antibody - coupled dynabeads overnight and separated by
sds - page analysis . following protein transfer on pvdf blot ,
melanogenic
proteins were detected by corresponding antibodies using ecl plus
( ge healthcare ) or supersignal west dura chemiluminescent substrate
( thermo fisher scientific ) . | 2,3,5,4-tetrahydroxystilbene-2-o--d - glucopyranoside ( 1 ) , isolated
from polygonum
multiflorum , is a noncompetitive inhibitor of tyrosinase
in cell - free kinetics ; it reduced the vmax values in a dose - dependent manner .
compound 1 inhibited
pka - induced melanogenesis , reduced the protein expression of tyrosinase
and its transcription factor , the microphthalmia - associated transcription
factor , and lowered the complex formation between tyrosinase and tyrosinase - related
protein 1 ( trp-1 ) .
immunofluorescence microscopy revealed no association
of tyrosinase with the endoplasmic reticulum or lysosomes , implying
the absence of a direct effect of 1 on the maturation
process of the enzyme .
the antimelanogenic activity of 1 is likely mediated through a noncompetitive inhibition on tyrosinase ,
down - regulation of the expression of melanogenic proteins , and reduction
of tyrosinase / trp-1 complex formation . |
in africa , the health impact of schistosomiasis , whether caused by schistosoma haematobium or by s. mansoni , is a well - known public health problem , one that is now receiving well - deserved attention .
this attention is focused primarily on the most vulnerable part of the community , that part of the population that is heavily debilitated by the disease and will benefit from mass drug administration . for the purpose of control and local elimination of the parasites , a quick sensitive test that may be low in specificity is acceptable but there is need to improve the detection of infection in chronic stages of disease or when parasitaemia is low .
the detection of infection among adults with long standing chronic infections is important particularly in the hospital environment when sequelae of the infection are suspected .
bladder damage and even bladder cancer are common problems in endemic areas and a definitive diagnosis which has high sensitivity , specificity , and can be carried out in a diagnostic laboratory with adequate facilities is needed .
definitive diagnosis of schistosomiasis is dependent on the demonstration of parasite eggs in urine or stool , and more recently the detection of circulating antigens ; however , these tests have not been assessed in adults .
detection of parasite - specific dna is an option that is being used with several infections for example , malaria , and it presents an opportunity with schistosoma haematobium and perhaps with s. mansoni . the importance of this has been shown in a recent study involving s. haematobium , it was evaluated using latent class modeling and was shown to detect parasite - specific dna fragments in adults both when eggs were present in urine and in 10% of cases , where eggs were not present .
gelfand in a very careful clinical analysis of mainly adults infected with bilharzia ( schistosomiasis ) concluded that in rhodesia ( now zimbabwe ) bilharziasis , both the urinary and intestinal is to be regarded as having serious consequences , he was speaking about the infection in adults as well as children . in a more recent review , king and dangerfield - cha
reiterate this importance although they do not cover the material in clinical detail as does gelfand ( loc cit ) .
more specifically and focusing on s. haematobium the role of this species in bladder cancer is well studied , not only in egypt but also in kenya , ghana , and zimbabwe . whereas in children
this infection causes haematuria and frequently bladder polyposis , these problems ameliorate following treatment ; however , the more severe squamous cell carcinoma appears in the 3rd and 4th decades of life .
the methods we have used to diagnose schistosomiasis decrease in sensitivity in adulthood and the question arises , are the current diagnostic tests sufficiently sensitive to detect infection in all age groups ?
community - based surveys done using the presence of schistosome eggs in urine or faeces as positive infection always show a similar population trend .
prevalence rises to a peak during the years 1015 , then declines through the 20s , 30s and 40s to well less than half of the childhood peak .
this surely indicates that the parasite causes the greatest health impact among children , but does it ?
as lesions form around the schistosome eggs , particularly in the bladder , granulomas develop and egg passage to the exterior becomes hindered .
there is a strong inflammatory response from the host and over time metaplasia sets in and eventually the chronic inflammation initiates the development of cancer .
ultrasound examinations done in the ghana study certainly show the extent of bladder damage in adult bladders ; yet there were numerous people in ages over 30 years who showed severe damage but no evidence of eggs in the urine .
a detailed study of the sensitivity and specificity of various diagnostic tests used in this study included haematuria , antigen detection , egg detection , and antibody detection but the results were equivocal and indicated the need of a more sensitive , yet specific test for improved diagnosis of schistosome infection .
detection of parasite dna in blood specimens is now an accepted procedure , whether the dna is intracellular or extracellular , the presence of trace but detectable quantities of specific fragments provides evidence of the organism .
malaria parasites can be detected in haemolysed blood specimens , but an advance occurred when plasmodium falciparum - specific dna was also demonstrated in saliva and urine .
this dna was free in saliva and in the urine clearly passed through the kidneys prior to excretion and was undamaged .
collecting urine for subsequent pcr examination has logistic issues as the urine must be fixed or frozen rapidly to 20c for storage and transport .
operating on the hypothesis that schistosome - specific dna is passed in urine , we proposed that it could be trapped in a convenient paper filter .
as such , this would obviate considerable handling problems and it was shown to be the case .
a 50 ml specimen of urine was passed through coarse filter paper ( whatman no .
3 ) ge healthcare , bucks , uk . the paper is sturdy and will hold a cone when folded , filtration could be processed in the neck of a disposable vessel , the paper subsequently dried away from aerial and insect contamination and if maintained dry , the dna would be preserved .
workers in kenya and israel had identified a specific fragment of dna ( dra1 ) that was detectable from snails infected with s. haematobium miracidia . the fragment is specific for s. haematobium and was shown to be more sensitive than egg detection or haematuria with high specificity , particularly among adults where egg detection versus pcr showed a sensitivity of 59% .
this infers that among adults , egg detection is unsatisfactory and supports the comment made above .
analysis of results from a large - scale epidemiological study comparing three measurements , haematuria , presence of parasite eggs , and detectable parasite - specific dna using latent class modeling was undertaken .
this is a statistical technique that models the probability of each combination of test results to give the true infection status ( this is the latent class variable true infection which is unobservable ) .
this model provides response probabilities for sensitivity ( se ) and specificity ( sp ) for each of the test procedures and finally indicates statistically which test is the most sensitive ( i.e. , with fewest false positives ) and most specific ( with fewest false negatives ) .
it was shown that presence of dra1 in males exceeded haematuria ( se 87.6% and sp 34.7% ) and detection of eggs ( se 70.1% and sp 100% ) . in females ,
presence of dra1 exceeded haematuria ( se 86.7% and sp 77% ) and presence of eggs ( se 70.1% and sp 100% ) .
this suggests that detection of dra1 is a definitive test for the presence of s. haematobium infection .
analysis of the dataset described above ( figure 1 ) shows that the proportion of positive cases detected for each age group in the study was higher when dra1 was detected by dna amplification than when parasite eggs were observed for all age groups ( p = 0.0005 ) , although when stratified across age groups , significant differences were only seen in the 2029- ( p = 0.004 ) and 4049- ( p = 0.02 ) year - old age groups .
the message from these studies is that if adult populations are examined for schistosome infection , if the only test applied is examination of urine for eggs , a significant number of people will be declared uninfected , yet they may yet be infected .
this example has shown that , for schistosomiasis , detection of parasite - specific dna in urine is feasible , highly sensitive , and specific . to make this applicable in endemic countries where it may be difficult to operate thermocyclers and electrophoretic equipment , there is an alternative method that is already being promoted , namely , the loop - mediated isothermal amplification ( lamp ) technique .
this method is well established as a viable and economical approach to dna amplification and detection .
work needs to be done on refining the process for both species and developing it in a way that it can be put to use in the field as well in the hospital and diagnostic centre
. the introduction of tests of high sensitivity and specificity will be valuable in monitoring the elimination programmes that are being carried out in many parts of the endemic world .
in particular , when interventions are introduced , monitoring of treated individuals with very low parasitaemia will be necessary because of the risk of maintaining transmission to snails , and reinfection of the community .
detection of parasite - specific dna will become an important means of identifying and hopefully eliminating risk foci .
there is no inference here that dna detection is promoted to supersede other standard means of diagnosing schistosome infection .
they are well tried and serve a role in most circumstances ; however , as the need for more sensitive tests arise as outlined above , dna detection adds another diagnostic which will expand the ability of the epidemiologist to collect data pertinent to any programme designed to eliminate the disease or warn clinicians of the potential problem of bladder cancer or some other sequel of this debilitating parasitic infection . | schistosomes are long - lived parasites , hence schistosomiasis is a chronic disease with severe long - term implications .
however , definitive diagnosis of active infection has been difficult because demonstration of infection has depended on detecting parasite eggs in urine and/or stool . in the case of schistosoma haematobium which parasitizes the urinogenital system ,
this method has low sensitivity in adults .
detection of parasite - specific dna in urine has been demonstrated and this has similar specificity but improved sensitivity .
the implications of this new procedure and the impact on diagnosis are discussed . |
the online version of this article ( doi:10.1007/s11032 - 015 - 0192-x ) contains supplementary material , which is available to authorized users .
supplemental table 1 primer pairs used in this study ( xls 32 kb )
supplemental fig
. 1 graphical genotype of a bc2f5 line ( hk114 ) used for substitution mapping of rbg2 .
positions of marker loci used for genotyping are shown as horizontal lines and were obtained from the linkage map of bils derived from a cross between kele and hitomebore ( mizobuchi et al .
the arrowhead shows rm11727 , the nearest marker detected by qtl analysis of an f2 population derived from hitomebore hk19 ( described in fig . 2 ) .
white boxes indicate regions homozygous for hitomebore marker alleles , black boxes indicate regions homozygous for kele marker alleles , and the gray box indicates a heterozygous region .
| bacterial grain rot ( bgr ) , caused by the bacterial pathogen burkholderia glumae , is a destructive disease of rice . at anthesis ,
rice panicles are attacked by the pathogen , and the infection causes unfilled or aborted grains , reducing grain yield and quality .
thus , increasing the level of bgr resistance is an important objective for rice breeding .
a quantitative trait locus ( qtl ) on rice chromosome 1 that controls bgr resistance was previously detected in backcross inbred lines ( bils ) derived from a cross between kele , a resistant traditional lowland cultivar ( indica ) that originated in india , and hitomebore , a susceptible modern lowland cultivar ( temperate japonica ) from japan .
further genetic analyses using a bc3f6 population derived from a cross between a resistant bil ( bc2f5 ) and hitomebore confirmed that a qtl for bgr resistance was located on the long arm of chromosome 1 . to define more precisely the chromosomal region underlying this qtl , we identified nine bc2f6 plants in which recombination occurred near the qtl .
substitution mapping using homozygous recombinant and nonrecombinant plants demonstrated that the qtl , here designated as resistance to burkholderia glumae 2 ( rbg2 ) , was located in a 502-kb interval defined by simple sequence repeat markers rm1216 and rm11727.electronic supplementary materialthe online version of this article ( doi:10.1007/s11032 - 015 - 0192-x ) contains supplementary material , which is available to authorized users . |
vigabatrin ( vgb ) is an antiepileptic drug that irreversibly inhibits brain -aminobutyric acid ( gaba ) transaminase ( 1 ) .
vgb is used as first - line treatment for infantile spasms , especially those secondary to tuberous sclerosis , and as an adjunct therapy for medically resistant localization - related childhood epilepsies ( 2 ) . following an initial report of 3 patients who experienced concentric visual field losses following vgb treatment ( 3 ) , several additional studies showed visual field constriction during vgb therapy .
cross - sectional studies have estimated the prevalence of vgb - associated visual field defects ( vfds ) as 20 - 50% in patients receiving vgb mono- or adjunct therapies .
various groups have found that longer treatment ( 4,5 ) and higher total doses ( 6 - 8 ) are associated with greater visual field losses in adults , while other studies dispute these correlations ( 9,10 ) . a characteristic pattern of ocular dysfunction has been observed in vgb - treated patients , including peripheral visual field constriction primarily in the nasal area , which is rarely seen in other conditions ( 11 - 13 ) .
although these studies have shown evidence of vgb - associated vfd , vgb is highly effective and has a low occurrence of side effects or metabolic interactions .
thus , it is still commonly used in the clinic , especially for treatment of infantile spasms and tuberous sclerosis with intractable epilepsy .
most studies on vgb - associated vfd to date have been performed in adult patients , in part because it is difficult to estimate the frequency of this condition in pediatric patients , who are generally unable to cooperate in visual field testing .
we therefore sought to estimate the prevalence , type , severity , and risk factors of vgb - attributed vfds in pediatric patients .
we retrospectively investigated the medical records of 67 pediatric epileptic patients who were treated with vgb at the department of pediatrics of the asan medical center and had undergone ophthalmological examinations . for inclusion , each patient was required to have a diagnosis of epilepsy , no evidence of ophthalmologic disease upon initial examination , and a history of treatment with antiepileptic drugs , including vgb , during the observation period .
patients were excluded if they had poor compliance or non - concentric vfds , which corresponded to brain organic lesions ( e.g. intracranial hemorrhage ) .
the enrolled patients had been given full ophthalmologic examinations , including visual acuity , ophthalmoscopy , measurement of intraocular pressure , and visual field examination using program 30 - 2 of the humphrey field analyzer .
the severities of the vgb - attributed vfds were scored as grade i ( minimal , 25 - 30 ) , grade ii ( moderate , 20 - 30 ) , grade iii ( moderate to severe , < 20 ) , or grade iv ( severe , ring scotoma)(fig .
data were analyzed by fisher 's exact test and student 's t - test using spss , version 10.01 .
the patient population consisted of 67 vgb - treated patients , 41 males ( 61.2% ) and 26 females ( 38.8% ) , with a mean age of 13 yr ( range ; 6 6/12 yr to 21 yr old ) .
of the 67 patients , 15 ( 22% ) had vfds , which we attributed to vgb , and 52 ( 78% ) did not .
of the patients with vgb - attributed vfds , 13 had nasal arcuate type , 1 had nasal and temporal constricted type , and 1 had nasal constricted type ( table 1 ) .
seven patients had grade i vgb - attributed vfds , 5 had grade ii , 2 had grade iii , and 1 had grade iv ( table 2 ) . to identify potential risk factors for vgb - attributed vfds , we compared the vfd and non - vfd patient groups with respect to medications and other clinical parameters .
there were no significant differences between the vfd and non - vfd patients in terms of mean age at the beginning of treatment , total treatment duration , cumulative vgb dose , maximum vgb dose , sex distribution , structural abnormalities in the brain , or whether vgb was given alone or in conjunction with another drug ( p>0.05 each ) ( table 3 ) .
however , there were no cases of vgb - attributed vfds in patients with total treatment durations < 2 yr ( table 4 and cumulative doses < 10 g / kg ( table 5 ) .
our results indicate that the prevalence of vfds in vgb - treated children is 22% .
this is consistent with recent reports of vfd prevalences ranging from 15 to 20% in western children ( 7,13 - 17 ) , but is lower than the about 30% previously observed in vgb - treated children .
this may be due to our patient population , which was limited to older patients capable of cooperating with ophthalmologic and visual field examinations .
the exclusion of younger patients may have decreased the estimated prevalence of vgb - associated vfds , because these younger patients , especially those with infantile spasms , require high dose , sustained treatment with vgb and might be more prone to vfds .
in addition , we used automated static perimetry to detect vgb - attributed vfds , which may have influenced our results .
the utilized 30 - 2 humphrey field analyzer has the advantages of being generally available , investigator independent , easy to use , simple to standardize , and highly effective at detecting nasal field defects . however , because of its limited spatial context , the humphrey field analyzermay miss mild to moderate visual field defects in the temporal fields .
these are more reliably demonstrated by other techniques , such as kinetic ( goldmann ) perimetry ( 10,18 ) .
however , vgb - associated vfds typically present as concentric peripheral losses that are most severe nasally , with relatively milder temporal losses ; these characteristics indicate that although some mild - to - moderate defects may be underestimated using this technique , the static humphrey field analyzer should reliably detect vgb - attributed vfds .
a final limitation in the present work is our inability to conclusively identify a causal link between vgb therapy and the observed vfds ; for this to be possible , we would have to enroll only patients who had completed vfd testing prior vgb therapy , which is rare .
however , this is a limitation in most , if not all , retrospective studies on pediatric vbg - attributed vfds . here ,
we believe that the noted vfds are vgb - attributed vfds because they have the nasal - dominated pattern that is characteristic of vgb - attributed vfds but is not seen in vfds due to other causes .
interestingly , our analysis did not reveal any correlations between the incidence of vgb - associated vfds and the mean age at the beginning of treatment , total treatment duration , cumulative dose or maximum dose .
however , we did note that no vgb - attributed vfds were seen in patients with total treatment durations < 2 yr and cumulative doses < 10 g / kg .
the latter trends are consistent with previous findings in adults , wherein longer treatment durations ( 5 ) and higher total doses of vgb ( 7 ) were associated with greater losses in visual fields .
these possible associations are particularly important in pediatric patients , because high dose , sustained vgb treatment is commonly used to treat infantile spasms , especially those from tuberous sclerosis ( 19 ) .
if the treatment duration and cumulative dose of vgb are related to vfd , shorter treatments or lower doses should be investigated by linear regression analysis and prospective trials for their abilities to be therapeutically effective while minimizing the occurrence of vfds .
another important finding was that the most common type of vgb - attributed vfd in our study population was the nasal arcuate type , and that severity grades i and ii were most frequent .
these findings are consistent with previous reports that vgb - associated vfds involve the nasal field ( 10 ) , and indicate that automated perimetry is likely to be more reliable for identifying vgb - associated vfds
. however , automated perimetry tends not to lend itself to assessment of pediatric patients , suggesting the need for more reliable and cooperation - independent testing methods .
while vgb - attributed vfd was originally considered irreversible ( 20 ) , more recent studies have shown that this condition can be reversed in adult ( 21 - 23 ) and pediatric ( 13,24,25 ) patients after discontinuation of vgb therapy .
these reports suggested that patients can recover their retinal function despite apparently permanent damage to retinal cells .
although the mechanism by which vgb causes vfd is still not known , studies have suggested that vgb causes a general defect in the retinal network rather than damaging the outer retinal layer ( 26 ) .
theoretically , such a defect could functionally recover , at least to some extent , in younger people whose neuronal systems still possess significant plasticity . in the future
, it may be possible to perform follow - up visual field examinations on our study population , in an effort to examine reversibility in pediatric patients . in sum
, the results of the present study suggest that vfd occurs in vgb - treated children , but at a lower prevalence than that seen in adults ( 5,9 - 11,14,26 - 29 ) .
our results also suggest that there is a relationship between vgb - attributed vfds and the duration and total dose of vgb .
therefore , we recommend that the total dose and duration of vgb treatment should be reduced as much as possible , and that patients receiving vgb should be given visual field examinations prior to vgb treatment , and at regular intervals thereafter .
overall , the clinical decision to use vgb should be based on a risk - benefit analysis of each individual case . | we studied the prevalence , type and severity of vigabatrin ( vgb)-attributed visual field defects ( vfds ) , and used these data to assess the associated risk factors in pediatric patients .
medical records were retrospectively reviewed for 67 pediatric patients who received vgb alone or in combination with other antiepileptic drugs , and who had undergone visual field examinations using a humphrey visual field analyzer .
of the 67 patients , 15 had vgb - attributed vfds : 13 had nasal arcuate type , 1 had nasal and temporal constricted type and 1 had nasal constricted type . in terms of severity , 7 patients had grade i vgb - attributed vfds , 5 had grade ii , 2 had grade iii , and 1 had grade iv . although there were no significant differences between the vfd and non - vfd groups with regards to all tested parameters , there were no cases of vgb - attributed vfds in patients with total treatment durations < 2 yr and cumulative doses < 10 g / kg . in conclusion , the prevalence of vgb - attributed vfds in vgb - treated pediatric epilepsy patients was 22% .
the high frequency of vgb - attributed vfds indicates that physicians should inform all patients of this risk prior to vgb treatment and perform periodic visual field examinations . |
paraneoplastic pemphigus ( pnp ) is an extremely severe autoimmune blistering disease associated with neoplasms ; the disease is most commonly of lymphoproliferative origin and presents a high mortality 1 .
follicular dendritic cell ( fdc ) sarcoma is rare and classified either as a conventionaltype or as an inflammatory pseudotumor ( ipt)like variant .
iptlike fdc sarcoma is a rare neoplasm with different histologic appearance and behavior from those of the classical fdc sarcoma 2 .
a review of 38 cases of iptlike fdc sarcoma reveals that this tumor occurs predominantly in females with a predilection for liver and spleen , with a strong association with epstein
less than 30 cases of fdc sarcoma have been reported , in which the patients were diagnosed with myasthenia gravis ( mg ) or pnp 3 , 4 .
moreover , two cases of castleman 's disease were associated with mg and pnp , suggesting a putative alternative linkage of pnp , mg , and the associated neoplasms 6 , 7 . to date , we have not found any reports on iptlike fdc sarcoma associated with mg or pnp .
a 60yearold chinese woman presented a 6month history of recurrent weakness and increasing upper extremity fatigue , fluctuating diplopia , and ptosis of both eyes , finally showing dysarthria and dysphagia . during 6 months
several masses had been found in her left axillary region and neck for 15 years , and the masses were resected every 4 years without radiotherapy or chemotherapy . the last surgery was 2 years prior to the study . after admission , thoracic computed tomography ( ct ) showed several masses in the left axillary and neck , and the diameter of the maximal tumor was 6.4 cm .
core needle biopsy revealed spindle tumor cells arranged in a storiform pattern with scattered lymphocytes ( fig .
spindle tumor cells contained vesicular chromatin , small nucleoli , and thin smooth nuclear membranes ( fig .
immunohistochemical studies revealed that the tumor cells were positive for ki67 ( + 20% ) ( fig .
2a ) , vimentin , cd21 , cd35 , cd20 , egfr , cd68 , s100 ( fig .
2b ) , cd1a , cd3 , and cd79a and negative for ema , ck , cd23 , cd34 , hmb45 , cd30 , mpo , and lysozyme . however , ebvencoded rna was detected by in situ hybridization and was negative .
( a ) microphotograph showing spindle tumor cells arranged in a storiform pattern with scattered lymphocytes ( 10 ) .
( b ) spindle tumor cells contained vesicular chromatin , small nucleoli , and thin smooth nuclear membranes ( 20 ) .
( b ) some of the spindle cells are positive for s100 ( 10 ) .
after admission , treatment was immediately started with pyridostigmine at a dose of 60 mg three times per day following a subtle improvement of muscle weakness .
five days later , the patient developed severe dyspnea , and then , an emergency intubation and mechanical ventilation were administered .
immunoglobulin ( 0.4 g / kg / day 5 days ) and steroid pulse therapy ( methylprednisolone sodium succinate , 1000 mg daily 3 days , gradually tapered off ) were given .
sixteen days after treatment with immunoglobulin and steroids , the patient was successfully detached from the respirator .
mechanical ventilation was again administered . after steroid and immunoglobulin treatment failure , rituximab at a dose of 500 mg ( 330 mg / m/week )
after four courses of rituximab treatment , the skin lesions and muscle weakness were almost recovered , and ventilator weaning was successful .
three weeks after discharge , she was hospitalized with severe generalized blistering skin eruptions and polymorphic erythemas and mucosal ulcerations ( fig .
3 ) , which were considerably worse than those in the previous occasion but without myasthenia symptoms .
then , she was treated with immunoglobulin ( 1 mg / kg per day ) without improvement of mucocutaneous lesions .
we describe the extremely unusual association of an iptlike fdc sarcoma with pnp and mg .
the pathogenesis is thought to be due to the production of antibodies against tumorderived antigens ; the antibodies may attack components sharing the same or a similar epitope 8 .
10 reported a case in which pnp associated with fdcs showed a good response to a treatment plan that included dexamethasone , surgery , and chemotherapy .
our observations on this case indicate that rituximab could be an effective alternative therapy in the treatment of pnp and mg . however , pnp recurred catastrophically , perhaps because the standard treatment dose was not achieved and surgical resection of tumors was not performed .
the patient presented multiple local recurrences without systemic dissemination for a long period , suggesting an indolent clinical course .
considering the high mortality of pnp , we should pay more attention on the diagnosis and treatment of iptlike fdc sarcoma .
| key clinical messageparaneoplastic pemphigus ( pnp ) is an autoimmune blistering disease associated with neoplasms .
the disease is most commonly of lymphoproliferative origin and presents high mortality .
we describe a patient with pnp and myasthenia gravis associated with inflammatory pseudotumorlike follicular dendritic cell sarcoma , as well as the response to rituximab . |
the 2-year survival of patients starting rrt has progressively increased for cohorts from 2001 to 2006 , measured either as overall survival for patients starting rrt ( to 82.2% for the cohort starting in 2006 reported in the era - edta registry 2012 report ) , on dialysis ( 79.7% ) or receiving living - related kidney transplantation ( 98.3% ) ( figure 1 ) [ 13 ] .
in contrast , 2-year survival for cadaveric kidney transplantation remains stable at 96.096.1% for cohorts starting from 2003 to 2006 pointing to a plateau that would need novel approaches to be surpassed .
these data are in agreement with other registries reporting increasing overall survival of patients starting rrt in the last decade [ 47 ] .
similar improvements were observed for 5-year survival , pointing to an overall improvement in the standard of care , unlike the us situation in the 1990s , in which progressive increases in survival at 12 and 24 months were associated with decreasing survival at 5 years [ 4 , 8 ] .
drop in longer - term survival and whether a similar phenomenon occurred outside this country still need detailed studies , in search of lessons that might be learned and applied to improve the current standard of care .
on the dark side , data point to persistent inequalities between european citizens in incidence and prevalence of rrt and in access to transplantation .
living in greece , belgium ( both french- and dutch - speaking ) or portugal ( the gbp countries ) is associated with higher chances of initiating rrt than living in other european countries .
very high incidence of rrt was also reported in israel and georgia in 2012 and in turkey in 2011 and 2010 .
the adjusted rrt incidence for gbp countries was 188 , 201 - 174 and 220 pmp in 2012 , respectively ( although portugal data are unadjusted ) .
this compares with 122 , 114 and 97 pmp in the netherlands or spanish regions ( galicia , castilla - leon ) bordering portugal ( figure 2a ) .
moreover , these differences persist when prevalence is examined ( figure 2c ) , although prevalence / incidence values are lower for high incidence countries ( figure 3 ) .
one potential explanation is more stringent patient selection in low incidence countries , although better on - rrt care may also contribute . in lower income countries ,
the low incidence of rrt may represent lack of access to needed healthcare ( e.g. montenegro 26 pmp ) .
however , how can the high incidence and prevalence of rrt in the gbp countries be explained ?
do gbp citizens have access to rrt techniques that are denied to citizens in other high income countries , e.g. adjusted incidence of rrt 100 pmp in scotland and 77 pmp in finland ?
are there local genetic or environmental factors that favour progression of ckd and have not been tackled by public health authorities ? are there perverse incentives or forces that favour ckd progression or initiation of rrt ? or it is just a matter of reporting methods or of nephrologist beliefs regarding the timing of initiation of rrt when hard evidence is lacking ? unravelling the underlying reasons for the observed inequalities is an urgent research need : only when the causes are known can corrective measures be applied .
table 1.potential causes of differences in rrt incidence and prevalencevery concerning overuse of rrt denial of access to rrt financial incentives to initiate rrtconcerning different incidence or progression of ckd public health system fails to prevent development of ckd healthcare system structure or access results in suboptimal ckd care different views on the relative value of rrt versus conservative management of esrdother different data reporting methods refusal of rrt by an informed population when rrt is offered potential causes of differences in rrt incidence and prevalence several countries with low incidence and prevalence of rrt have higher incomes than countries with higher prevalence ( figure 4a ) .
the differences do not appear to depend on a better public health system or success in preventing ckd progression in those countries with high income .
thus , some countries with higher income and low rrt incidence start rrt at a median younger age than countries which have both lower income and higher incidence of rrt . in this
regard , low overall incidence and prevalence of rrt is frequently associated with low incidence and prevalence of rrt in those aged > 75 years ( figures 2b , d and 4c ) .
thus , the age pattern is consistent with denial of rrt to the elderly , refusal of rrt by the elderly or different criteria for indicating conservative treatment versus rrt in younger or elderly patients in countries with low rrt incidence . in these high income countries , low rrt incidence in the elderly
does not appear to depend on economic constraints , as is the case in low income countries .
the low rrt incidence may be based on the genuine belief that conservative treatment is the best option for elderly patients with esrd [ 9 , 10 ] . in this regard
, there is an urgent need for hard evidence on the relative merits of conservative treatment versus rrt in the elderly as well as on the optimal timing of rrt start .
if indeed conservative management has clear advantages , then in some countries overdialysis might be an issue . in this regard ,
a recent survey of nephrologists from 11 european countries disclosed very different practices regarding start of rrt in uncomplicated relatively young ( 60-year - old ) patients : the estimated glomerular filtration rates ( egfrs ) at which rrt would be started by different nephrologists ranged from 5 to 20 ml / min/1.73 m and this was the most important parameter to initiate rrt in such a patient for a majority of nephrologists .
obviously , starting at an egfr of 5 or 20 ml / min/1.73 m may have a great impact on the incidence of rrt .
if the same range would be applied to those over 75 years of age ( which was not the subject of the study ) , it would mean the difference between initiating and never initiating rrt for a great number of patients .
interestingly , nephrologists from countries with a high incidence of rrt more often believed that starting at a gfr > 10.5 ml / min/1.73 m is always beneficial compared with respondents from low incidence countries ( 18 versus 6% ) .
furthermore , the target egfr was higher for nephrologists working at for - profit centres .
further information is needed about the gfr at initiation of rrt in different countries as well as on the use of and acceptance by patients and doctors and outcomes of conservative management of esrd in the elderly .
the fact that differences in prevalence are lower than differences in incidence is concordant with prevalence / incidence value data ( figure 3 ) and can be interpreted as a high rate of patient loss in some high incidence countries . whether these shorter periods on rrt in some countries with a high incidence of rrt result in improved patient quality of life and are indeed appreciated by the patient
there are additional hypotheses potentially explaining the lower incidence of rrt coupled to a younger mean age at start of rrt in some high income countries .
patients may be dying earlier from cardiovascular disease and , thus , not living long enough to need rrt .
however , life expectancy does not differ much between countries selected for this commentary , except for low income montenegro ( figure 4b ) .
public health may also impact the incidence and prevalence of ckd and , thus , of esrd .
factors potentially involved include the presence of toxins in the environment or food ( i.e. the exposome , as an example , aristolochic nephropathy ) , over - the - counter medication policies ( allowing unrestricted access to nephrotoxic drugs ) or country - level policies regarding salt or phosphate content in the diet .
in addition , the healthcare system may also impact , as an example , whether there is universal unrestricted access to healthcare or to nephrologists .
the percentage of patients on rrt who have a functioning kidney graft ranges from 7% in bosnia and herzegovina to 60% in norway , finland and iceland .
also worrisome is the fact that rrt epidemiology data from some countries big and small ( e.g. germany , ireland , hungary and italy , among others ) at the core of europe are not available , as it was the case for 2011 and , except for some parts of italy , for 2010 [ 13 ] .
this virtual black - out makes comparison with other european countries difficult that could improve the standard of care and to detect pockets of substandard care or endemic ckd hotspots . in summary , the era - edta registry continues to provide a wealth of data on the epidemiology of rrt .
key points of this year 's report include ( i ) the improving outcomes for patients on rrt , with exception of recipients of cadaveric donor kidneys , ( ii ) the persistence of inequalities in rrt incidence and prevalence and transplantation availability in different european countries that may hide ckd hotspots , substandard ckd care , overuse of rrt or lack of access to needed rrt ; and ( iii ) a preoccupying lack of data from key countries .
there is an urgent need to study the factors underlying the inequalities in rrt incidence and prevalence and to identify and correct potential substandard care for some european citizens .
renal replacement therapy in europe : a summary of the 2012 era edta registry annual report . | the 2012 era - edta registry annual report contains both good news and bad news . on the bright side ,
the 2-year survival of patients starting renal replacement therapy ( rrt ) for chronic kidney disease ( ckd ) , on dialysis or receiving a living - related kidney transplantation , has progressively increased to 82.2 , 79.7 and 98.3% , respectively , whereas for cadaveric kidney transplantation it remains stable ( 96.096.1% ) . on the dark side ,
inequalities persist between european citizens in access to renal transplantation and in incidence and prevalence of rrt . living in greece , belgium ( french- or dutch - speaking ) or portugal ( the gbp countries )
is associated with higher chances of initiating rrt than living in other european countries .
the adjusted rrt incidence for gbp countries was 188 , 201 - 174 and 220 * ( * unadjusted ) pmp in 2012 , respectively ( versus 122 , 114 and 97 pmp in the netherlands or two spanish regions bordering portugal ) . in lower income countries ,
a low rrt incidence may represent lack of access to needed healthcare ( e.g. montenegro 26 pmp ) .
however , how can the high incidence and prevalence of rrt in the gbp countries be explained ? do gbp citizens have access to rrt that is denied , rejected or considered unnecessary in other high income countries ?
does the gbp healthcare system fail to prevent progression of ckd ?
do local genetic or environmental factors
favour ckd progression ?
unravelling the underlying reasons is an urgent research need : only an understanding of the causes will allow correction of the problem .
unavailability of data from some large countries ( e.g. germany and italy ) is not helpful . |
since serum prostate specific antigen ( psa ) was introduced as an early detection marker of prostate cancer ( pca ) in the 1980s , several trials involving novel and more precise biomarkers have failed to provide new markers with definitive clinical relevance and applicability .
considering the low sensitivity of psa and the importance of distinguishing latent from clinically significant pca to optimize treatment , the need for specific diagnostic markers is apparent .
recent investigations have pointed to caveolin-1 , exosomes , and pca-3 as diagnostic markers . however , their clinical applicability is still to be determined .
this pilot study was designed to investigate the potential of chemokines as biomarkers for pca .
chemokines are low molecular weight secretory proteins divided into four groups ( cxc , cc , c , and the cx3c family ) according to the arrangement of conserved cysteine ( c ) residues of the mature proteins , which interact as ligands ( cl ) with their corresponding receptors ( cr ) . due to their proinflammatory properties ,
chemokines regulate chemotaxis and metabolic activity of leukocytes circulating in the body and therefore significantly affect normal development , angiogenesis and atherosclerosis .
they are involved in many aspects of oncogenesis , including regulation of cancer cell growth , dissemination and host - tumor response .
hence , various tumor types differentially trigger a complex chemokine network determining the qualitative and quantitative degree of immunocellular infiltration .
alterations of the chemokine expression level are significantly associated with more aggressive disease and poorer prognosis in diverse malignancies . however
preliminary work pointed out the potential of cxc chemokines as prognostic biomarkers for pca . to assess
the diagnostic relevance of chemokines in pca , the expression of a broad panel of chemokines in tumor tissue and their serum levels in patients with pca was investigated and associations tested with clinical and pathological parameters .
patients undergoing radical prostatectomy for biopsyproven pca between january 2008 and october 2010 in the department of urology , goethe university , frankfurt , germany , were included in the study after signing informed consent for utilization of biomaterials and further scientific assessment .
the tumor stage and grade was determined according to the sixth edition of the tnm classification .
clinical parameters , including the patient s age and preoperative and postoperative psa values , were documented .
ten milliliters of peripheral blood was drawn from patients the day before surgery and 5 to 7 days after surgery . as controls ,
10 ml blood was drawn from 15 healthy , male , age - matched volunteers .
blood samples were allowed to coagulate and then centrifuged at 3,000 rpm at 4c for 10 minutes .
the human cytokine multiplex bead assay kit ( millipore , darmstadt , germany ) was used to determine cxcl12 , cxcl13 , cx3cl1 , ccl2 , ccl5 , and ccl20 in serum , according to the manufacturer s instructions .
anti - chemokine antibody coated beads ( 25 l ) were pipetted into each well .
samples ( serum ) or standards ( 25 l ) were then added to the wells and incubated with the beads for 2 hours .
after washing , biotin conjugated detector antibody was added and incubated for 1 hour , followed by a 30-minute incubation with r - phycoerythrin conjugated streptavidin .
analysis was carried out with a luminex 100 ( gurce , nivelles , belgium ) and expressed as pg / ml . immediately after resection , tissue was transferred to the department of pathology and reviewed by a pathologist .
tumor tissue , as well as adjacent normal tissue , was fresh - frozen in liquid nitrogen .
total rna from solid tumor tissue of patients with pca and adjacent normal tissue was extracted using rneasy kit ( qiagen , hilden , germany ) , according to the manufacturer s instructions .
solid tissues were sliced into small pieces and transferred to a precellys tube pre - filled with 1.4-mm ceramic beads ( bertin technologies , saint - quentin - en - yvelines cedex , france ) containing lysis buffer .
cells were lysed under rapid agitation at 6,500 rpm for 1 minute in three cycles using a precellys 24 homogenizer ( bertin technologies ) .
cdna was synthesized from 1 g of total rna with oligo(dt ) primer using an affinityscript qpcr cdna synthesis kit ( stratagene , santa clara , ca ) , according to the manufacturer s instructions .
the amount and quality of rna were analyzed using nanovue ( ge healthcare , piscataway , nj ) and bioanalyzer ( agilent technologies , palo alto , ca ) , respectively .
real - time quantitative pcr ( qrt - pcr ) was performed using rt sybr - green / roxqpcr master mix and gene specific primers for cl and cr ( both from sabioscience , madison , wi ) ( table 1 ) , according to the manufacturer s protocol .
gene expression ( fold change ) was calculated using the ct method , after normalizing the tumor sample with a corresponding normal sample .
statistical significance of differences between parameters within the same group was tested by the wilcoxon matched pairs test and in different groups by the wilcoxon mann - whitney test .
subgroup analyses employed the kruskal - wallis test with the iman - conover method ( bonferroni- holm correction ) .
null hypotheses of no difference were rejected if p - values were less than 0.05 .
patients undergoing radical prostatectomy for biopsyproven pca between january 2008 and october 2010 in the department of urology , goethe university , frankfurt , germany , were included in the study after signing informed consent for utilization of biomaterials and further scientific assessment .
the tumor stage and grade was determined according to the sixth edition of the tnm classification .
clinical parameters , including the patient s age and preoperative and postoperative psa values , were documented .
ten milliliters of peripheral blood was drawn from patients the day before surgery and 5 to 7 days after surgery . as controls ,
10 ml blood was drawn from 15 healthy , male , age - matched volunteers .
blood samples were allowed to coagulate and then centrifuged at 3,000 rpm at 4c for 10 minutes .
the human cytokine multiplex bead assay kit ( millipore , darmstadt , germany ) was used to determine cxcl12 , cxcl13 , cx3cl1 , ccl2 , ccl5 , and ccl20 in serum , according to the manufacturer s instructions .
anti - chemokine antibody coated beads ( 25 l ) were pipetted into each well .
samples ( serum ) or standards ( 25 l ) were then added to the wells and incubated with the beads for 2 hours .
after washing , biotin conjugated detector antibody was added and incubated for 1 hour , followed by a 30-minute incubation with r - phycoerythrin conjugated streptavidin .
analysis was carried out with a luminex 100 ( gurce , nivelles , belgium ) and expressed as pg / ml .
immediately after resection , tissue was transferred to the department of pathology and reviewed by a pathologist .
tumor tissue , as well as adjacent normal tissue , was fresh - frozen in liquid nitrogen .
total rna from solid tumor tissue of patients with pca and adjacent normal tissue was extracted using rneasy kit ( qiagen , hilden , germany ) , according to the manufacturer s instructions .
solid tissues were sliced into small pieces and transferred to a precellys tube pre - filled with 1.4-mm ceramic beads ( bertin technologies , saint - quentin - en - yvelines cedex , france ) containing lysis buffer .
cells were lysed under rapid agitation at 6,500 rpm for 1 minute in three cycles using a precellys 24 homogenizer ( bertin technologies ) .
cdna was synthesized from 1 g of total rna with oligo(dt ) primer using an affinityscript qpcr cdna synthesis kit ( stratagene , santa clara , ca ) , according to the manufacturer s instructions .
the amount and quality of rna were analyzed using nanovue ( ge healthcare , piscataway , nj ) and bioanalyzer ( agilent technologies , palo alto , ca ) , respectively .
real - time quantitative pcr ( qrt - pcr ) was performed using rt sybr - green / roxqpcr master mix and gene specific primers for cl and cr ( both from sabioscience , madison , wi ) ( table 1 ) , according to the manufacturer s protocol .
glyceraldehyde 3-phosphate dehydrogenase and -actin were used as housekeeping gene and for normalization . gene expression
( fold change ) was calculated using the ct method , after normalizing the tumor sample with a corresponding normal sample .
all experiments were performed three to six times . statistical significance of differences between parameters within the same group was tested by the wilcoxon matched pairs test and in different groups by the wilcoxon mann - whitney test .
subgroup analyses employed the kruskal - wallis test with the iman - conover method ( bonferroni- holm correction ) .
null hypotheses of no difference were rejected if p - values were less than 0.05 .
the study population consisted of 39 patients ( table 2 ) with a mean age at tumor diagnosis of 68.95.6 years .
none of the patients had evident clinical signs of infection or acute or chronic inflammation at the time of surgery .
histologically , all tumors were conventional acinar adenocarcinomas . in four patients the final histopathological analysis revealed positive margins ( r1 ) .
all patients with microscopic residual disease ( r1 ) and five other patients ( 2 pt3a , 2 pt3b and 1 pt2c with biochemical relapse after surgery ) received radiation therapy .
the mean follow - up time was 18.115.1 months . at the study endpoint , all patients were alive without signs of biochemical or clinical recurrence . using qrt - pcr , significantly higher expression of ccl2 and ccr6 in tumor tissue was observed , compared to normal adjacent tissue ( fig .
the expression of cxcr4 , cx3cr1 , ccr2 , ccr5 , ccr7 , ccl5 , and ccl20 was higher and that of cxcr3 , cxcr5 , cxcr7 , cxcl12 , cxcl13 , and cx3cl1 lower in the tumor tissue without reaching significance .
the association of gene expression of the chemokines in the tumor tissue was tested with clinical and oncologic parameters ( fig .
no association was found between the other investigated chemokines and t stage , gleason score , grade , r1- and pn1-status , preoperative and postoperative psa or biochemical relapse .
ccl2 was significantly over - expressed both preoperatively and postoperatively in pca patients , compared to controls .
significantly diminished concentrations of ccl5 in preoperative and postoperative samples were detected , compared to controls .
the concentration of ccl20 increased significantly in the control group , compared to both preoperative and postoperative serum concentration in pca patients .
the serum concentration of cx3cl1 also increased significantly in the controls compared to that in patient serum , both preoperatively and postoperatively .
the level of cxcl12 and cxcl13 did not significantly differ among any of the groups .
the association of chemokine serum concentrations with t stage , gleason score , grade , r1- and pn1-status , preoperative psa as well as biochemical relapse were then tested .
ccl2 was significantly negatively correlated with the preoperative psa value ( spearman s correlation coefficient rho , 0.48 ; p=0.035 ) .
chemokines are small secreted chemotactic cytokines that coordinate immunological machinery by promoting leukocyte migration and cross - communication among different cell types of the immune system . due to similarities between leukocyte trafficking and cancer cell dissemination ,
recent studies regarding different tumor entities point to significant involvement of chemokines in facilitating tumor promotion and dissemination .
based on the evaluation of 15 members of the chemokine family , evidence is provided here that the gene activity of particular molecules is altered in pca , compared to normal tissue .
ccl2 and ccr6 mrna were overexpressed in the tumor specimens , compared to adjacent normal tissue .
ccl2 , but not ccr6 , was also significantly augmented in serum from tumor patients compared to healthy controls , indicating a critical role of ccl2 in neoplastic progression . a meta - analysis of gene - expression data has identified ccl2 as a primary driver in pca development .
immunohistochemistry and immunofluorescence analysis of the chemokine expression profile reveal ccl2 to be predominantly expressed at the tumor site , more than for other chemokines .
tissue microarray analysis shows that levels of ccl2 expression in pca tissue are greater than in non - neoplastic epithelia .
therefore , ccl2 could serve as a specific diagnostic marker , closely associated with pca , even though ccl2 did not reach control values postoperatively .
a possible explanation is that immunologic processes are still active five to seven days after surgical intervention .
studies incorporating blood sampling at later post - surgical dates should , therefore , more clearly define the specific diagnostic value of ccl2 .
one problem in employing ccl2 to diagnose pca is the lack of association with tumor stage and grade , meaning that ccl2 may not allow evaluation of tumor dissemination .
shirotake et al . found differences in ccl2 levels only between very early localized , and advanced castration resistant pca .
in contrast , the report from lu et al . pointed to a significant relationship between ccl2 level and gleason score .
different analytic techniques ( microarray ) or a selected patient cohort ( asian ) may account for this discrepancy . obviously , further patient studies are necessary to clarify the diagnostic value of ccl2 for pca .
ccl5 , ccl20 , and cx3cl1 were all reduced in patient sera , whereas no differences were seen in the gene analyses .
therefore , pca progression seems accompanied by alterations in ccl5 , ccl20 , and cx3cl1 protein release but not in transcriptional modification .
, this study was limited to pca with serum psa values of < 10 ng / ml , which can not be applied to our cohort with 41% of patients with a preoperative psa > 10 ng / ml .
immunohistochemistry of 80 pca cases revealed absent or weak ccl20 expression in 61% , whereas strong staining was evident in only 10% of patients .
this may corroborate our ccl20 data , although serum probes were not analyzed in this study .
cx3cl1 is down - regulated in high - risk pca patients who develop biochemical recurrence following prostatectomy .
the authors of this case - control study recommend cx3cl1 as a potential indicator of future recurrence .
cxcl12 is suggested to facilitate tumor cell recruitment to the bone marrow and a high cxcl12 expression is associated with lymph node metastatic prostate carcinoma compared with non lymph - node metastatic cancer in an immunohistochemical analysis .
semiquantitative pcr evaluation points to an enhanced cxcl12 mrna expression level in prostate tumor , compared to adjacent ' normal ' tissue .
serum cxcl12 levels are significantly higher for men who were biopsy positive compared to those who were biopsy negative for pca . in contrast , agarwal et al . , who performed multiplex enzyme - linked immunosorbent assay assays on 272 men , found no association between cxcl12 and pca status .
a final assessment of the role of cxcl12 in pca is difficult since different techniques have been applied in relevant studies .
the ccl2 changes dominate other chemokine changes , since pca related ccl2 alterations were found at both gene and protein level . however , ccr6 , ccl5 , ccl20 , and cx3cl1 might be further candidates pointing to neoplastic transformation .
further large - volume biomarker studies are required to expand and validate the preliminary findings of this pilot study . | purposeprostate specific antigen is not reliable in diagnosing prostate cancer ( pca ) , making the identification of novel , precise diagnostic biomarkers important . since chemokines are associated with more aggressive disease and poor prognosis in diverse malignancies , we aimed to investigate the diagnostic relevance of chemokines in pca.materials and methodspreoperative and early postoperative serum samples were obtained from 39 consecutive pca patients undergoing radical prostatectomy .
serum from 15 healthy volunteers served as controls .
concentrations of cxcl12 , cxcl13 , cx3cl1 , ccl2 , ccl5 , and ccl20 were measured in serum by luminex .
the expression activity of cxcr3 , cxcr4 , cxcr5 , cxcr7 , cxcl12 , cxcl13 , cx3cr1 , cxcl1 , ccr2 , ccr5 , ccr6 , ccr7 , ccl2 , and ccl5 mrna was assessed in tumor and adjacent normal tissue of prostatectomy specimens by quantitative real - time polymerase chain reaction .
the associations of these chemokines with clinical and histological parameters were tested.resultsthe gene expression activity of ccl2 and ccr6 was significantly higher in tumor tissue compared to adjacent normal tissue .
ccl2 was also significantly higher in the blood samples of pca patients , compared to controls .
ccl5 , ccl20 , and cx3cl1 were lower in patient serum , compared to controls .
ccr2 tissue mrna was negatively correlated with the gleason score and grading.conclusionchemokines are significantly modified during tumorigenesis of pca , and ccl2 is a promising diagnostic biomarker . |
homozygous loss - of - function mutations in the gba1 gene cause gaucher disease ( gd ) , the commonest lysosomal storage disorder .
this gene encodes glucocerebrosidase ( gcase ) , a lysosomal enzyme responsible for the hydrolysis of the glycolipid substrate glucosylceramide ( glccer ) to ceramide and glucose .
reductions in enzymatic activity result in the lysosomal accumulation of glccer , as well as glucosylsphingosine , within reticulo - endoendothelial cells , leading to the systemic sequelae of gd , including organomegaly , bone disease , anaemia and thrombocytopenia .
the clinical spectrum of gd has historically been sub - divided into type 1 gd , the so - called non - neuronopathic milder form of the disease , and types ii and iii gd , which are severe acute and chronic neuronopathic forms respectively ( cox , 2010 ) . in recent years
heterozygous gba1 mutations have been identified as key genetic risk factors for parkinson 's disease ( pd ) , and increase the risk of developing pd by approximately 20-fold ( sidransky et al . , 2009 ; migdalska - richards and schapira , 2016 ) .
the neuropathological hallmark of pd is the presence of proteinacious intraneuronal inclusions , known as lewy bodies , predominantly composed of aggregated -synuclein ( -syn ) , in addition to other proteins such as ubiquitin and p62 ( zatloukal et al . , 2002 ; shults , 2006 ) .
-syn is a presynaptic protein of unknown function , which is thought to play a central role in the pathogenesis of pd .
moreover , there is mounting evidence that soluble -syn aggregation intermediates ( so - called oligomeric or protofibrillar species ) represent the most toxic form of the protein ( volles and lansbury , 2003 ) .
-syn is removed from the cell by both macroautophagy and chaperone - mediated autophagy ( cma ) ( cuervo et al . , 2004 ;
watanabe et al . , 2012 ) , and indeed -syn aggregates accumulate in response to the pharmacological inhibition of autophagy in mice ( klucken et al . , 2012 ) .
-syn is a well - characterised substrate of cma due to the presence of a cma - specific pentapeptide sequence motif .
it is selectively translocated across the lysosomal membrane in a complex with the heat shock cognate protein 70 ( hsc70 ) , a process dependent on lysosomal - associated membrane protein 2a ( lamp-2a ) .
furthermore pathogenic forms of -syn inhibit cma through the blockade of receptor - mediated uptake into the lysosome ( cuervo et al . , 2004 ) .
macroautophagy is a less selective degradative pathway , which is responsible for the bulk removal of defective organelles and mis - folded cytoplasmic proteins , including -syn , from the cell .
it involves the sequestration of the cytosolic contents into double - membrane autophagosomes , which are then delivered to the lysosome to form a single - membrane autophagolysosome .
macroautophagy dysregulation is increasingly being recognized as a pathogenic factor in neurodegeneration , including in pd ( ravikumar et al . , 2010 ) .
consistent with this , the selective suppression of autophagy , through the neuronal loss of the autophagy genes atg7 or atg5 , results in a number of phenotypes in mice , including locomotor defects , accumulation of polyubiquitinated proteins and neurodegeneration ( hara et al .
both loss - of - function and toxic gain - of - function hypothesizes have been put forward to explain the link between gba1 mutations and pd ( kinghorn , 2011 ; migdalska - richards and schapira , 2016 ) .
there is growing evidence supporting the role of gcase loss - of - function in pd .
the majority of gba1 mutations , including missense , nonsense and frame - shift mutations , insertions or deletions , are associated with reduced lysosomal gcase levels ( montfort et al . ,
moreover , milder mutations , associated with slightly diminished gcase levels , confer a much lower risk of pd than more severe mutations resulting in severe enzymatic dysfunction ( swan and saunders - pullman , 2013 ) .
moreover , gcase protein levels and enzymatic activity are both decreased in the post - mortem brain tissue from patients with both idiopathic and gba1-linked pd ( gegg et al . , 2012 ) .
a number of mechanisms linking gcase loss - of - function with neurodegeneration have been demonstrated ( kinghorn , 2011 ; migdalska - richards and schapira , 2016 ) .
loss of gcase activity has been shown to lead to glccer accumulation , resulting in stabilisation of -syn oligomers and -syn accumulation .
the subsequent increase in -syn may inhibit gcase er - golgi trafficking and lysosomal gcase levels , thus creating a bidirectional pathogenic loop ( mazzulli et al . , 2012 ) .
we recently investigated the role of the autophagy - lysosomal ( als ) system in neuronopathic gd using drosophila melanogaster ( kinghorn et al . , 2016 ) . employing homologous recombination techniques we developed a fly model lacking gcase in the brain .
gcase - deficient flies displayed reduced lifespan , age - dependent locomotor abnormalities , as well clear evidence of synaptic loss and neurodegeneration . in keeping with the hallmark lysosomal dysfunction seen in gd cells , staining with lysotracker revealed numerous abnormally enlarged lysosomes in the brains of flies lacking gcase .
this abnormal lysosomal pathology was associated with the accumulation of glccer , similar to that seen in neuronopathic gd brains ( conradi et al . , 1984 )
glucocerebrosidase ( gcase ) deficiency results in autophagy - lysosomal system ( als ) dysfunction . under normal physiological conditions gcase
normal lysosomal function is required for the autophagic clearance of defective cellular organelles and mis - folded proteins .
mutations in the gba1 gene result in gcase loss - of - function and accumulation of its substrates .
this leads to lysosomal dysfunction and subsequent defects in both macroautophagy and chaperone - mediated autophagy ( cma ) , with the consequent accumulation of -synuclein ( -syn ) .
als dysfunction in a fly model of neuronal gcase deficiency was associated with the accumulation of abnormal giant mitochondria ( representative giant mitochondria in gba1 knockout ( gba1b ) and healthy mitochondria in wild type control fly brains are shown with yellow asterisks , shown under identical magnification ) , accumulation of autophagy substrates ( p62 and polyubiquitinated proteins ) , in addition to neurodegeneration and synaptic loss .
these neuropathological defects resulted in a number of neurotoxic phenotypes , including reduced lifespan , locomotor abnormalities and decreased resistance to oxidative stress .
likely as a compensatory response to the autophagy block , mammalian target of rapamycin ( mtor)-complex 1 ( mtorc1 ) activity was decreased and mitf / tfeb gene expression was up - regulated in the brains of gcase - deficient flies .
the mtor inhibitor rapamycin was able to functionally rescue the lifespan , locomotor and oxidative stress phenotypes of the gcase - deficient flies , highlighting the potential therapeutic benefits of rapamycin and other inhibitors of mtorc1 in gaucher disease ( gd ) and parkinson 's disease ( pd ) ( kinghorn et al . , 2016 ) .
normal lysosomal functioning is required for the formation of autolysosomes and thus the autophagic removal of misfolded proteins and defective organelles from the cell ( ravikumar et al . , 2010 ) . during macroautophagy lc3-i
is converted into its lipidated form , lc3-ii , which is recruited to autophagosomal membranes ( ravikumar et al . , 2010 ) .
we therefore probed macroautophagy function by measuring the levels of the fly homologue of lc3 , atg8 , by western blot analysis .
this demonstrated that both atg8(lc3)-i and atg8(lc3)-ii are increased in the gcase - deficient fly brain , in addition to the ratio of atg - ii / atg - i , suggestive of increased autophagosome number and a block in autophagy flux .
this mirrors the increased lc3-ii levels seen in the substantia nigra of pd patients ( dehay et al . , 2010 ) .
in addition , we demonstrated increased accumulation in gcase - deficient fly brains of autophagy substrates , namely polyubiquinated proteins and ref(2)p , the drosophila homologue of p62 .
both ubiquitin and p62 are abundant constituents of protein inclusions associated with neurodegenerative diseases , including lbs in pd ( zatloukal et al .
. further interrogation of autophagy at the regulatory level in fly brains revealed a decrease in the phosphorylation of p70s6 kinase , a substrate of the energy sensing mammalian target of rapamycin ( mtor)-complex 1 ( mtorc1 ) .
mtorc1 plays a central role in macroautophagy by initiating the formation and elongation of the autophagosomal membrane ( ravikumar et al . , 2010 ) .
thus the decrease in mtorc1 activity seen in gcase - deficient fly brains likely represents a compensatory response to the autophagy block and is similar to that seen in fibroblasts derived from pd patients harbouring gba1 mutations ( magalhaes et al . ,
, abnormal giant mitochondria were observed in the brains of gcase - deficient flies , a reflection of the inability of the cells to clear aged and defective mitochondria by autophagy ( the process of mitophagy ) .
mitf ( microphthalmia transcription factor ) is the fly homologue of mammalian tfeb ( transcription factor eb ) , the master regulator of lysosomal biogenesis .
it has many conserved functions in the fly including regulation of the lysosomal - autophagy pathway and lipid metabolism ( bouche et al . , 2016 ) .
gcase deficiency in the fly was also associated with mitf gene up - regulation , likely in response to the lysosomal - autophagy block .
lastly treatment of gcase - deficient flies with the mtor inhibitor rapamycin resulted in a significant rescue of the lifespan and locomotor phenotypes , as well as resistance to oxidative stress ( figure 1 ) .
als dysfunction is increasingly being implicated in the pathogenesis of gd and pd , and has been identified in several gd and gcase - deficient pd models .
autophagy defects , in association with inflammasone activation , were observed in gd macrophages ( aflaki et al . , 2014 )
. pharmacological inhibition of gcase in mice , through the chronic systemic treatment with conduritol--epoxide , lead to the accumulation of insoluble -syn aggregates and neurodegeneration , in addition to an increase in the cma - associated protein lamp-2a and the macroautophagy marker lc3-ii ( rocha et al .
gcase sirna knockdown in cell culture also inhibited macroautophagy flux with a rise in lc3-ii and p62 , and gba1 knockout in mouse embryonic fibroblasts lead to a compromise in cma .
furthermore the autophagy defects were associated with loss of autophagic lysosomal reformation and maturation of endosomes .
they also observed a decrease in functional acidified lysosomes within gcase - deficient cells ( magalhaes et al . , 2016 ) .
enlargement of the lysosomal compartment , as observed in gcase - deficient fly brains , was also seen in gba1 mutant pd induced pluripotent stem cell ( ipsc)-derived dopamine neurons , in association with autophagy defects and accumulation of intracellular p62 ( fernandes et al . , 2016 ) .
it has been suggested that both gd patients and gba1 mutation carriers display a similar risk of developing pd ( schapira , 2015 ) .
consistent with this , in vitro work has shown that the degree of autophagy inhibition and -syn accumulation is comparable in ipsc - derived neurones from gd patients and gba1 mutation carriers with pd ( schndorf et al . , 2014 ) . moreover ,
autophagic lysosomal reformation defects in gcase - deficient cellular models were also similar in heterozygote and homozygote mutant gba1 models ( magalhaes et al . , 2016 ) .
these observations are in keeping , at least in part , by the complex interplay between the loss and toxic gain - of - functions of gcase activity , the ageing cellular degradative machinery , including the als , and additional genetic risk factors predisposing to pd .
work in our gcase knockout fly model , as well as in other cellular and mouse models , has demonstrated that loss of gcase activity is sufficient to cause neurodegeneration , even in the absence of -syn , when gcase levels are sufficiently low ( kinghorn et al 2016 ) .
as already mentioned , -syn can further potentiate gcase deficiency through the generation of a bidirectional feedback loop , leading to lysosomal dysfunction and the generation of toxic oligomeric -syn species ( mazzulli et al . ,
in addition to the effects of gcase loss - of - function , the cellular sequelae of aging undoubtedly play a role .
. during ageing gcase activity also declines in the substantia nigra and putamen of healthy controls , and is comparable to the enzymatic activity in pd patients with gba1 mutations ( rocha et al . , 2015a ) .
this suggests that an age - dependent reduction in gcase activity may lower the threshold for developing pd , and contribute to the lysosomal dysfunction that occurs in ageing and pd
. therefore moderate reductions in gcase activity in gba1 mutation carriers , in addition to the age - related decline in the als , and other cellular perturbations secondary to additional genetic and environmental risk factors , may be sufficient to promote -syn accumulation and neurodegeneration .
further studies are required to better characterize the relationship between specific pathogenic gba1 mutations , their corresponding gcase activity and als function , in addition to their dose effect and clinical pd phenotypes .
such studies will undoubtedly aid our understanding of the variable penetrance of gba1 mutations , in both the heterozygous and homozygous state , in causing pd .
the beneficial effects of rapamycin , demonstrated in our gcase knockout fly model , suggest that the inhibition of mtorc1 may represent an additional therapeutic strategy in the treatment of gd and pd , as well as other neurodegenerative disorders , in which there is significant als dysfunction . in support of this
, rapamycin increased the clearance of exogenously expressed human wild - type and mutant -syn in an inducible cell model ( webb et al . , 2003 ) as well as
endogenous -syn in gba1-knockdown primary cortical neurons ( du et al . , 2015 ) .
rapamycin has also been shown to mediate neuroprotective effects in other neurodegenerative diseases , including in a tdp-43-expressing amyotrophic lateral sclerosis mouse model ( bov et al . , 2011 ; wang et al . ,
however , inhibition of mtor by rapamycin is not invariably protective in all gba1 mutant models , and indeed rapamycin was found to trigger cell death in gd ipsc - derived neuronal cells harboring a gba1 mutation ( awad et al . , 2015 ) .
furthermore chronic rapamycin treatment aggravated the pathology and muscle weakness in a mouse model of vcp ( valosin containing protein)-associated myopathy , despite a defect in mtor signaling ( ching and weihl , 2013 ) .
thus the effects of inhibiting mtorc1 activity appear to vary depending on disease pathology , the model organism , and also likely the timing of the therapy .
further studies in mammalian models are now required to verify the benefits of rapamycin , and other inhibitors of mtor , in ameliorating the neuropathology in gd and pd .
it is likely that the clinical success of pharmacological mtor inhibitors will rely on the precise titration of mtor activity , in order to stimulate autophagy appropriately in the face of als dysfunction , minimalizing unwanted side effects . | gaucher disease ( gd ) , the commonest lysosomal storage disorder , results from the lack or functional deficiency of glucocerebrosidase ( gcase ) secondary to mutations in the gba1 gene .
there is an established association between gba1 mutations and parkinson 's disease ( pd ) , and indeed gba1 mutations are now considered to be the greatest genetic risk factor for pd .
impaired lysosomal - autophagic degradation of cellular proteins , including -synuclein ( -syn ) , is implicated in the pathogenesis of pd , and there is increasing evidence for this also in gd and gba1-pd .
indeed we have recently shown in a drosophila model lacking neuronal gcase , that there are clear lysosomal - autophagic defects in association with synaptic loss and neurodegeneration .
in addition , we demonstrated alterations in mechanistic target of rapamycin complex 1 ( mtorc1 ) signaling and functional rescue of the lifespan , locomotor defects and hypersensitivity to oxidative stress on treatment of gcase - deficient flies with the mtor inhibitor rapamycin .
moreover , a number of other recent studies have shown autophagy - lysosomal system ( als ) dysfunction , with specific defects in both chaperone - mediated autophagy ( cma ) , as well as macroautophagy , in gd and gba1-pd model systems .
lastly we discuss the possible therapeutic benefits of inhibiting mtor using drugs such as rapamycin to reverse the autophagy defects in gd and pd . |
data were derived from the third national health and nutrition examination survey ( nhanes iii , 19881994 ) , a representative survey of the civilian , noninstitutionalized us population conducted by the national center for health statistics ( nchs ) of the center for disease control and prevention .
details of the survey design and methodology are available elsewhere ( nchs , 2006a ) . briefly , individuals were interviewed at home , then invited to a mobile examination centre ( mec ) for further interviews , tests and examinations .
details of the oral health component of nhanes iii are published elsewhere ( drury et al . 1996 ) .
the teeth were divided into two maxillary and two mandibular halves and measurements were taken from two sites per tooth ( midbuccal and mesiobuccal ) for all teeth ( excluding third molars ) in one randomly chosen upper and lower quadrant .
these measurements included periodontal probing depth ( ppd ) , gingival recession and bleeding on probing ( bop ) .
clinical attachment loss ( cal ) was calculated as the sum of the recession and ppd .
individuals receiving renal replacement therapy ( through dialysis or kidney transplant ) were excluded from periodontal examination .
periodontitis was defined using the 2007 cdc / aap ( centre for disease control and prevention / american academy of periodontology ) classification ( page & eke 2007 ) .
in addition , continuous periodontal parameters were also employed such as mean ppd , mean cal , cumulative periodontal probing depth ( cppd ) , number of teeth present and proportion of sites that bled upon probing .
cumulative ppd was calculated as the sum of the maximum probing pocket depths 4 mm of each tooth and as such is a surrogate measure of the potential extent of biofilm exposed connective tissues ( dietrich et al .
edentulous individuals were included in the analyses but formed a group distinct from individuals with periodontitis .
the serum creatinine levels recorded in the nhanes iii survey were recalibrated to be traceable to an isotopederived mass spectroscopy method using the equation below ( nchs , 2006b ) :
standardized creatinine=(0.960serum creatinine)0.18
age , sex , ethnicity and standardized serum creatinine were incorporated in the ckd epidemiology collaboration ( ckdepi ) equation to calculate estimated glomerular filtration rate ( egfr ) ( levey et al .
this equation improves mortality risk stratification in individuals with ckd compared with the modification of diet in renal disease ( mdrd ) equation ( shafi et al . 2012 ) .
based on an egfr<60 ml / min/1.73 m , individuals were classified as having stage 35 ckd , hitherto referred to as
urinary albumin and creatinine levels were employed to calculate the albumincreatinine ratio ( acr ) .
albuminuria was classified as acr < 30 mg / g ; acr 30 mg / g and < 300 mg / g ; and acr 300 mg / g .
individuals were classed as hypertensive if their mean ( of three consecutive measurements ) systolic blood pressure ( bp ) was 140 mmhg or mean diastolic bp was 90 mmhg .
individuals were classed as diabetic by selfreporting ( excluding gestational diabetes ) or if their glycated haemoglobin ( hba1c ) was 6.5% .
individuals ' smoking status was determined from selfreporting and classified into current , former or never smokers ( cigarettes only ) .
data on covariates employed in the statistical analyses included information on age , sex , ethnicity ( nonhispanic white , nonhispanic black , mexican american or other ) , alcohol consumption ( never , not in last year , between 014 drinks / week , more than 14 drinks / week ) and selfreported history of previous cardiovascular events ( stroke , heart attack or heart failure ) . pulse pressure was calculated as the difference between the mean systolic and diastolic bp .
self reported measures of socioeconomic status ( household income , marital status and educational attainment ) were coded as follows .
household income ( less than $ 20,000 or $ 20,000 or more ) ; marital status ( married or living as married , never married , divorced or separated or widowed ) ; educational attainment ( less than high school , high school diploma or more than high school ) .
body mass index ( bmi ) was coded as a categorical variable with bmi
< 18.5 kg / m as underweight ; 18.5 kg / m and < 25 kg / m as normal ; 25 kg / m and < 30 kg / m as overweight and 30 kg / m as obese .
laboratory tests including serum cholesterol ( total and highdensity lipoprotein / hdl ) were performed .
serum cholesterol levels were classified into binary variables ( total serum cholesterol 24 mg / l or < 24 mg / l and serum hdl cholesterol 3.5 mg
physical activity was selfreported by individuals and reclassified as recommended or more if they reported moderate activity five or more times a week or vigorous activity three or more times a week .
physical activity was also classified as recommended or more if individuals reported moderate physical activity four or more times a week and vigorous activity one or more times a week or reported moderate activity three or more times a week and vigorous activity two or more times a week .
individuals ' physical activity was classified as none if they reported no leisure time physical activities .
individuals who reported some level of physical activity but less than recommended were classed as
the nchs provide mortality data for nhanes iii participants up to 31st december 2006 , linked by probabilistic record matching with the national death index ( ndi ) .
the publicly available data set contains information on the mortality status of individuals aged 17 years or older . for individuals who are classified as
assumed deceased , information is available on 113 underlying cause of death categories , based on the ninth and tenth revisions of the international classification of diseases ( icd9 and icd10 ) .
cvd mortality was limited to cases where the underlying cause of death was coded between 53 and 75 ( inclusive ) ( anderson et al . 2001 ) .
details of the linked mortality data have been published elsewhere ( nchs , 2010 ) .
analyses performed followed guidelines for nhanes iii ( nchs , 1996 ) , accounting for the complex survey design and sampling weights to yield estimates generalizable to the us population .
differences in categorical and continuous data were assessed for statistical significance using pearson chisquare , ttest , fisher 's exact test and analysis of variance ( anova ) as appropriate .
cox proportional hazards ( ph ) regression models were fitted to evaluate the association between periodontal status , traditional risk factors ( diabetes , hypertension and smoking status ) and allcause and cvd mortality , independent of potential confounders .
the fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) .
the ph assumption was tested using schoenfeld residuals , scaled schoenfeld residuals and graphical methods .
were not included in the analyses ( listwise deletion ) . thus out of a possible 13,784 individuals eligible for analyses , 1379 ( 10% ) individuals were excluded due to incomplete covariate data ( table s1 ) .
we considered the effect measure modification of mortality ( allcause and cardiovascular ) in individuals with ckd according to their periodontal health status .
we conducted formal tests of interaction between periodontal variables and ckd case definition by entering interaction terms in the model .
further formal tests of interactions between ckd , periodontitis or edentulism and age , gender and ethnicity were also carried out .
analyses were carried out using stata / ic version 12.1 ( statacorp lp , college station , tx , usa ) .
data were derived from the third national health and nutrition examination survey ( nhanes iii , 19881994 ) , a representative survey of the civilian , noninstitutionalized us population conducted by the national center for health statistics ( nchs ) of the center for disease control and prevention .
details of the survey design and methodology are available elsewhere ( nchs , 2006a ) . briefly , individuals were interviewed at home , then invited to a mobile examination centre ( mec ) for further interviews , tests and examinations .
details of the oral health component of nhanes iii are published elsewhere ( drury et al . 1996 ) .
the teeth were divided into two maxillary and two mandibular halves and measurements were taken from two sites per tooth ( midbuccal and mesiobuccal ) for all teeth ( excluding third molars ) in one randomly chosen upper and lower quadrant .
these measurements included periodontal probing depth ( ppd ) , gingival recession and bleeding on probing ( bop ) . clinical attachment loss ( cal )
individuals receiving renal replacement therapy ( through dialysis or kidney transplant ) were excluded from periodontal examination .
periodontitis was defined using the 2007 cdc / aap ( centre for disease control and prevention / american academy of periodontology ) classification ( page & eke 2007 ) .
in addition , continuous periodontal parameters were also employed such as mean ppd , mean cal , cumulative periodontal probing depth ( cppd ) , number of teeth present and proportion of sites that bled upon probing .
cumulative ppd was calculated as the sum of the maximum probing pocket depths 4 mm of each tooth and as such is a surrogate measure of the potential extent of biofilm exposed connective tissues ( dietrich et al .
edentulous individuals were included in the analyses but formed a group distinct from individuals with periodontitis .
the serum creatinine levels recorded in the nhanes iii survey were recalibrated to be traceable to an isotopederived mass spectroscopy method using the equation below ( nchs , 2006b ) :
standardized creatinine=(0.960serum creatinine)0.18
age , sex , ethnicity and standardized serum creatinine were incorporated in the ckd epidemiology collaboration ( ckdepi ) equation to calculate estimated glomerular filtration rate ( egfr ) ( levey et al . 2009 ) .
this equation improves mortality risk stratification in individuals with ckd compared with the modification of diet in renal disease ( mdrd ) equation ( shafi et al .
based on an egfr<60 ml / min/1.73 m , individuals were classified as having stage 35 ckd , hitherto referred to as
urinary albumin and creatinine levels were employed to calculate the albumincreatinine ratio ( acr ) .
albuminuria was classified as acr < 30 mg / g ; acr 30 mg / g and < 300 mg / g ; and acr 300 mg / g .
individuals were classed as hypertensive if their mean ( of three consecutive measurements ) systolic blood pressure ( bp ) was 140 mmhg or mean diastolic bp was 90 mmhg .
individuals were classed as diabetic by selfreporting ( excluding gestational diabetes ) or if their glycated haemoglobin ( hba1c ) was 6.5% .
individuals ' smoking status was determined from selfreporting and classified into current , former or never smokers ( cigarettes only ) .
data on covariates employed in the statistical analyses included information on age , sex , ethnicity ( nonhispanic white , nonhispanic black , mexican american or other ) , alcohol consumption ( never , not in last year , between 014 drinks / week , more than 14 drinks / week ) and selfreported history of previous cardiovascular events ( stroke , heart attack or heart failure ) .
self reported measures of socioeconomic status ( household income , marital status and educational attainment ) were coded as follows .
household income ( less than $ 20,000 or $ 20,000 or more ) ; marital status ( married or living as married , never married , divorced or separated or widowed ) ; educational attainment ( less than high school , high school diploma or more than high school ) .
body mass index ( bmi ) was coded as a categorical variable with bmi < 18.5 kg / m as underweight ; 18.5 kg / m and < 25 kg / m as normal ; 25 kg / m and < 30 kg / m as overweight and 30 kg / m as obese .
laboratory tests including serum cholesterol ( total and highdensity lipoprotein / hdl ) were performed .
serum cholesterol levels were classified into binary variables ( total serum cholesterol 24 mg / l or < 24 mg / l and serum hdl cholesterol 3.5 mg
physical activity was selfreported by individuals and reclassified as recommended or more if they reported moderate activity five or more times a week or vigorous activity three or more times a week .
if individuals reported moderate physical activity four or more times a week and vigorous activity one or more times a week or reported moderate activity three or more times a week and vigorous activity two or more times a week .
individuals ' physical activity was classified as none if they reported no leisure time physical activities .
individuals who reported some level of physical activity but less than recommended were classed as
the nchs provide mortality data for nhanes iii participants up to 31st december 2006 , linked by probabilistic record matching with the national death index ( ndi ) .
the publicly available data set contains information on the mortality status of individuals aged 17 years or older . for individuals who are classified as
assumed deceased , information is available on 113 underlying cause of death categories , based on the ninth and tenth revisions of the international classification of diseases ( icd9 and icd10 ) .
cvd mortality was limited to cases where the underlying cause of death was coded between 53 and 75 ( inclusive ) ( anderson et al .
details of the linked mortality data have been published elsewhere ( nchs , 2010 ) .
analyses performed followed guidelines for nhanes iii ( nchs , 1996 ) , accounting for the complex survey design and sampling weights to yield estimates generalizable to the us population .
differences in categorical and continuous data were assessed for statistical significance using pearson chisquare , ttest , fisher 's exact test and analysis of variance ( anova ) as appropriate .
cox proportional hazards ( ph ) regression models were fitted to evaluate the association between periodontal status , traditional risk factors ( diabetes , hypertension and smoking status ) and allcause and cvd mortality , independent of potential confounders .
the fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) .
the ph assumption was tested using schoenfeld residuals , scaled schoenfeld residuals and graphical methods .
any individuals with missing covariate data were not included in the analyses ( listwise deletion ) . thus out of a possible 13,784 individuals eligible for analyses , 1379 ( 10% ) individuals were excluded due to incomplete covariate data ( table s1 ) .
we considered the effect measure modification of mortality ( allcause and cardiovascular ) in individuals with ckd according to their periodontal health status .
we conducted formal tests of interaction between periodontal variables and ckd case definition by entering interaction terms in the model .
further formal tests of interactions between ckd , periodontitis or edentulism and age , gender and ethnicity were also carried out .
analyses were carried out using stata / ic version 12.1 ( statacorp lp , college station , tx , usa ) .
we analysed data from individuals in nhanes iii aged 20 years and older with complete data on serum creatinine , periodontal status and mortality followup ( n = 13,784 ) and with a median followup time of 14.3 years ( mean 13.5 years , range 1 month18.2 years ) .
of the 13,794 individuals included in the analyses , 861 ( 6% ) were classified as ckd and 12,923 as nonckd .
individuals with ckd were more likely to be older , have different ethnic and socioeconomic mix , nonsmokers ( never or ex smokers ) , diabetic , hypertensive , with higher total serum cholesterol and lower levels of serum hdl , report lower levels of physical activity and consume less alcohol and report a history of cvd ( stroke , heart attack and congestive heart failure ) compared to those without ckd .
individuals with ckd were more likely to suffer from periodontitis ( or be edentulous ) and have fewer teeth compared to individuals without ckd .
when examining continuous variables of periodontal health , patients with ckd were more likely to have a greater mean cal and greater bop ( table 1 ) .
values are percentages ( standard error ) unless stated acr , albumincreatinine ratio ; bmi , body mass index ; bop , percentage of sites that bleed on probing ; cal , clinical attachment loss ; cppd , cumulative probing depth ; egfr , estimated glomerular filtration rate ; hdl , highdensity lipoprotein ; ppd , periodontal probing depth . comparing no ckd and ckd . within individuals with ckd , comparing healthy and periodontitis . among individuals with ckd ,
those with periodontitis were more likely to be older , of nonwhite ethnicity , current smokers , diabetic and hypertensive and have a lower egfr compared to periodontally healthy individuals .
these individuals also had lower household incomes and educational attainments compared to periodontally healthy individuals .
periodontally healthy individuals were similar to those with periodontitis in terms of their sex , alcohol consumption , marital status , physical activity , history of cvd events and bmi ( table 1 ) .
after adjusting for covariates , individuals with ckd had a 44% ( 95% ci : 2863% ) increased rate of allcause mortality compared to those without ckd ( table 2 ) .
individuals with periodontitis had a 36% ( 2251% ) increased rate of allcause mortality compared to individuals who were periodontally healthy .
results from cox proportional hazards regression analyses for allcause and cardiovascular mortality using an age and sexadjusted and a fully adjusted model bop , proportion of sites that bleed on probing ; cal , clinical attachment loss ; ckd , chronic kidney disease ; cppd , cumulative periodontal probing depth ; ppd , periodontal probing depth .
fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) .
the association between periodontitis and allcause mortality was similar between individuals with or without ckd ( pvalue for interaction = 0.57 ) .
similarly , the associations between ckd and allcause mortality did not vary by age , sex or diabetes status ( pvalues for interaction 0.14 , 0.99 and 0.09 respectively ) .
furthermore , the association between periodontitis and allcause mortality did not vary by age , gender or diabetes status ( pvalues for interaction 0.73 , 0.51 and 0.51 respectively ) . in edentulous individuals
edentulous individuals under the age of 65 had a significantly increased rate of allcause mortality compared to edentulous individuals 65 years and older , hazard ratio ( hr ) 1.85 ( 1.412.44 ) and 1.18 ( 1.041.33 ) respectively ( tables s2 , s3 and fig .
for example a 1 mm increase in mean ppd was associated with a 17% ( 628% ) increase in incident rate of allcause mortality ( table 2 ) .
edentulousness was associated with a 32% ( 1750% ) increased rate of allcause mortality compared with periodontally healthy dentate individuals .
diabetes ( hr 1.41 ; 1.271.57 ) , hypertension ( hr 1.06 ; 0.931.20 ) and current smoking ( hr 2.12 ; 1.822.48 ) were associated with an increased rate of allcause mortality although this increase was not significant for hypertension ( table 2 ) .
the 10year allcause mortality for individuals with ckd ( but without periodontitis or other traditional risk factors ) was 32% ( 2935% ) .
addition of periodontitis to the risk profile increased 10year mortality to 41% ( 3647% ) .
this increase in mortality was comparable with that seen in individuals with ckd who had diabetes instead of periodontitis ( 43% ; 3849% ) .
a similar cumulative effect on mortality is seen with periodontitis and other traditional risk factors ( table 3 ) .
these estimates are based on the demographic features of individuals with ckd within nhanes iii ( e.g. a mean age of 73 years ) .
estimated survival curves for individuals with ckd and different risk factor profiles is given in fig . 1 .
tenyear allcause mortality ( percentages ) of individuals with ckd by risk factors ( along with the addition of periodontitis to the risk factor ) fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) .
cox proportional hazard regression graphs ( adjusting for age , sex , ethnicity , pulse pressure , history of cvd , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity , household income , marital status and educational attainment ) of survival in patients with ckd stratified using periodontitis and other traditional risk factors ( diabetes , hypertension and smoking ) .
individuals with ckd had a 60% ( 3295% ) increased rate of cvd mortality compared to those without ckd ( table 2 ) , independent of confounders specified .
individuals with periodontitis had a 38% ( 1665% ) increased rate of cvd mortality compared to individuals who were periodontally healthy .
the association between periodontitis and cvd mortality was similar between individuals with or without ckd ( pvalue for interaction = 0.27 ) .
the associations between ckd and cvd mortality did not vary by age , sex or diabetes status ( pvalues for interaction 0.39 , 0.82 and 0.34 respectively ) .
the association between periodontitis and cvd mortality did not vary by gender or diabetes status ( pvalues for interaction 0.77 and 0.17 respectively ) .
there was a trend in patients with ckd and periodontitis to have an increased hr of cvd mortality if they were under the age of 65 compared with 65 and over but this was not significant .
edentulous individuals under the age of 65 having a significantly increased rate of cvd mortality , hr 2.03 ( 1.313.13 ) , compared to edentulous individuals 65 years and older who had comparable rates of cvd mortality compared to periodontally healthy individuals , hr 0.89 ( 0.711.10 ) ( tables s4 , s5 and fig .
mean ppd and percentage of sites that bleed on probing were associated with a statistically significant increase in the rate of cvd mortality ( table 2 ) .
edentulous and periodontally healthy dentate individuals had comparable rates of cvd mortality ( table 2 ) .
diabetes ( hr 1.45 ; 1.241.70 ) , hypertension ( hr 1.32 ; 1.061.63 ) and current smoking ( hr 2.10 ; 1.692.62 ) were associated with an increased rate of cvd mortality ( table 2 ) .
the 10year cvd mortality for individuals with ckd ( and combinations of risk factors ) highlights the similarity in the magnitude of increase in cvd mortality associated with diabetes ( 24% ; 1930% ) compared with periodontitis ( 22% ; 1927% ) ( table 4 ) .
estimated cvd survival for individuals with ckd and different risk factor profiles is given in fig . 2 .
tenyear cvd mortality ( percentages ) of individuals with ckd by risk factors ( along with the addition of periodontitis to the risk factor ) fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) . for cardiovascular mortality .
cox proportional hazard regression graphs ( adjusting for age , sex , ethnicity , pulse pressure , history of cvd , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity , household income , marital status and educational attainment ) of survival in patients with ckd stratified using periodontitis and other traditional risk factors ( diabetes , hypertension and smoking ) .
we analysed data from individuals in nhanes iii aged 20 years and older with complete data on serum creatinine , periodontal status and mortality followup ( n = 13,784 ) and with a median followup time of 14.3 years ( mean 13.5 years , range 1 month18.2 years ) .
of the 13,794 individuals included in the analyses , 861 ( 6% ) were classified as ckd and 12,923 as nonckd .
individuals with ckd were more likely to be older , have different ethnic and socioeconomic mix , nonsmokers ( never or ex smokers ) , diabetic , hypertensive , with higher total serum cholesterol and lower levels of serum hdl , report lower levels of physical activity and consume less alcohol and report a history of cvd ( stroke , heart attack and congestive heart failure ) compared to those without ckd .
individuals with ckd were more likely to suffer from periodontitis ( or be edentulous ) and have fewer teeth compared to individuals without ckd .
when examining continuous variables of periodontal health , patients with ckd were more likely to have a greater mean cal and greater bop ( table 1 ) .
values are percentages ( standard error ) unless stated acr , albumincreatinine ratio ; bmi , body mass index ; bop , percentage of sites that bleed on probing ; cal , clinical attachment loss ; cppd , cumulative probing depth ; egfr , estimated glomerular filtration rate ; hdl , highdensity lipoprotein ; ppd , periodontal probing depth . comparing no ckd and ckd . within individuals with ckd , comparing healthy and periodontitis . among individuals with ckd ,
those with periodontitis were more likely to be older , of nonwhite ethnicity , current smokers , diabetic and hypertensive and have a lower egfr compared to periodontally healthy individuals .
these individuals also had lower household incomes and educational attainments compared to periodontally healthy individuals .
periodontally healthy individuals were similar to those with periodontitis in terms of their sex , alcohol consumption , marital status , physical activity , history of cvd events and bmi ( table 1 ) .
after adjusting for covariates , individuals with ckd had a 44% ( 95% ci : 2863% ) increased rate of allcause mortality compared to those without ckd ( table 2 ) .
individuals with periodontitis had a 36% ( 2251% ) increased rate of allcause mortality compared to individuals who were periodontally healthy .
results from cox proportional hazards regression analyses for allcause and cardiovascular mortality using an age and sexadjusted and a fully adjusted model bop , proportion of sites that bleed on probing ; cal , clinical attachment loss ; ckd , chronic kidney disease ; cppd , cumulative periodontal probing depth ; ppd , periodontal probing depth . fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) .
the association between periodontitis and allcause mortality was similar between individuals with or without ckd ( pvalue for interaction = 0.57 ) .
similarly , the associations between ckd and allcause mortality did not vary by age , sex or diabetes status ( pvalues for interaction 0.14 , 0.99 and 0.09 respectively ) .
furthermore , the association between periodontitis and allcause mortality did not vary by age , gender or diabetes status ( pvalues for interaction 0.73 , 0.51 and 0.51 respectively ) . in edentulous individuals , there was a significant difference in allcause mortality by age .
edentulous individuals under the age of 65 had a significantly increased rate of allcause mortality compared to edentulous individuals 65 years and older , hazard ratio ( hr ) 1.85 ( 1.412.44 ) and 1.18 ( 1.041.33 ) respectively ( tables s2 , s3 and fig .
for example a 1 mm increase in mean ppd was associated with a 17% ( 628% ) increase in incident rate of allcause mortality ( table 2 ) .
edentulousness was associated with a 32% ( 1750% ) increased rate of allcause mortality compared with periodontally healthy dentate individuals .
diabetes ( hr 1.41 ; 1.271.57 ) , hypertension ( hr 1.06 ; 0.931.20 ) and current smoking ( hr 2.12 ; 1.822.48 ) were associated with an increased rate of allcause mortality although this increase was not significant for hypertension ( table 2 ) .
the 10year allcause mortality for individuals with ckd ( but without periodontitis or other traditional risk factors ) was 32% ( 2935% ) .
addition of periodontitis to the risk profile increased 10year mortality to 41% ( 3647% ) .
this increase in mortality was comparable with that seen in individuals with ckd who had diabetes instead of periodontitis ( 43% ; 3849% ) .
a similar cumulative effect on mortality is seen with periodontitis and other traditional risk factors ( table 3 ) .
these estimates are based on the demographic features of individuals with ckd within nhanes iii ( e.g. a mean age of 73 years ) .
estimated survival curves for individuals with ckd and different risk factor profiles is given in fig . 1 .
tenyear allcause mortality ( percentages ) of individuals with ckd by risk factors ( along with the addition of periodontitis to the risk factor ) fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) . for allcause mortality .
cox proportional hazard regression graphs ( adjusting for age , sex , ethnicity , pulse pressure , history of cvd , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity , household income , marital status and educational attainment ) of survival in patients with ckd stratified using periodontitis and other traditional risk factors ( diabetes , hypertension and smoking ) .
after adjusting for covariates , individuals with ckd had a 60% ( 3295% ) increased rate of cvd mortality compared to those without ckd ( table 2 ) , independent of confounders specified .
individuals with periodontitis had a 38% ( 1665% ) increased rate of cvd mortality compared to individuals who were periodontally healthy .
the association between periodontitis and cvd mortality was similar between individuals with or without ckd ( pvalue for interaction = 0.27 ) .
the associations between ckd and cvd mortality did not vary by age , sex or diabetes status ( pvalues for interaction 0.39 , 0.82 and 0.34 respectively ) .
the association between periodontitis and cvd mortality did not vary by gender or diabetes status ( pvalues for interaction 0.77 and 0.17 respectively ) .
there was a trend in patients with ckd and periodontitis to have an increased hr of cvd mortality if they were under the age of 65 compared with 65 and over but this was not significant .
edentulous individuals under the age of 65 having a significantly increased rate of cvd mortality , hr 2.03 ( 1.313.13 ) , compared to edentulous individuals 65 years and older who had comparable rates of cvd mortality compared to periodontally healthy individuals , hr 0.89 ( 0.711.10 ) ( tables s4 , s5 and fig .
mean ppd and percentage of sites that bleed on probing were associated with a statistically significant increase in the rate of cvd mortality ( table 2 ) .
edentulous and periodontally healthy dentate individuals had comparable rates of cvd mortality ( table 2 ) .
diabetes ( hr 1.45 ; 1.241.70 ) , hypertension ( hr 1.32 ; 1.061.63 ) and current smoking ( hr 2.10 ; 1.692.62 ) were associated with an increased rate of cvd mortality ( table 2 ) .
the 10year cvd mortality for individuals with ckd ( and combinations of risk factors ) highlights the similarity in the magnitude of increase in cvd mortality associated with diabetes ( 24% ; 1930% ) compared with periodontitis ( 22% ; 1927% ) ( table 4 ) .
estimated cvd survival for individuals with ckd and different risk factor profiles is given in fig .
tenyear cvd mortality ( percentages ) of individuals with ckd by risk factors ( along with the addition of periodontitis to the risk factor ) fully adjusted model adjusted for age , sex , ethnicity , ckd status , periodontal status , diabetic status , hypertensive status , smoking status , pulse pressure , history of cvd ( heart attack or stroke or heart failure ) , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity and measures of socioeconomic status ( household income , marital status and educational attainment ) . for cardiovascular mortality .
cox proportional hazard regression graphs ( adjusting for age , sex , ethnicity , pulse pressure , history of cvd , alcohol consumption , acr , hypercholesterolaemia and lowhdl , bmi , physical activity , household income , marital status and educational attainment ) of survival in patients with ckd stratified using periodontitis and other traditional risk factors ( diabetes , hypertension and smoking ) .
in this large cohort , representative of the us population from which it was derived , ckd was associated with increased rates of allcause mortality and cvd mortality , independent of periodontitis , traditional risk factors and other confounders .
periodontitis was associated with increased rates of allcause and cvd mortality comparable with , but independent of , that associated with diabetes ( tables 2 , 3 , 4 ; figs 1 and 2 ) .
there was an increased rate of allcause mortality but not cvd mortality in edentulous individuals with ckd compared with periodontally healthy dentate individuals .
the association between edentulousness and cvd mortality was significant in a subgroup of edentulous individuals under the age of 65 . given the high prevalence of chronic periodontitis in patients with ckd ( chambrone et al .
2013 ) , our results suggest that periodontitis may be an important nontraditional risk factor for cvd and allcause mortality in these patients , and interestingly contributing to the increased risk to a similar extent as diabetes .
the strengths of this study are its large populationbased sampling with robust sampling methodology which allow the results from this analysis to be generalized to the us population . the detailed clinical , demographic and anthropomorphic data collected allows for many of the known covariates to be accounted for in the cox proportional hazards regression model , generating more accurate point estimates .
the length of followup for this study is its final strength and allows for the pragmatic assessment of long term , hard outcomes ( allcause and cvd mortality )
. the limitations of this study include the lack of longitudinal examination of individuals .
unfortunately , in nhanes , the longitudinal data is limited to the mortality status of patients derived from the national death index .
data on variables were only gathered at inception and therefore changes in variables ( periodontal , diabetes , smoking status , etc ) are not ascertainable .
analyses were carried out on the assumption that characteristics did not change between inception and time to death or censoring .
some individuals with periodontitis are likely to have received treatment and/or lost teeth during followup , resulting in disease misclassification over time .
furthermore , periodontal measurements from nhanes iii are known to underestimate the prevalence of periodontitis by 13.4% ( absolute ) or 60% ( relative ) ( eke et al .
the results of this study may therefore underestimate the association between periodontitis and mortality in ckd .
also , as with any multivariable regression analysis , the issue of residual confounding from inaccurate measurement or categorization of variables or confounding from variables not included in the analysis can not be ruled out .
previous studies investigating the link between mortality and periodontitis in patients with ckd have done so in patients on haemodialysis ( kshirsagar et al . 2009 , chen et al .
apart from the small sample sizes ( 122253 patients ) and shorter followup period ( 18 months to 6 years ) , these studies differed significantly from the present analysis as individuals receiving rrt ( through chronic dialysis or a functioning kidney transplant ) were not included in the present analysis ( rrt was an exclusion criteria for periodontal examination in nhanes iii ) .
hence , even though these studies demonstrate an association between periodontitis and mortality , thereby lending support to the current findings , the results can not be directly compared .
a putative mechanism for a possible link between periodontitis and increased allcause and cvd mortality is via the increased systemic acutephase and oxidative stress burden .
this increased burden is seen in individuals with periodontitis and ckd ( ioannidou et al .
2011 ) and individuals with periodontitis who do not have ckd ( d'aiuto et al .
2004 , chapple & genco 2013 ) . increased systemic inflammatory and oxidative stress burdens increase the incidence of cvd events in patients with ckd ( arici & walls 2001 , mathew et al .
this mechanism is supported by the association demonstrated here between increased risk of cvd mortality and measures of active periodontitis ( periodontitis case definition , mean ppd and bop ) , as opposed to measures of historical periodontitis ( edentulousness and mean cal ) , where there was a lack of association ( table 2 ) . however
, at least part of the association between periodontitis and cvd may also be due to common risk factors such as smoking and diabetes ( dietrich et al .
the increase in allcause mortality in edentulous individuals compared to periodontally healthy dentate individuals , as reported here and also by other investigators in nonckd cohorts ( brown 2009 ) , may be due to several factors .
patients are rendered edentulous for a variety of reasons including periodontitis , with approximately 50% of teeth being extracted due to periodontal disease ( phipps & stevens 1995 ) .
as approximately half of all tooth extractions are for reasons other than periodontal disease , edentulousness may act as a surrogate marker of general health attitudes and/or behaviours , limited healthcare access or other socioeconomic measures ( joshipura & ritchie 2005 ) .
this might also explain the association between edentulousness and cvd mortality in patients under the age of 65 who might have such characteristics and attitudes towards healthcare that render them edentulous before the age of 65 .
the biological mechanisms underpinning the relationship between periodontitis and increased mortality in individuals with ckd form a promising area of research and may produce mechanistic targets leading to risk stratification and novel interventions .
ongoing longitudinal studies ( stringer et al . 2013 ) investigating large cohorts of patients with predialysis ckd may provide confirmation of this association and shed light upon explanatory mechanisms .
successful treatment of periodontitis has been shown to improve surrogate markers of cvd risk , including serum markers of systemic inflammation ( crp , il6 ) ( d'aiuto et al .
2004 ) , endothelial function as measured by flowmediated dilatation ( fmd ) and endothelialactivation markers such as soluble eselectin and von willebrand factor ( tonetti et al .
two randomized controlled trials of periodontal interventions in patients with ckd have been carried out but limited to cohorts of haemodialysis patients .
these have produced conflicting results either not demonstrating changes in inflammatory markers following periodontal intervention ( wehmeyer et al .
2013 ) or demonstrating that significant reductions in inflammatory markers can be achieved following periodontal therapy ( fang et al .
currently , patients with ckd are managed to strict targets concerning glycaemic control ( diabetes ) and control of hypertension and smoking cessation to improve outcomes .
if a causal link is established between periodontitis and increased rates of adverse outcomes in ckd patients , then establishing and maintaining periodontal health may become an important part of the care pathway of patients with ckd .
figure s1 . cox ph regression graphs for allcause mortality in individuals with periodontitis / edentulism and ckd by age category click here for additional data file .
figure s2 cox ph regression graphs for cvd mortality in individuals with periodontitis / edentulism and ckd by age category .
table s1 numbers of participants ( and percentage ) with missing data in variables included in statistical model .
table s2 exploring the interactions between ckd ( stage 35 ) , periodontal variables and age , gender and diabetes status for allcause mortality .
table s3 hazard ratios ( 95% ci ) and 10year survival ( 95% ci ) of allcause mortality by subgroups of age ( < 65 years of age and 65 years of age ) .
table s4 exploring the interactions between ckd ( stage 35 ) , periodontal variables and age , gender and diabetes status for cvd mortality .
table s5 hazard ratios ( 95% ci ) and 10year survival ( 95% ci ) of cvd mortality by subgroups of age ( < 65 years of age and 65 years of age ) . | abstractintroductionperiodontitis may add to the systemic inflammatory burden in individuals with chronic kidney disease ( ckd ) , thereby contributing to an increased mortality rate .
this study aimed to determine the association between periodontitis and mortality rate ( allcause and cardiovascular diseaserelated ) in individuals with stage 35 ckd , hitherto referred to as
ckd.methodssurvival analysis was carried out using the third national health and nutrition examination survey ( nhanes iii ) and linked mortality data .
cox proportional hazards regression was employed to assess the association between periodontitis and mortality , in individuals with ckd .
this association was compared with the association between mortality and traditional risk factors in ckd mortality ( diabetes , hypertension and smoking).resultsof the 13,784 participants eligible for analysis in nhanes iii , 861 ( 6% ) had ckd .
the median followup for this cohort was 14.3 years . adjusting for confounders ,
the 10year allcause mortality rate for individuals with ckd increased from 32% ( 95% ci : 2935% ) to 41% ( 3647% ) with the addition of periodontitis . for diabetes ,
the 10year allcause mortality rate increased to 43% ( 3849%).conclusionthere is a strong , association between periodontitis and increased mortality in individuals with ckd .
sources of chronic systemic inflammation ( including periodontitis ) may be important contributors to mortality in patients with ckd . |
syndrome research reveals detailed insight into different aspects of congenital malformation of several body components .
such a wide - spread field of interest requires multidisciplinary teamwork , including clinicians and researchers with different backgrounds . in the last decade
a complete overview of craniofacial syndromes would involve not only the dental approach , but also several medical , psychological , and neurological clinical and theoretical approaches .
these are definition and classification of dental deviations ( part 1 ) ; appearance of malformations before birth ( part 2 ) and a specific new dental approach to craniofacial syndromes ( part 3 ) .
defined syndromes accordingly : a syndrome is a pattern of multiple anomalies thought to be pathogenetically related and not known to represent a single sequence or a polytopic field defect . furthermore , spranger et al . defines
sequence as a pattern of multiple anomalies derived from a single known or presumed prior anomaly or mechanical factor .
a polytopic field defect is defined as a pattern of anomalies derived from the disturbance of a single developmental field .
dorland 's illustrated medical dictionary defines syndrome as a set of symptoms that occur together ; the sum of signs of any morbid state ; a symptom complex . in genetics , a pattern of multiple malformations thought to be pathogenetically related .
a craniofacial syndrome is characterized by morphological and developmental deviations in the cranial tissue components , including teeth .
it is difficult to determine whether the deviation has developed at a primary location that subsequently causes secondary alterations or whether there are general deviations in many structures and at many locations . in some craniofacial syndromes ,
even though the genotype is known in a craniofacial syndrome , it is not understood why that particular genotype causes the malformations observed .
a known genotype deviation can manifest itself in the cranium by many different phenotypic expressions , from severe to minor affection .
how and why the genotype affects the cranial development and why it affects the cranial components differently are far from explained in full . in the foreword to the textbook syndromes of the head and neck by gorlin et al .
f. clarke fraser stated that the symptoms in craniofacial syndromes may not be limited to the head and neck .
it can therefore be questioned whether there are craniofacial syndromes that are limited to the head and neck .
focus is given to studies on craniofacial syndromes in which abnormal traits in the dentition are associated symptoms .
such abnormal traits could be deviations in tooth number ( agenesis , supernumerarity ) , tooth morphology ( size , dimensions , crown invaginations , and abnormal shapes of crowns and roots ) , tooth eruption ( delayed eruption , ankylosis ) , and resorption ( crowns and roots ) .
schalk - van der weide reported that specific patterns of agenesis was associated with specific syndromes . thus
, absence of the following teeth was observed in these syndromes : bk 's syndrome : premolars ; rieger syndrome : maxillary incisors ( constant ) , mandibular incisors , and premolars ( occasional ) ; ellis - van creveld syndrome : mandibular incisors and canines ; gorlin - chaudry - moss syndrome : deciduous molars , secondary premolars , and molars ; lipoid proteinosis : maxillary lateral incisors , canines , and premolars ; coffin - lowry syndrome : maxillary lateral incisors , mandibular incisors ; orofaciodigital syndrome type i : mandibular lateral incisors and canines ; down syndrome : third molars , mandibular second premolars , and maxillary lateral incisors ; holoprosencephaly : maxillary incisors ; hypoglossia - hypodactylia syndrome : mandibular incisors and canines ; glossopalatine ankylosis syndrome : incisors .
deviations in tooth dimensions such as short roots and malformations of crowns and roots have been observed in turner syndrome , in klinefelter syndrome , and in rothmund - thomson syndrome .
delayed resorption of primary teeth has been observed in cleidocranial dysplasia and in hyper ige syndrome .
these symptoms of deviations in the dentition are all phenotypic traits of the specific syndromes mentioned .
craniofacial research is currently focused especially on determination of genotypes of different syndromes [ 6 , 1821 ] .
research is also devoted to phenotypic descriptions of craniofacial syndromes [ 2224 ] . from a treatment point of view
yap and klineberg stated that the success rate is lower for implants in patients with ectodermal dysplasia than it is in patients with tooth agenesis but without ectodermal dysplasia .
in the central textbook on craniofacial syndromes , gorlin et al . stated that one - third of children born with birth defects display anomalies in the head and neck .
he also reported that 72 different syndromes with orofacial cleft were registered in 1971 and that this number had increased to 250 syndromes in 1990 .
this development involves all craniofacial phenotypes and new findings document that craniofacial phenotypes are gradually associated with symptoms outside the craniofacial area . in 2010 , trainor reported that more than 700 craniofacial malformations have been described .
the goal for future research on craniofacial syndromes must be to understand the connection between symptoms in the dentition and other symptoms in the craniofacial area .
how can the initial malformation sites in these syndromes be studied and what can we learn from it ? prenatal syndromes can be studied by ultrasound or by autopsy after spontaneous or provoked abortion .
research on human prenatal autopsy material for demonstration of craniofacial development has a morphological character and is predominantly based on histological , histochemical , and radiographic studies .
these studies depend entirely on autopsy legislation in different countries and represent primarily the embryonic period and the foetal period before the 16th week of gestation .
in contrast to human studies , experimental studies on the craniofacial development in animals allow for a variety of advanced procedures , such as cell cultures and labelling of migrating cells , and may cover the complete foetal period [ 28 , 29 ] . in a series of human autopsy studies , the initial sites of malformation in craniofacial syndromes have been investigated . of specific interest
are studies on holoprosencephaly [ 30 , 31 ] , anencephaly [ 3235 ] , amniotic band rupture , down syndrome ( trisomy 21 ) [ 3740 ] , other trisomies [ 4145 ] , meckel syndrome [ 46 , 47 ] , orofacial clefts [ 4850 ] , and cranial encephalocele . in order to evaluate these pathologies ,
normal development has been described in several studies and comprised in the textbook the prenatal human cranium . when normal development is compared with the pathologic conditions mentioned above , the following is observed .
the deviation is located in a midsagittal field between the eyes and spans backwards to the sella turcica .
the cranial deviation corresponds to the desmal - formed theka crania , but the cartilaginous originated part of the occipital squama is not malformed .
other cranial structures may be deformed due to the absent cerebrum , but the structures are present .
though amniotic band rupture in the cranium appears as a craniofacial syndrome , it is not a congenital malformation and not a syndrome .
analyses of the condition reveal that rupture of a normal developmental course has occurred and caused extensive disruptions , not limited to developmental regions ( fields ) .
the cranial phenotype in foetuses with down syndrome is characterized by short or absent nasal bone , short and abnormal , often cleft , palate , deviations in size and dimensions of the occipital area ( the occipital or cerebellar field ) , and malformations in the sella turcica .
thus , deviations occur in many regions of the cranium . the craniofacial skeleton in trisomy 18 and 13 and triploidy has been studied and it was shown that the developmental deviations are completely different in these trisomies .
this indicates an early and specific interrelationship between genotype and phenotype . in meckel syndrome ,
the nasal bone is short in cleft lip , but not in cleft palate and combined cleft lip and palate .
abnormal findings were seen in the cranial base / sella turcica and the nasal bone / maxilla .
interestingly , the same type of malformation occurs in the sella turcica / pituitary gland region in different types of encephalocele .
a general conclusion from craniofacial studies in the foetal period is that an association exists between the development of the cranium and the body axis / cervical spine and between the cranium and the central and peripheral nervous systems ( cns / pns ) .
this is not surprising as studies on the early gastrulation in the embryonic period have shown an axial midline structure in the body , the primitive streak , which will later involve the notochord and the head formation .
the rostral extent of the notochord appears in the posterior wall of the sella turcica .
concerning cranial development , the main conclusion is that different regions of the cranium are malformed in different genotypes .
experimental studies performed by le douarin et al . [ 28 , 55 ] on animal tissue have revolutionized the embryological understanding of the craniofacial development . in these studies
the neural crest cells from different specific regions were labelled and the authors discovered that cells from different regions on the neural tube migrate to different specific parts of the cranial face and dentition , thus forming different developmental fields . in order to understand the human craniofacial development , it is necessary to combine morphological human studies with knowledge acquired through experimental studies regarding cell migration .
such combination studies have led to insight into developmental fields in humans [ 5 , 7 ] .
the developmental fields in the human cranium are illustrated in figures 1 and 2 and in the dentition in figures 3 and 4 .
defined a developmental field as a region or part of an embryo which responds as a coordinated unit to embryonic interaction and results in complex or multiple anatomical structures .
an instrinsic , nondisruptive disturbance of a developmental field will lead to a field defect .
the developmental fields in the maxilla described in the present paper are the frontonasal field , the maxillary field , and the palatal field .
these three bilateral fields arise from different regions on the neural crest and have three different main nerve supplies : nervus nasopalatinus , nervus maxillaris , and nervus palatines , respectively .
three similar developmental fields exist in the mandible , innervated by different nerve branches , which are connected during development and mandibular growth in the bundle of peripheral nerves named the inferior alveolar nerve .
the interrelationship between the central and peripheral nervous systems has been studied intensively in human foetal pathological studies .
developmental fields in the cranium extend in a triangular shape from the pituitary gland / sella turcica region to an outer region of the face .
the sella turcica / pituitary gland region is the end region for the rostral extension of the notochord .
it is therefore necessary , when describing a craniofacial developmental field , to describe the complete field in its 3-dimensional extent .
the field that has been described most in detail based on foetal pathological observations is the frontonasal field [ 30 , 34 ] ( figure 5 ) .
this field includes the anterior wall of the sella turcica , the anterior cranial base , the interocular region , nose , and philtrum ( figure 5 ) . within this field ,
the involvement of the vomeronasal organs producing luteinizing hormone - releasing hormone , lhrh , and the sella turcica / pituitary gland [ 60 , 61 ] can often explain hormone - related deviations in body height and maturity associated with deviations in the frontonasal field .
it has been shown that syndromes , for example , velocardiofacial syndrome located in the palatal field in the cranium , also involve organ structures such as the brain , thymus , thyroid , and heart septum .
it is not obvious that malformations of these organs are caused by the same field defect , but it may be explained from the neural crest cell migration in this region .
another example of craniofacial malformations interrelated with organ malformations can be seen in cri du chat syndrome where the cerebellum and larynx , located far apart , are developmentally associated with the posterior cranial base .
analysis of the primary and permanent dentitions can determine whether a syndrome affects a single craniofacial field or several fields .
the previous section showed how prenatal studies are useful for analysis of the initial site of organ and osseous malformations , but due to the early period of development , less useful for analysis of the dentition . only early traces in the development of the primary teeth
in contrast to the prenatal tissue analysis , postnatal analysis on radiographic material is highly useful for analysis of primary and permanent teeth , but not useful at all for analysis and limitation of developmental fields .
a combination of prenatal and postnatal insight is therefore necessary in order to understand how developmental fields are affected in different craniofacial syndromes .
he also states that processes in developmental fields are self - organizing spatially coordinated and ordered , epimorphically hierarchical , temporarily synchronized , epigenetically interactive , developmentally constrained , and phylogenetically conserved . in a craniofacial syndrome where deviations occur in a specific field , it is expected that all structures , including teeth , within the field can be deviant .
, focus will be on the smmci syndrome ( single median maxillary central incisor ) with gene location 7q36.3 . in this syndrome
the craniofacial examination of this condition reveals a highly significantly shorter anterior cranial base . in smmci , only one maxillary central incisor exists in both the primary and permanent dentitions [ 67 , 68 ] .
the malformation has been associated with sonic hedgehog gene , which normally defines the midaxial part of the frontonasal field [ 31 , 69 ] . as a result ,
the midaxial structures of the face , maxilla , nasal cavity , nasal bone , anterior cranial fossa , and sella turcica are malformed .
the sella turcica is often tiny and malformed and the growth hormone production in the pituitary gland deviates .
this is a new finding that expands the craniofacial diagnostics to include brain diagnostics as well .
the smmci condition is demonstrated by clinical and radiographic images ( figures 6 , 7 , 8 , and 9 ) .
the illustrations show that the postnatal findings are in complete concurrence with the prenatal findings stating that only a single field ( frontonasal field ) is involved in the syndrome . in several craniofacial syndromes ,
ageneses are registered in all craniofacial fields [ 70 , 71 ] in locations where ageneses are normally registered [ 70 , 72 ] , that is , lateral incisors in the frontonasal fields ; second premolars in the maxillary field ; third molars in the palatal field . in the mandible ,
ageneses are seen most often in the central incisor region , the second premolar region , and third molar region .
compared to normal conditions , the occurrence of agenesis is about 10 times higher in patients with down syndrome , and especially high in the mandibular incisor region . also narrow crowns and short roots
the craniofacial morphology reveals different malocclusions and different malformation signs in the cranium , such as absent or short nasal bone , enlarged thickness of the theka crania , and malformations in the cervical column .
the down condition is demonstrated by clinical and radiographic images ( figures 10 and 11 ) .
the illustrations show that the postnatal findings are in complete concurrence with the prenatal findings stating that symptoms occur in several fields in down syndrome .
this distinction between the location of dental deviations in a single field and in several fields is essential for our insight into craniofacial syndromes .
it is believed that the dentition will become central in diagnostics and evaluation of the pathogenesis behind craniofacial syndromes . in a recent paper ,
trainor has highlighted the role of neural crest cells in the aetiology and pathogenesis of treacher collins syndrome and furthermore for the potential for prevention .
the pathogenesis of smmci is associated with the sonic hedgehog gene that is expressed in the midcranial region anterior to the pituitary gland [ 31 , 69 ] . in
down syndrome / trisomy 21 , a possible gene dosage effect associated with the extra chromosome may influence the phenotype .
future research in craniofacial syndromology should include extended studies on developmental fields in order to establish a more sufficient background for elucidating the pathogenesis . in the frontonasal segment
it would be interesting to focus on fields with dental deviations in the maxillary incisor region such as kallmann syndrome , rieger syndrome , kgb syndrome [ 3 , 76 ] , and cleft lip syndrome [ 77 , 78 ] .
in the anterior incisor field in the mandible , it would be interesting to focus on ellis - van creveld syndrome described by gorlin et al . .
also williams syndrome and turner syndrome may contribute to new knowledge on the pathogenesis .
it is important to realize that a syndrome has different phenotypic appearances , ranging from mild to severe .
it is also obvious that syndromes that are normally confined to one field may have symptoms outside that specific field , as observed in rieger syndrome .
there remains still a need for a systematic analysis of the interrelationship between dental deviations , skeletal deviations , associated organ deviations , body growth , and genotypes . in recent years
, extensive experimental animal studies have focused in particular on the neural crest cell migration in the craniofacial region .
these studies are highly important for understanding human development as well . a study such as the previously described study on velocardiofacial syndrome , also known as chromosome 22q 11.2 deletion syndrome , with widespread symptoms in the same neural crest developmental field , may influence the classification by spranger et al . from 1982 .
part 3 shows how developmental fields can explore and advance the concept of dental approaches to craniofacial syndromes .
the great advantage of dental analyses compared to all other analyses performed on human tissue is that deviations in the hard tissue persist and remain stable during the developmental course . as dental tissues
. therefore , a dental approach to craniofacial syndromes by analysis of developmental fields contributes to an understanding of the pathogenesis of craniofacial syndromes .
a problem raised by hennekam in 2007 is which clinical condition can be called a syndrome and which can not .
there are arguments in favour for using aetiology and pathogenesis as the core issue , but hennekam also states that there are arguments to make the patient 's phenotype decide the syndrome definition .
another problem that should be solved in the craniofacial analysis is the genetics behind the craniofacial fields .
is there a signalling gradient involved in the cranial pattern formation , such as suggested in limb development ? .
from an embryological and pathological point of view , it can be presumed that the notochord activates the neural crest cells to migration and that different genes are responsible for the different locations of neural crest cells at the neural tube .
this adds another aspect to craniofacial development and syndromology and calls for scientific attention in the future . | the paper consists of three parts .
part 1 : definition of syndromes .
focus is given to craniofacial syndromes in which abnormal traits in the dentition are associated symptoms .
in the last decade , research has concentrated on phenotype , genotype , growth , development , function , and treatment .
part 2 : syndromes before birth . how can the initial malformation sites in these syndromes be studied and what can we learn from it ? in this section , deviations observed in syndromes prenatally will be highlighted and compared to the normal human embryological craniofacial development .
specific focus will be given to developmental fields studied on animal tissue and transferred to human cranial development .
part 3 : developmental fields affected in two craniofacial syndromes .
analysis of primary and permanent dentitions can determine whether a syndrome affects a single craniofacial field or several fields .
this distinction is essential for insight into craniofacial syndromes .
the dentition , thus , becomes central in diagnostics and evaluation of the pathogenesis .
developmental fields can explore and advance the concept of dental approaches to craniofacial syndromes . discussion .
as deviations in teeth persist and do not reorganize during growth and development , the dentition is considered useful for distinguishing between syndrome pathogenesis manifested in a single developmental field and in several fields . |
unilateral ventriculomegaly is well - described in children secondary to neoplastic or infectious causes , and also due to congenital atresia , with or without the presence of a membrane .
idiopathic occlusion of the foramen of monro in adulthood is exceedingly rare , with only 14 cases previously described .
these patients present with clinical features of raised intracranial pressure ( icp ) necessitating early treatment .
presentation as chronic hydrocephalus without clinical and radiological features of raised icp poses a dilemma regarding surgical treatment .
there is a possibility that the patient may have a primary headache , and imaging findings are incidental . in such scenario
we describe a case of unilateral hydrocephalus due to foramen of monro occlusion , presenting as chronic headache , successfully treated with neuroendoscopy .
the headache used to be left - sided accompanied with vomiting , and intolerance to noise and light .
she was diagnosed with migraine headaches , and took the following drugs for migraine : flunarizine , propranolol , tramadol , and naproxen .
she had partial relief with flunarizine . however , she developed episodic retro - orbital pain with nausea .
there was no history suggestive of neither prior central nervous system infection nor intraventricular hemorrhage .
a combination of above - mentioned drugs was tried sequentially but did not relieve headache .
she was evaluated with a magnetic resonance imaging ( mri ) of brain , which revealed the dilatation of left lateral ventricle , with no periventricular lucency . as she did not have any classical clinical features of raised icp ,
she underwent mri twice at the annual interval , with no change in her ventriculomegaly .
the constructed interference in steady state three - dimensional sequence revealed a membrane blocking the foramen of monro but no lesion [ figure 1 ] .
lumbar puncture was not done to find the etiology of the occlusion of foramen as the patient had unilateral ventriculomegaly , and it was deemed as a contraindication . a final diagnosis of idiopathic occlusion of the left foramen of monro was made .
the proposed mechanism of headache was probably raised icp due to obstruction of the foramen of monro . as she did not have adequate relief of headache , a decision for surgery
( b ) t1-weighted axial acquisition she underwent left precoronal burr hole , and neuroendoscopy .
the right side ventricle was small , and negotiation of endoscope in the right ventricle would have been technically difficult . moreover , the aim of surgery was also to inspect the occluded foramen and dilate it if possible , hence left side approach was used . after making the burr hole
a ventricular cannula was inserted , and icp was measured by attaching it to a transducer that was connected to monitor . the opening pressure was 22 mm hg .
the fenestration of the septum pellucidum was performed at the level of avascular zone , between anterior and posterior septal veins .
, the endoscope was introduced into the stoma to inspect the contralateral ventricle and confirm the adequacy of septostomy .
there were membranes at ipsilateral foramen of monro which were fenestrated [ figure 2 ] .
the ventricular cerebrospinal fluid ( csf ) analysis was normal , hence infection was ruled out .
she was discharged with instruction to take paracetamol for headache as and when required . at 3 months of follow - up
, she was free from headache without medications . at follow - up of 15 months , she was still free from headaches .
an mri done at 15 months showed septostomy defect ; however , the membranes at foramen monro were persistent [ figure 3 ] .
( b ) dilated foramen after formanioplasty magnetic resonance imaging at follow - up showing patency of septostomy
though unilateral ventriculomegaly due to the occlusion of the foramen of monro is described in the literature , our case is unique in terms of clinical and radiological presentation .
our patient did not have classical clinical as well as radiological features of raised icp leading to dilemma of surgical treatment .
there were no classical symptoms of raised icp such as headache with vomiting and blurring of vision and papilledema in present case . though raised icp can present as chronic headache , it was difficult to ascribe headache to intracranial pathology in present case .
the inherent risks of procedure including bleeding and subdural hygroma were kept in mind . hence a conservative treatment in our case
as her headache was not responding to medications , the option of surgical treatment was considered .
a septostomy was done to establish communication between ventricles so that csf can drain through opposite side normal ventricle .
though there was no appreciable reduction in the size of ventricles at follow - up mri , the treatment was still considered successful as the patient had headache relief .
one may argue that the cause of relief of headache is not surgery as there was no reduction in ventricular size .
though comparison ventricular measurements are not available in cases of unilateral ventriculomegaly , it is known that in the cases of endoscopic third ventriculostomy , ventricular size is not always reduced . the proportion of patients who do not have ventricular size reduction ranges between 11% and 60% .
the ventricular size reduction is easier to appreciate in patients with acute hydrocephalus , whereas in chronic hydrocephalus the ventricular size changes are subtler and in some cases are only perceived when detailed measurements or volumetric studies are performed .
currently , the consensus is that ventricular size reduction does not seem to correlate with clinical outcome . in patients with chronic hydrocephalus
, there may not be reduction in the size of ventricles after neuroendoscopic procedure , in such cases the success of treatment should be measured by clinical rather than radiological improvement .
the neuroendoscopic options for unilateral occlusion of the foramen of monro are septostomy and/or foraminoplasty .
though fenestration of membranes at the foramen of monro was done in our patient , the patency could not be established as evident by the persistence of membranes visible on mri at follow - up . however , the patency of septostomy helped the circulation of csf , and relief of headache .
the early success rate of more than 95% and a long - term success rate of 92% has been described with septostomy .
the septostomy allows the obstruction to be bypassed by creating a csf circulation between the obstructed ventricle and the opposite ventricle communicating with the third ventricle .
the same aim can be achieved by performing a foraminoplasty , but it poses higher risk of injuring the adjacent fornix , veins , and hypothalamus .
unilateral hydrocephalus due to the occlusion of foramen of monro can present with chronic headache .
| asymmetric ventriculomegly due to idiopathic occlusion of the foramen of monro is rare .
such patients present with clinical features of raised intracranial pressure ( icp ) . presentation as chronic headache has not been previously described . in the absence of raised icp ,
pursuing surgical treatment raises a clinical dilemma as the headache may be a primary headache with no improvement after surgery .
a 21-year - old woman presented with chronic headache .
she was found to have asymmetric ventriculomegaly due to the occlusion of the foramen of monro .
she underwent endoscopic septostomy and widening of the foramen of monro .
her headache subsided after surgery . at 15 months of follow - up
, she was free from headache without medications .
unilateral occlusion of the foramen of monro can present with asymmetric ventriculomegaly resulting in chronic headache .
though the symptoms of raised icp may not be present , still endoscopic relief of ventriculomegaly leads to cure of headache . |
although even the complete absence of attached gingiva is compatible with oral health , certain clinical situations require definitive gingival augmentation .
an epithelized autogenous masticatory mucosal graft procured from palate is preferred by most of the clinicians to increase the zone of keratinized tissue .
leading to compromised esthetics , also postoperative sequela on palate at times can be significant .
this procedure aimed at investigating the potential of buccal marginal gingiva as a donor tissue to augment gingiva .
two patients ( 25-year - old male , female aged 32 years ) with miller 's class iii gingival recession along with narrow keratinized gingiva in the mandibular anterior region reported to the department of periodontics , post graduate institute of dental sciences , rohtak , india in may 2010 [ figure 1a and b ] .
initial periodontal therapy included sessions of oral hygiene instructions , scaling , and root planing . despite their efforts and professional guidance ,
as both the patients had shallow palatal valves , it was decided to procure donor tissue from maxillary buccal marginal gingiva .
preoperative view ( case 1 ) : gingival recession with shallow vestibular depth and narrow band of keratinized gingiva in respect to 31 , 41 ( a ) .
preoperative situation ( case2 ) : gingival recession along with shallow vestibular and narrow band of keratinized gingiva depth in respect to 41 ( b )
. recipient bed preparation apical to the available keratinized tissue ( case 1 ) ( c ) .
donor tissue was harvested from buccal marginal gingiva of maxillary first molar ( case 1 ) ( d ) .
graft was sutured on the recipient bed with silk sutures ( case 1 ) ( e ) following local anesthesia ( 2% lignocaine , adrenaline 1:80,000 ) the exposed root surfaces were planed using gracey 12 curette .
a horizontal incision was made with no 15 scalpel apical to the available keratinized tissue to prepare recipient bed [ figure 1c ] .
muscle and loose connective tissue ( ct ) fibers were thoroughly scraped to prevent subsequent graft mobility .
a horizontal incision was given at the base of gingival sulcus and at least 1 mm coronal to alveolar crest in relation to right and left maxillary first molars ( 16 , 26 ) for case 1 and case 2 respectively [ figure 1d ] to harvest donor tissue .
tissue measuring 3 mm 9 mm was made free by sulcular incision and was trimmed to remove sulcular epithelium and to standardize its thickness .
it was positioned on the recipient bed and secured in place with silk sutures ( 3 - 0 ethicon non absorbable silk suture ) [ figure 1e ] and covered with coe pack .
patients were refrained from brushing at the surgical sites and were instructed not to pull the lip or manipulate operated area for 2 weeks .
patients were recalled at 2 and 4 week and then after every 3 months to monitor healing .
parameters recorded at recipient sites included plaque index ( pi ) , gingival index ( gi ) , width of keratinized tissue ( wkt ) that is , corono apical width of graft at baseline ; increased width of keratinized gingiva on recalls .
at donor site pi , gi , probing depth and location of the gingival margin with respect to cementoenamel junction were recorded using williams probe .
esthetics was evaluated at the end of 3 years using a qualitative questionnaire given to five masked examiners who rated the result as excellent , very good , good , fair , and poor . at 3 months postoperatively when keratinization is expected to be complete , graft blended well with the adjacent tissues and was not clearly distinguishable [ figure 2a and b ] . increased gingival width following surgical procedure [ table 1 , figure 2c and d ] improved oral hygiene and decreased gingival inflammation [ tables 2 and 3 ] .
gingiva at donor sites regained its normal form and contour at 3 months [ figure 3a and b ] .
evaluation of esthetics demonstrated acceptable esthetic results wherein 80% and 60% examiners rated results as excellent for case 1 and case 2 , respectively , while rest rated it as very good [ table 4 ] .
( a ) final healing at 36 months ( case 1 ) : note the presence of increased keratinized tissue and color match .
( b ) final healing at 36 months ( case 2 ) : note the presence of enough keratinized tissue and color match .
( c ) 2 mm gain in the width of keratinized gingiva following soft tissue graft at 36-month follow - up ( case 1 ) .
( d ) a gain of 2.5 mm in the width of keratinized gingiva following gingival augmentation was recorded at 36-month follow - up ( case 2 ) ( a ) healing at donor site at 36 months : note the normal form and harmony of gingiva with the adjacent teeth ( case 1 ) .
( b ) maxillary first molar on the contralateral site : note the comparable contour of the gingival margin ( case 1 ) wkt ( mm ) at baseline and subsequent recall visits pi scores at recipient sites at baseline and various follow - up gi scores at recipient sites at baseline and various follow - up color evaluation at the recipient site at 3 years
two patients ( 25-year - old male , female aged 32 years ) with miller 's class iii gingival recession along with narrow keratinized gingiva in the mandibular anterior region reported to the department of periodontics , post graduate institute of dental sciences , rohtak , india in may 2010 [ figure 1a and b ] .
initial periodontal therapy included sessions of oral hygiene instructions , scaling , and root planing . despite their efforts and professional guidance ,
as both the patients had shallow palatal valves , it was decided to procure donor tissue from maxillary buccal marginal gingiva .
preoperative view ( case 1 ) : gingival recession with shallow vestibular depth and narrow band of keratinized gingiva in respect to 31 , 41 ( a ) .
preoperative situation ( case2 ) : gingival recession along with shallow vestibular and narrow band of keratinized gingiva depth in respect to 41 ( b )
. recipient bed preparation apical to the available keratinized tissue ( case 1 ) ( c ) .
donor tissue was harvested from buccal marginal gingiva of maxillary first molar ( case 1 ) ( d ) .
graft was sutured on the recipient bed with silk sutures ( case 1 ) ( e )
following local anesthesia ( 2% lignocaine , adrenaline 1:80,000 ) the exposed root surfaces were planed using gracey 12 curette .
a horizontal incision was made with no 15 scalpel apical to the available keratinized tissue to prepare recipient bed [ figure 1c ] .
muscle and loose connective tissue ( ct ) fibers were thoroughly scraped to prevent subsequent graft mobility .
a horizontal incision was given at the base of gingival sulcus and at least 1 mm coronal to alveolar crest in relation to right and left maxillary first molars ( 16 , 26 ) for case 1 and case 2 respectively [ figure 1d ] to harvest donor tissue .
tissue measuring 3 mm 9 mm was made free by sulcular incision and was trimmed to remove sulcular epithelium and to standardize its thickness .
it was positioned on the recipient bed and secured in place with silk sutures ( 3 - 0 ethicon non absorbable silk suture ) [ figure 1e ] and covered with coe pack .
patients were refrained from brushing at the surgical sites and were instructed not to pull the lip or manipulate operated area for 2 weeks .
patients were recalled at 2 and 4 week and then after every 3 months to monitor healing .
parameters recorded at recipient sites included plaque index ( pi ) , gingival index ( gi ) , width of keratinized tissue ( wkt ) that is , corono apical width of graft at baseline ; increased width of keratinized gingiva on recalls .
at donor site pi , gi , probing depth and location of the gingival margin with respect to cementoenamel junction were recorded using williams probe .
esthetics was evaluated at the end of 3 years using a qualitative questionnaire given to five masked examiners who rated the result as excellent , very good , good , fair , and poor . at 3 months postoperatively
when keratinization is expected to be complete , graft blended well with the adjacent tissues and was not clearly distinguishable [ figure 2a and b ] . increased gingival width following surgical procedure [ table 1 , figure 2c and d ] improved oral hygiene and decreased gingival inflammation [ tables 2 and 3 ] .
gingiva at donor sites regained its normal form and contour at 3 months [ figure 3a and b ] .
evaluation of esthetics demonstrated acceptable esthetic results wherein 80% and 60% examiners rated results as excellent for case 1 and case 2 , respectively , while rest rated it as very good [ table 4 ] .
( a ) final healing at 36 months ( case 1 ) : note the presence of increased keratinized tissue and color match .
( b ) final healing at 36 months ( case 2 ) : note the presence of enough keratinized tissue and color match .
( c ) 2 mm gain in the width of keratinized gingiva following soft tissue graft at 36-month follow - up ( case 1 ) .
( d ) a gain of 2.5 mm in the width of keratinized gingiva following gingival augmentation was recorded at 36-month follow - up ( case 2 ) ( a ) healing at donor site at 36 months : note the normal form and harmony of gingiva with the adjacent teeth ( case 1 ) .
( b ) maxillary first molar on the contralateral site : note the comparable contour of the gingival margin ( case 1 ) wkt ( mm ) at baseline and subsequent recall visits pi scores at recipient sites at baseline and various follow - up gi scores at recipient sites at baseline and various follow - up color evaluation at the recipient site at 3 years
in past few years , esthetic results for augmentation procedures have become a prime concern for many patients and clinicians . in both the cases ,
the boundary between grafted tissue and surrounding gingiva could not be easily distinguished as can be seen by comparing pre and postoperative photographs .
whereas the color match of free gingival graft ( fgg ) from palate is not always predictable and often appears whitish and thick ; with characteristic tire - patch appearance .
subepithelial connective tissue graft ( sectg ) often results in bulky tissue contour which mostly demands post healing gingivoplasty .
research also suggests that deep palatal ct lack the full potential of inducing transformation to keratinized epithelium .
allografts ( adma ) offer the advantage of predictable color match but suffer poor predictability owing to considerable postoperative shrinkage and inconsistent quality of tissue gained .
it has been suggested that most of the graft shrinkage occurs within the 1 year of healing . in the documented cases , most of the postoperative shrinkage was complete by 3 months after which the results were maintained .
a total shrinkage of 33.33% and 17.6% was observed for case 1 and case 2 , respectively .
postoperative shrinkage using donor tissue harvested from the palate has been reported to vary between 30% and 50% while significant shrinkage up to 89.5% is reported with adma .
patients in the presented cases did not complain of any postoperative discomfort at both recipient and donor sites at any point of time . on the other hand
, patients treated with fgg harvested from the palate frequently experience postoperative pain and discomfort as a palatal wound is most of the times painful and slow to heal .
another finding which supports this procedure is that the gingiva gained its normal form at donor sites in both the cases .
this could be attributed to the inherent characteristic of periodontium to form a new physiological supracrestal gingival unit .
gingiva in documented cases was of a thick biotype which is known to predictability grow coronally to acquire healthy gingival sulcus .
chief limitation of this technique is limited availability of harvestable tissue ; also tissue biotype should be cautiously evaluated .
it may be concluded that buccal marginal gingiva may provide a useful substitute to other donor tissues to augment gingiva . | the indications to increase the width of keratinized gingiva have not been proven beyond doubt ; however it becomes indispensable in certain clinical situations .
inspite of frequently encountered complications , palate is considered most preferred area to harvest the free gingival graft ( fgg ) .
this procedure aimed at investigating the potential of buccal marginal gingiva as a donor to augment keratinized gingiva . to the best of our knowledge ,
no such cases have been documented in the literature .
fgg harvested from maxillary buccal marginal gingiva was used to augment gingiva in the mandibular anterior region for two patients .
this not only improved plaque control but also resulted in acceptable esthetic results over 3 years .
furthermore , gingiva at donor sites gained its normal form and was in harmony with the neighboring teeth .
it may be concluded that buccal marginal gingiva may provide a predictable substitute to other donor tissues to augment gingiva . |
cysteine - rich peptides are of significant and growing interest due to the increasing use of tetracysteine tags for fluorescence imaging of living cells(1 ) and as tools for bioremediation and phytoremediation of toxic heavy metal contaminants such as cadmium and mercury.(2 ) a library of 205 designed tetracysteine peptides immobilized on cellulose has been successfully screened against biarsenical probes.(3 ) but , the drawback is that these spatially addressed libraries tend to be orders of magnitude smaller than biological or bead - based libraries .
cell - based cysteine - rich libraries have also been successfully used to identify optimized peptide tags for biarsenicals.(4 ) however , cytotoxicity is an intrinsic liability in the use of cell - based libraries to identify peptides that can sequester heavy metals .
in contrast , phage libraries can be screened under abiotic conditions and are less susceptible to cytotoxic effects .
they have been used to identify peptides that bind to immobilized metals , but immobilized metals present few open coordination sites for tight peptide binding of phage - displayed peptides.(5 ) a complementary method is needed for identification of cysteine - rich peptides that tightly bind to free metals and cytotoxic ligands .
one - bead one - compound ( oboc ) peptide libraries are powerful tools to simultaneously screen millions of peptides for exceptional reactivity and selectivity.(6 ) they can incorporate both ribosomal and nonribosomal amino acids and can be screened under conditions toxic to cells .
however , due to the problems caused by cysteine during synthesis , screening , and sequencing , cysteine is generally excluded from oboc libraries .
standard conditions ( 1:1 dmf / ch2cl2)(7 ) for the synthesis of polycysteine peptides using tritylcysteine are inefficient due to the bulk and hydrophobicity of the s - trityl group , leading to aggregation and ultimately to racemization .
alternative protecting groups such as s - acetamidomethyl and s - t - butylthiocysteine can be used to deter aggregation , but at the cost of reagent toxicity , additional side reactions , and additional deprotection steps .
thiols will form disulfides under ambient conditions , so screening and sequencing must be performed under reducing conditions to prevent disulfide formation .
free cysteine thiols can undergo side reactions during edman degradation and during cyanogen bromide cleavage chemistry used in the two traditional methods of direct sequencing of oboc - derived peptides .
in addition , cysteine can be difficult to distinguish from arginine by edman degradation.(8 ) the first cysteine - rich oboc library has recently appeared in the literature.(9 ) while this library was neither screened nor sequenced , randomly chosen metal - soaked beads were extracted and analyzed to reveal interesting variations in metal affinities .
clearly , a robust method for sequencing cysteine - rich oboc libraries would be invaluable .
a wide range of methods are available to identify the sequences of peptides on single beads . after screening an oboc library and isolating bead hits , there are either indirect or direct approaches to identify the sequences of these library hits .
the indirect approaches like recursive deconvolution are laborious while the use of encoding tags introduces various complications.(10 ) the direct sequencing of individual beads from oboc peptide libraries traditionally uses edman degradation(6 ) which is restricted to -amino acids(11 ) and requires access to a free n - terminus .
surprisingly , after two decades of research using oboc peptide libraries , free cysteines have been included in variable positions and identified by edman sequencing in only a handful of instances.(12 ) continuing advances in the efficiency and economy of mass spectrometry have led to powerful new methods of peptide sequencing based on chemical formation of mass ladders .
tandem mass spectrometry ( ms / ms)(15 ) is still the most direct approach to peptide sequence determination and does not share the structural limitations imposed by edman chemistry.(16 ) therefore it is suprising that there are so few examples of ms / ms sequencing applied to peptides obtained from single beads . in no case
have single - bead ms / ms analyses been applied to oligomers containing cysteine . despite the lack of precedence , we chose to use ms / ms as our method to sequence thiol - rich compounds from a highly diverse oboc library in order to reduce postscreening chemical manipulations and facilitate compatibility with capped n - termini , beta peptides , and peptoids.(19 ) a wide range of major and minor problems conspire to make the synthesis , screening , and sequencing of cysteine - rich oboc peptide libraries , at best , impractical and , at worst , impossible , using any combination of existing methods
. herein we describe the first synthesis , screening , and direct sequencing of a cysteine - rich oboc library and validate this methodology by screening for and discovering peptides that form highly fluorescent complexes with the biarsenical flashedt2 .
therefore , we first developed robust conditions for the synthesis of a cysteine - rich peptide library and optimized the cleavage and sequencing of peptides from individual beads from a cysteine - rich combinatorial library .
we then screened a cysteine - rich library in the presence of thiol and phosphine reducing agents to identify optimal ligands for the biarsenical flashedt2 .
cysteine is often incorporated into peptides with an s - acetamidomethyl protecting group that can be removed using mercuric acetate .
however , mercury is difficult to remove from cysteine - rich peptides(20 ) and can lead to beta elimination , generating dehydroalanine intermediates.(21 ) indeed , removal of acetamidomethyl groups from polycysteines like ac - ccpkwccckkm - nh2 using mild conditions ( 60 mm hg(oac)2 in 1:1 acoh / dmf ) led to low yields due to formation of dehydroalanines which were apparent in the mass spectra of the products .
the problems of mercury deprotection have been overcome in the synthesis of ac-(-glu - cys)2gly - nh2 using s - t - butylthiocysteine , but it required an additional deprotection step.(22 ) tritylcysteine is a good alternative for incorporation of cysteine since the s - trityl protecting group is removed under standard acidic deprotection conditions .
however , the standard conditions ( 1:1 dmf / ch2cl2)(23 ) for the synthesis of polycysteine peptides using tritylcysteine is inefficient due to the bulk and hydrophobicity of the s - trityl group , leading to aggregation and ultimately to racemization.(24 ) through experimentation , we found that using nmp as the solvent for all couplings during synthesis of polycysteines alleviates the problems with tritylcysteine .
as a prelude to construction and screening of a cysteine - rich library it was essential to demonstrate that we could cleave cysteine - rich sequences from tentagel using cyanogen bromide .
we synthesized a biarsenical - binding tetracysteine peptide ( ac - wdccpgcckm)(1 ) on tentagel in order to optimize conditions for cyanogen bromide cleavage .
the first problem we encountered was centered on the reactivity of the cysteine thiols . while there is precedent for chemoselective cyanogen bromide cleavage of full - length proteins containing cysteine,(25 ) we found that cyanogen bromide cleavage of the cysteine - rich peptide led to a wide range of side products ( figure 1 ) , presumably due to thiocyanate and disulfide formation . to overcome these problems with cyanogen bromide cleavage
, we first treated the beads with dithiothreitol ( dtt ) and then alkylated the cysteine thiols with iodoacetamide in a sodium carbonate buffer .
these extra steps led to a dramatic improvement in the purity of the cleavage products ( figure 2 ) .
lc - ms analysis of the crude products of cyanogen bromide cleavage of ac - wdccpgcckm from tentagel .
lc - ms analysis of the crude products of cyanogen bromide cleavage of ac - wdc*c*pgc*c*km from tentagel .
the alkylation / cleavage chemistry was readily applied to individual beads in millipore ultrafree - mc centrifugal filter units adapted for microcentrifuge tubes .
unfortunately , single beads are poorly solvated in the alkylation step and often float at the aqueousair interface , leading to incomplete reduction and alkylation .
to ensure reproducibility , we included methanol as a cosolvent in the alkylation procedure . under these conditions
the beads are well - solvated and sink to the bottom of the tube , leading to clean , reproducible alkylation . a cysteine - rich decapeptide library ( ac - x8-km - tentagel ) with a potential diversity of 18 ( all amino acids excluding methionine and isoleucine )
was synthesized on 5 g of 130 m tentagel ( approximately 6.5 million beads ) .
ile was excluded because leu and ile are isobaric ( have exactly the same mass ) .
lysine was included near the c - terminus to favor the formation of y - ions in the ms / ms spectrum of the doubly charged peptide .
the library was biased toward cysteine by coupling half of the resin to fmoc - tritylcysteine during each round of coupling .
fourteen beads were selected at random from the library and subjected to the cysteine alkylation and cyanogen bromide cleavage conditions .
the samples were then subjected to nanoflow lc - ms / ms for sequencing of the doubly charged molecular ions . for these cleaved peptides , which are different from traditional tryptic peptides , manual interpretation of the ms / ms spectra proved to be more reliable than automated interpretation .
the ms / ms for the peptide cleaved from bead cys01 ( figure 3 ) , and all beads from this library , exhibits a peak at m / z 230 corresponding to the y2-ion derived from the c - terminal lys - hsl . using m / z 230 as starting point one can readily compare successive peaks to a table of potential y3-ions.(26 ) by continuing this style of analysis the remaining y - ions were identified .
the ms / ms of the peptide cleaved from cys01 also exhibits a strong signal at m / z 203 , corresponding to the b1 ion derived from n - terminal ac - cys*. in cases where the y - series of ions was incomplete , b - ions could resolve ambiguities near the n - terminus . of the 14 crude cleavage samples , four did not exhibit a significant doubly charged ion required for good ms / ms fragmentation .
ten of the samples exhibited a doubly charged molecular ion amenable to sequencing , and nine of these samples yielded unambiguous sequences for cys01cys09 ( table 1 ) .
the main product from bead cys06 was found to be a truncated peptide , probably due to a problematic coupling step during synthesis .
the peptide from cys10 did not yield a complete set of y - ions and could not be fully sequenced .
ultimately , with this challenging class of polycysteine - containing peptides , 60% of the samples yielded complete sequences ( cys01cys09 ) . for comparison
, beads from a library of cysteine - free peptides ( ac - kx8km - tentagel ; x c , m , i ) , which were not subjected to reduction and alkylation , were found to yield unambiguous sequence data in 6 out of 8 ( 75% ) cases .
tsien and co - workers evaluated series of peptide ligands for biarsenicals and concluded that the ccxxcc hexapeptide motif is optimal for binding and fluorescence of the biarsenical flash .
subsequently , a mammalian cell library and a cellulose array were used to identify optimized ccxxcc tetracysteine peptide tags for flash .
two types of optimized tetracysteine motifs appear to be superior to all others : ccpgcc and cc(k / r)xcc.(3 ) the proven superiority of these flash tags provided us with the opportunity to validate our protocols for screening and sequencing of our cysteine - rich library .
our library was heavily biased toward cysteine by coupling 50% of the resin to cysteine at each stage of the library synthesis .
this kind of user - control over amino acid composition is difficult to achieve with biological libraries synthesized on a dna synthesizer , but is easy to achieve with oboc peptide libraries .
while the potential diversity of our library ( ac - x8 km ; x met , ile ) is around 10 billion ( 18 ) , the actual diversity of our library was limited by the amount of resin used ( about 6.5 million beads ) and the fact that 50% cysteine was incorporated at each and every position .
this amount of resin does not fully cover the potential sequence space , but the cysteine bias optimizes the coverage toward known tetracysteine sequences . in order to ensure the presence of an optimized flash sequence ( wdccpgcck),(27 )
three beads with the known biarsenical tag ac - wdccpgcckm were added to the library .
the beads ( 0.33 g resin ) were suspended in 50 ml of a low sodium / high potassium ph 7.2 phosphate buffer that approximates the ion content of mammalian cytoplasm.(28 ) several reducing agents , commonly used in cell staining with flash , were included in the screening buffer : beta - mercaptoethanol ( 1 mm ) , the bis - thiol bal(29 ) ( 0.5 mm ) , and tris - carboxyethylphosphine ( 2 mm ) .
the library , containing about 130 mol total peptide , was exposed to 0.005 mol flashedt2 over 6 h. the beads were then washed with phosphate buffer containing triton x-100 . to increase the stringency of the assay
, the beads were exposed to screening buffer containing 1 mm bal , but devoid of flashedt2 for 10 h. secondary thiol washes are a common method to improve contrast when staining mammalian cells with biarsenicals .
the suspended beads were screened under a long wave uv lamp , which has sufficient output within the flash excitation range to readily identify hits .
six beads exhibited strong to weak green fluorescence and were removed for sequencing ( figure 4 ) .
each bead was then subjected to reduction , alkylation , and cleavage , and the cleaved peptide was analyzed by tandem mass spectrometry .
three of the six cleaved peptides , fb1 , fb2 , and fb6 gave unambiguous sequences ( table 2 ) .
the other three samples contained very little peptide , insufficient for ms / ms analysis , possibly due to interference with residual flash . to our satisfaction ,
one of the three sequences matched the sequence of the three beads doped into the library .
surprisingly , this bead exhibited the weakest green fluorescence of the six beads selected for sequencing .
since the fluorescence of bead fb6 was on the borderline of our criteria for selection , the other two doped beads may have been passed over .
it is possible , but less likely , that the other two doped beads corresponded to the unsequencable samples fb3fb5 .
all three of the flash - binding sequences contain a ccxxcc tetracysteine motif and suggest that it is superior to other short tetracysteine patterns : e.g. , cxcxcxc , cxxccxxc , etc .
flash - binding peptide fb1 contains one of the two known efficient sequences ( cckxcc and ccpgcc ) ; peptide fb6 contains the other .
peptide fb2 contains a potential flash tag that has not previously been identified : ccpccc .
we have developed the first method for the synthesis , screening , and sequencing of cysteine - rich oboc peptide libraries .
a diverse , heavily biased cysteine - rich library ac-(xxx)8lysmet ( xxx = 50% cys met , ile ) was synthesized on tentagel resin with fmoc - tritylcysteine , using nmp to avoid tritylcysteine aggregation .
conditions were developed for iodoacetamide alkylation and cyanogen bromide cleavage of single beads , and the samples were analyzed by tandem mass spectrometry . over half of the peptide samples cleaved from beads gave unambiguous sequences .
cysteine - free peptides yielded slightly more unambiguous sequences than the cysteine - rich sequences .
the method was validated by synthesizing and screening a cysteine - rich library against the profluorescent reagent flash . using this method , we successfully identified sequences containing two of the known short biarsenical tag motifs : ccpgcc and cckxcc .
our synthesis and sequencing methodology expands the power of oboc libraries to include short , cysteine - rich peptides for applications such as biarsenical binding tags,(27 ) metal - chelating species,(9 ) and cysteine - rich pharmaceuticals.(30 )
all glassware used in the library synthesis was silylated with 5% dichlorodimethylsilane in dichloromethane , rinsed , and oven - dried .
solutions of 0.5 m fmoc - amino acids and 0.5 m hbtu / hobt in nmp were prepared for the coupling reactions .
beads were transferred between vessels with a silylated 10 ml electric pipettor as suspensions in nmp .
the couplings were performed with 5 g of 130 m rapp polymer tentagel hl nh2 resin ( 0.45 mmol / g , batch no .
124.782 ) with 3 equiv of each amino acid , 3 equiv hbtu , 3 equiv hobt , and 6 equiv dipea in nmp . the first two residues ( met and lys(boc ) ) were sequentially coupled to the entire batch of tentagel in a 500 ml solid phase synthesis vessel . to ensure 50% incorporation of cysteine at each randomized position ,
one - half of the resin was coupled to fmoc - cys(trt)-oh in the 500 ml solid phase synthesis vessel ; the remaining resin was equally divided between 17 20 ml scintillation vials .
the vials were gently agitated on a shaker during the coupling reaction . after coupling for 90 min ,
the resin was pooled in the spps vessel , rinsed with dmf ( 3 50 ml ) , and deprotected for 30 min with 50 ml of 20% piperidine in dmf .
after the final amino acid was coupled , the n - terminus of each peptide in the library was acetylated in the spps vessel with 5 ml acetic anhydride and 24 ml dipea in nmp .
the library was washed with dmf ( 3 50 ml ) , methanol ( 3 50 ml ) , and dichloromethane ( 3 50 ml ) .
the protecting groups were removed by treatment of the resin with 94% tfa , 2.5% ethanedithiol , 2.5% h2o , and 1% triisopropylsilane for 3 h. following cleavage , the beads were washed with methanol ( 3 50 ml ) and ph 7.2 buffer ( 3 50 ml ) . the bead library was suspended in 50 ml ph 7.2 aqueous solution . to select random beads from the library , 200 l of suspended beads were transferred to a 90 mm petri dish using a p200 pipetman .
individual beads were taken up with the p200 pipetman under a 50 dissecting microscope and transferred to a prewashed ( 2 250 l , methanol ) millipore ultrafree - mc spin tube with a 0.22 m durapore pvdf membrane .
the bead was separated from solvent by centrifugal filtration by spinning at 12 000 rpm in a benchtop microcentrifuge . to avoid loss of the single bead , it is essential to confirm by microscope that the bead is at the bottom of the spin tube between washes . beads were subsequently washed with 30% aqueous methanol ( 2 100 l ) .
small wash volumes and gentle agitation were used to confine the bead to the bottom of the spin filter .
the cysteine sidechains of beads isolated from cysteine - containing libraries were reduced and alkylated to prevent side reactions during cyanogen bromide cleavage : to each tube was added 100 l of 50 mm dtt buffered with 0.4 m nahco3 in 30% aqueous methanol , and the tube was shaken for 1 h. next , 40 l of 0.5 m iodoacetamide in 30% aqueous methanol was added to each tube . after shaking for 30 min ,
150 l of 50 mm dtt buffered with 0.4 m nahco3 in 30% aqueous methanol was added and the tube was shaken for 15 min .
finally , the alkylation mixture was spun through the spin tube and the tube progresses to the rinsing phase . still inside of the spin tube ,
the bead was rinsed with 3:7 methanol/0.1% aqueous formic acid ( 2 100 l ) and 1:1 ch2cl2/meoh ( 2 100 l ) .
the bead was then transferred as a suspension in 50 l meoh to a pcr tube .
the meoh was evaporated under vacuum , leaving the bead in bottom of the tube .
the presence and location of the bead was verified under the microscope . to each pcr tube
was added 30 l of a solution of 20 mg / ml cyanogen bromide in 0.1 m hcl .
each tube was covered from light and was then frozen at 78 c , and the solvent was removed by lyophilization .
a solution of aqueous formic acid in acetonitrile ( 30 l of 1:1 ( v / v ) 0.1% formic acid / mecn ) was added to the pcr tubes , that were then vortexed for one minute to dissolve the residue .
samples were analyzed on a waters qtof2 mass spectrometer in positive ion mode equipped with a waters nanoacquity hplc ( column : waters symmetry 100 100 mm c18 1.7 m particle size ) .
doubly charged ions were selected and isolated for sequencing by survey scanning during chromatographic analyses .
the flow rate was 350 nl / min ramped from 5% b to 80% b over 40 min .
the electrospray voltage was 3 kv and the cone voltage 30 v. to assist nebulization , nitrogen gas was used at approximately 5 psi .
argon was used as the collision gas at 4 10 torr on the analyzer s penning gauge .
the ms / ms spectra were interpreted manually , primarily by assigning the peaks corresponding to the c - terminal y - ions .
the y - ion peaks were abundant since every cleaved peptide had lyshsl ( hsl = homoserine lactone ) at the c - terminus .
the interpretation of the spectra was simplified because the formation of b - ions was suppressed as a result of n - terminal acetylation . a cysteine - rich oboc library ( 0.33 g ; ac-(xxx)8lysmet ; xxx = 50% cys ; met , ile ) was first prescreened to remove fluorescent beads .
the beads were transferred to a 50 ml solid phase synthesis vessel . once inside the 50 ml vessel ,
the library was washed with 70:30 100 mm potassium bicarbonate / meoh ( 2 30 l ) .
the beads were transferred to , and suspended in , a 90 mm petri dish using 30 ml of 100 mm potassium bicarbonate and observed in the dark under an 8 w long wave uv lamp ( spectroline enf-280c ) with a stereo microscope ( 1545 magnification ) .
the library was then transferred back to the solid phase synthesis vessel for screenng using 100 mm potassium bicarbonate . at this point
three beads bearing the sequence ac - wdccpgcckm , suspended in 10 ml deionized water , were added to the vessel using a 200 l pipetman .
the library was then washed ( 3 50 ml ) with a potassium - rich phosphate buffer ( 15 mm nacl , 0.5 mm mgcl2 , 100 mm potassium phosphate , ph 7.2 ) containing 0.01% ( v / v ) triton x-100 . to the washed library in the solid phase synthesis vessel
was added 50 ml of the potassium - rich phosphate buffer containing 0.03% ( v / v ) triton x-100 , 1 mm -mercaptoethanol , 0.5 mm bal , and 2.6 mm tris - carboxyethylphosphinehcl .
flash - edt2 ( 5 l ) was added as a 1 mm stock solution in dmso .
the vessel was then agitated on a shaker for 6 h. after 6 h , the screening solution was drained and the resin was extensively washed .
the library was first washed with potassium - rich phosphate buffer containing 0.01% triton x-100 ( v / v ) ( 3 30 l ) .
then , a phosphate buffer containing 0.03% ( v / v ) triton x-100 , 1 mm -mercaptoethanol , 1 mm bal , and 2.5 mm tris - carboxyethylphosphinehcl was added to the vessel , and the vessel was shaken for 10 h. after 10 h , the solution was drained and the resin was washed extensively with 100 mm potassium bicarbonate buffer ( 6 30 l ) until the odor of thiols from the resin was no longer diminished .
finally , the library was suspended in 200 ml of 100 mm potassium bicarbonate buffer and divided equally into four 90 mm petri dishes for visualization / bead selection .
each of the four library fractions 90 mm petri dish was observed under an 8 w long wave uv lamp , held 10 cm from the beads , using a stereo microscope to identify fluorescent beads .
fluorescent beads were photographed with a nikon coolpix e4500 digital camera , top - mounted on the microscope ( exposure times between 1 and 8 s were used ) .
a small amount of meoh was added by pipet to sink floating beads into the focal plane . after being photographed
, the fluorescent beads were isolated from the surrounding nonfluorescent beads using the tip of a 200 l pipetman .
once the surrounding beads were cleared , the fluorescent bead was drawn into the tip of the 200 l pipetman with about 10 l of the screening solution . in each case , the presence of a single fluorescent bead was confirmed by observing the pipet tip under the long wave uv lamp . the bead was transferred to a millipore ultrafree - mc spin tube by placing the bead - loaded tip of the pipetman near ( but not resting on ) the bottom of the tube before dispensing the contents .
the presence of the fluorescent bead in the spin tube was confirmed under a uv lamp using a stereo microscope .
one , the prewashing of the millipore ultrafree - mc spin tube consisted of a water wash ( 2 250 l ) , followed by a methanol wash ( 2 250 l ) , and finished with a dichloromethane wash ( 2 250 l ) .
two , just before adding the cnbr solution , the beads are placed in a drying oven for 5 min , then removed and allowed to cool for an additional 5 min . | cysteine - rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals . due to the inherent difficulties created by cysteine
, the power of one - bead one - compound ( oboc ) libraries has never been applied to the discovery of short cysteine - rich peptides .
we have developed the first method for the synthesis , screening , and sequencing of cysteine - rich oboc peptide libraries .
first , we synthesized a heavily biased cysteine - rich oboc library , incorporating 50% cysteine at each position ( ac - x8-km - tentagel ) .
then , we developed conditions for cysteine alkylation , cyanogen bromide cleavage , and direct ms / ms sequencing of that library at the single bead level .
the sequencing efficiency of this library was comparable to a traditional cysteine - free library . to validate screening of cysteine - rich oboc libraries
, we reacted a library with the biarsenical flash and identified beads bearing the known biarsenical - binding motif ( ccxxcc ) .
these results enable oboc libraries to be used in high - throughput discovery of cysteine - rich peptides for protein tagging , environmental remediation of metal contaminants , or cysteine - rich pharmaceuticals . |
cell culture and reagents293 t cells were maintained in
dulbecco 's modified eagle 's medium supplemented with 10% fetal bovine serum
and transfected in 6- or 12-well plates by using the calcium phosphate method
leading usually to > 70% transfection efficiency .
0.25 g of
htt73 encoding plasmid were transfected together with 0.125 , 0.25 , and 0.5
g of plasmid encoding 19s subunits except when indicated otherwise . where
indicated ,
transfections were performed after 8 days of culture in 6-well plates with 0.5
g of htt73-gfp plasmid with or without 0.5 g of rpt6 plasmid and
lipofectamine 2000 ( invitrogen ) .
rpt6 was kindly
provided by r. losson and subcloned in the hindiii and xhoi restriction sites
of pxj41 .
rpt6 was generated by pcr and cloned hindiii and
xhoi restriction sites of pxj41 .
the cdnas encoding rpt4 was amplified by
reverse transcription - pcr from hela cell total rna flanked with ecori and xhoi
sites and cloned into pxj41 . to generate gfp fusion , htt73 was pcr - amplified ,
flanked with bamhi and kpni sites , and cloned in the bglii and kpni sites of
pegfp - n2 ( bd biosciences ) . to produce gst fusions , rpt6 and
rpt6 were subcloned into the bamhi and xhoi restriction sites
of pgex-4t3 vector in - frame with gst ( ge healthcare ) .
the cdna sequence coding
for the last 52 amino acids of ornithine decarboxylase ( odc ) was pcr - amplified
using pzs proteasome sensor ( clontech ) as a template , flanked with xhoi and
xbai restriction sites , and cloned in pcdna1/amp vector ( invitrogen )
containing htt73 to produce htt73odc . in the odc - encoding sequence , cysteine
441 was mutated in alanine to generate htt73odcc441a .
the cdna
encoding the first 108 amino acids of huntingtin with an expansion of 50 cag
was cloned in pgex-4 t vector in - frame with gst ( ge healthcare ) generating
gst - htt50 , similar to gst - hd51 described in scherzinger et al .
( 38 ) . expression of
recombinant proteins was performed as described in scherzinger et al .
small interfering rnas80 pmol of double - stranded rna
oligonucleotides directed against the target rat rpt6 ( rpt6 sirna_1 ,
ggattgacaggaaaattga ; rpt6 sirna_2 , ggagatcaaagaagtgatt ) or rpt4 ( rpt4 sirna_1 ,
tggacagattgtaggcgaa ; rpt4 sirna_2 , tgaggtatctgccgagaga ) or control sirna
silencer negative control # 1 ( ambion ) were transfected in primary
hippocampal neurons 8 days after their plating in a 6-well plate using
lipofectamine 2000 ( invitrogen ) .
micrographs were taken at 100 magnification on a leica tcs
sp2aobs confocal microscope or leica dmrb fluorescence microscope .
frozen brain samples ( parietal cortex ,
brodmann area number 7 ) from two individuals with juvenile hd were obtained
from the harvard brain tissue resource center .
the two cases were a
14-year - old , grade 3 female with 97 cag repeats and a 18-year - old grade 4
female with 75 cag repeats , previously described in hoffner et al .
sections were fixed with
4% paraformaldehyde and labeled with a polyclonal antibody directed against
the first 17 amino acids of human htt ( 1:400 dilution )
( 39 ) , 7 ( pw8110 biomol ,
1:1000 dilution ) , and rpt6 antibody ( sug-1b8 , euromedex , 1:5000 dilution ) . 100
inclusions were examined , 50 nuclear and 50 cytoplasmic , in the two tissues .
micrographs were taken at 63 magnification combined with digital
zooming on a zeiss confocal microscope ( carl zeiss , inc . ,
routinely , 70%
confluent cells from a well of a 12-well plate were lysed in 140 l of
boiling laemmli buffer ( 25 mm tris - hcl , ph 6.8 , 1% sds , 25
mm dithiothreitol , 7.5% glycerol , 0.05% bromphenol blue ) for
immunoblot analysis or 140 l of ice - cold nonidet p-40 buffer ( 25
mm tris - hcl ph 7.5 , 1% nonidet p-40 , 300 mm nacl , 5
mm edta , 1 mm phenylmethylsulfonyl fluoride , 5 g / ml
pepstatin , leupeptin , and aprotinin ) for fractionation experiments .
18 l
of protein extracts were loaded on 10% sds - page and transferred to optitran
ba - s 83 reinforced nitrocellulose membrane ( whatman and schleicher &
schuell ) .
equal loading of protein extracts analyzed by immunoblot was
controlled by ponceau red staining ( data not shown ) and vimentin .
membranes were saturated
in 5% dried skimmed milk in phosphate - buffered saline and probed with htt 2b4
antibody unless specified .
the appropriate secondary antibody coupled to
peroxidase was revealed using the supersignal west pico chemiluminescent kit
( pierce ) .
chemiluminescent images were acquired using the chemi - smart 5000
( vilber - lourmat ) allowing quantitative detection of chemiluminescence .
antibodies for immunocytochemistry , primary antibodies were
diluted as follows : 1:1000 dilution for tau-1 antibody ( chemicon ) , 7
( pw8110 biomol ) , rpt1 ( ab3322 , abcam ) , rpt2 ( ab3317 ) , rpt3 ( ab22634 ) , and rpt5
( ab22635 ) ; 1:5000 dilution for rpt6 antibody ( sug-1b8 , euromedex ) or rpt4
( ab22639 ) . for immunoblots ,
antibodies were used as follows : 1:5000 for htt
2b4 antibody and 1:1000 for antibodies against proteasome subunits .
vimentin
antibody v 6630 ( sigma ) was used at a 1:500 dilution , p21 antibody ( bd
pharmingen 556431 ) at a 1:500 dilution , and e2f-1 ( santa cruz ) sc-251 at a
1:200 dilution .
immunoprecipitation293 t cells were plated 24 h before
transfection in 10-cm dishes at a density of 80000 cells / ml .
transfection were
performed with 2.5 g of htt73 alone or with 5 g of rpt6-flag - encoding
plasmid .
48 h post - transfection , cells were washed with ice - cold
phosphate - buffered saline , lysed in 1 ml of 0.2% nonidet p-40 buffer ( 0.2%
nonidet p-40 , 50 mm tris - hcl , ph 7.5 , 300 mm nacl , 5
mm edta , 1 mm phenylmethylsulfonyl fluoride , 5 g / ml
pepstatin , leupeptin , and aprotinin ) , left 10 min on ice , and clarified by
centrifugation at 16,000 g for 10 min .
soluble proteins were
immunoprecipitated with 2 l of the anti - rpt6 , anti - gfp , or anti - htt
monoclonal antibodies together with 10 l of protein g - sepharose overnight
at 4 c .
beads were then washed 3 times in 0.2% nonidet p-40 buffer and
resolved by 10% sds - page under reducing conditions , transferred to
nitrocellulose membranes , and revealed with corresponding antibodies .
gst pulldown assay gst , gst - rpt6 , and
gst - rpt6 were expressed in escherichia coli strain
bl21 ( de3 ) plyss and purified by glutathione - sepharose chromatography . for
each pulldown experiment , about 0.1 g of gst or gst - rpt6 fusion protein
was incubated with htt73 produced in 293 t cells and extracted in 1% nonidet
p-40 buffer .
after 3 h of incubation at room temperature , beads were washed 3
times for 5 min in 1% nonidet p-40 buffer , and complexes were analyzed by
sds - page followed by immunoblots .
figure 2.overexpressed and endogenous rpts are subunits of two distinct
complexes , the 26 s proteasome and apis .
a - c , lysates of
mock - transfected cells or cells overexpressing rpt6-flag or rpt4-flag were
analyzed by glycerol gradients sedimentation .
an aliquot of each fraction was
analyzed by immunoblots and for peptidase activity with
n - succinyl - leu - leu - val - tyr - aminomethylcoumarin as indicated below the
immunoblots ( 41 ) .
d , fractions
2 - 4 ( apis ) and 8 - 9 ( 26 s ) of glycerol gradients similar to those shown in
( a and c ) were pooled , immunoprecipitated , and revealed with
indicated antibodies .
horseradish peroxidase - conjugated goat anti - mouse
were used to reveal the immunoblots , as rpts co - migrate with the heavy
chain of the immunoglobulins .
overexpressed and endogenous rpts are subunits of two distinct
complexes , the 26 s proteasome and apis .
a - c , lysates of
mock - transfected cells or cells overexpressing rpt6-flag or rpt4-flag were
analyzed by glycerol gradients sedimentation .
an aliquot of each fraction was
analyzed by immunoblots and for peptidase activity with
n - succinyl - leu - leu - val - tyr - aminomethylcoumarin as indicated below the
immunoblots ( 41 ) .
d , fractions
2 - 4 ( apis ) and 8 - 9 ( 26 s ) of glycerol gradients similar to those shown in
( a and c ) were pooled , immunoprecipitated , and revealed with
indicated antibodies .
horseradish peroxidase - conjugated goat anti - mouse
were used to reveal the immunoblots , as rpts co - migrate with the heavy
chain of the immunoglobulins .
glycerol - gradient sedimentation225000 cells were plated in
a 5-cm dish and transfected with empty vector or the indicated constructs 24 h
after plating .
cells were lysed 40 h post - transfection in 200 l of
proteasome lysis buffer ( 50 mm tris - hcl , ph 8.0 , 1% triton x-100 , 1
mm edta , 1.5 mm mgcl2 , 1 mm
dithiothreitol , and 5 mm atp ) .
extracts were clarified by
centrifugation and fractionated through linear 20 - 40% glycerol gradients in
polyallomer tubes of 11 60 mm for 13 h at 40,000 rpm in a sw60 rotor
( beckman coulter ) .
note that all the proteasome complexes sedimented below their
expected molecular weights , as previously reported
( 25 ) .
assays for proteasome activity were
performed using the substrate
n - succinyl - leu - leu - val - tyr - aminomethylcoumarin as described in
kisselev and goldberg
( 41 ) .
aggregation assays gst - htt50 ( 0.5 m )
was incubated with or without purified 19s proteasome ( 70 nm ;
boston biochem ) in the presence of 10 units of thrombin ( amersham biosciences )
at 37 c with 300 rpm shaking in 50 mm tris - hcl , ph 7.5 , 150
mm nacl , 10% glycerol , 20 mm mgcl2 with or
without 5 mm atp or 15 mm atps .
reactions were
stopped at the indicated times by adding an equal volume of laemmli buffer and
boiling for 5 min .
aliquots corresponding to 50 ng of gst fusion protein were
analyzed by sds - page stained with coomassie brilliant blue , and filter
retardation assays revealed with htt antibody .
a , lysates of cells expressing increasing amounts of htt73 were
either boiled in 1% sds or fractionated into np40 supernatant and pellet and
analyzed by immunoblotting with htt antibody .
b - d , htt73 was
co - expressed with increasing amounts of rpt4 or rpt6 , and whole cell lysates
or supernatants and pellets were analyzed by immunoblotting with htt , rpt6 , or
rpt4 antibodies .
confocal micrographs of cells expressing htt73-gfp
either alone or together with rpt6 or rpt4 are shown .
arrows show cells with small inclusions , and
asterisks show cells with large inclusions .
f , micrographs
of primary neurons expressing htt17-gfp and htt73-gfp alone or together with
rpt6 .
neuronal identity was confirmed by staining with tau-1 antibody , and
nuclei are revealed with h33258 .
a , lysates of cells expressing increasing amounts of htt73 were
either boiled in 1% sds or fractionated into np40 supernatant and pellet and
analyzed by immunoblotting with htt antibody .
b - d , htt73 was
co - expressed with increasing amounts of rpt4 or rpt6 , and whole cell lysates
or supernatants and pellets were analyzed by immunoblotting with htt , rpt6 , or
rpt4 antibodies .
confocal micrographs of cells expressing htt73-gfp
either alone or together with rpt6 or rpt4 are shown .
arrows show cells with small inclusions , and
asterisks show cells with large inclusions .
f , micrographs
of primary neurons expressing htt17-gfp and htt73-gfp alone or together with
rpt6 .
neuronal identity was confirmed by staining with tau-1 antibody , and
nuclei are revealed with h33258 .
aggregation of peptides with a long poly(q ) stretch can occur spontaneously
in vitro , but in yeast aggregation of the amino - terminal fragment of
htt requires the presence of the chaperone hsp104
( 42 ) . although chaperones are
widely conserved throughout evolution , there is no mammalian orthologue of
hsp104 .
the typical feature of hsp104 and the bacterial atpase clpb proteins
is the presence of two aaa domains and a middle region or m - domain , adopting a
coiled - coil structure essential for the hsp104/clpb protein remodeling
function ( 43 ,
44 ) .
aaa atpases in available
databases were examined aiming to identify human proteins containing both a
coiled - coil and an aaa domain .
we found that only the atpase subunits of the
19s proteasome , rpt6 , rpt4 , rpt3 , rpt2 , and rpt1 , exhibit these specific
features .
interestingly , an immunohistochemical study of brain tissue from
patients with spinocerebellar ataxia type 3 ( sca-3 ) has revealed that poly(q )
inclusions co - localize with subunits of the 19s proteasome but rarely of the
20s catalytic particle ( 45 ) .
these observations together with the unfoldase function of the 19s atpases in
protein degradation prompted us to investigate whether rpts contribute to
inclusion formation . to determine whether co - localization of regulatory subunits of the
proteasome with poly(q ) inclusions was a generic feature of distinct poly(q )
disorders , we analyzed the localization of several proteasome subunits in
nerve cells containing mutant htt inclusions .
in primary neurons , rpt6
staining was extremely intense around poly(q ) aggregates , in contrast to its
diffuse and mostly nuclear localization in untransfected cells
( fig .
the other 19s atpases were
also enriched around poly(q ) inclusions
( fig .
in
contrast , the 20s proteasome was not enriched around the poly(q ) inclusions ,
as revealed by immunostaining with antibodies directed against the 20s
subunit , 7 .
these observations reveal that rpt subunits co - localize
with mutant htt inclusions in neuronal cells independently of the 20s
proteasome . to determine whether these observations were relevant to hd , we
stained cortical sections of post - mortem hd brains with htt and rpt6 or
7 ( 20s ) antibodies .
we performed this analysis on brain tissue from two
juvenile hd patients to examine a large number of cytoplasmic and nuclear
inclusions .
like primary neurons expressing mutant htt , all the inclusions
from both hd patients were intensely labeled with rpt6 regardless of their
subcellular localization ( fig .
in contrast , only a small
fraction of htt inclusions ( 13% ) exhibited a light staining for the 20s
subunit ( fig .
these results indicate that the chaperone subunits co - localize with hd
inclusions largely independently of the 20s subunits .
figure 4.the coiled - coil region is essential for the activity of rpt6 on
htt73 .
a , schematic representation of rpt6 domain organization
and coiled - coil mutant analyzed in this study .
b , lysates of cells
expressing htt73 alone or together with increasing amounts of rpt6 wild type
or rpt6 deletion mutant were analyzed by immunoblots and
filter retardation assays .
the coiled - coil region is essential for the activity of rpt6 on
htt73 . a , schematic representation of rpt6 domain organization
and coiled - coil mutant analyzed in this study .
b , lysates of cells
expressing htt73 alone or together with increasing amounts of rpt6 wild type
or rpt6 deletion mutant were analyzed by immunoblots and
filter retardation assays .
seeking further evidence for the existence of rpt subunits independently of
the proteasome , we analyzed the distribution of proteasome subunits from cell
lysates by glycerol gradient fractionation .
19s and 20s subunits as well as
the proteolytic activity of the proteasome were localized in high molecular
weight fractions 8 and 9 , indicating that these fractions contained the 26s
proteasome ( fig . 2a ) .
in addition to the 26s proteasome , 19s subunits were also found in lower
molecular weight fractions devoid of 20s subunits and proteolytic activity
( fig .
this sedimentation profile is reminiscent of the gel filtration profile
of the free 19s - like complex
( 25 ) , also called apis complex
( atpase proteins independent of 20s )
( 22 ) .
thus , in mammalian cells
rpts are subunits of 2 distinct complexes , the 26s proteasome and apis or free
19s - like complex .
the observation that inclusions of expanded poly(q)-containing proteins
co - localize with rpts independently of the proteasome
( fig . 1 and
( 45 ) suggested that apis could
be involved in inclusion formation .
we , therefore , attempted to increase the
abundance of this complex by overexpressing some of its subunits .
the
sedimentation profile of overexpressed rpt4 and rpt6 was similar to the
sedimentation profile of the endogenous subunits . in fractions 8 and 9 of the
glycerol gradients , rpt6 antibody revealed 2 bands corresponding to the
endogenous protein and the overexpressed flag - tagged rpt6 , indicating that
overexpressed rpt6 was a subunit of the 26s proteasome complex
( fig .
a large
fraction of overexpressed rpt6 was detected in fractions 2 - 4 of the gradient
together with the apis complex .
similarly , a fraction of overexpressed rpt4
entered the 26s proteasome , whereas most of the protein was found in the
smaller apis complex ( fig .
2c ) .
note that overexpression of rpt4 or rpt6 did not
alter the sedimentation of the endogenous proteasome nor its proteolytic
activity ( fig .
2 , a - c ,
and data not shown ) . to confirm these observations ,
immunoprecipitations were
carried out with rpt6 antibodies on glycerol gradient fractions containing
either apis ( fractions 2 - 4 ) or the 26s complex ( fractions 8 - 9 ) . both
endogenous and overexpressed
rpt4 were detected in rpt6 immunoprecipitates
performed on fractions 2 - 4 as well as fractions 8 - 9 of the glycerol gradients
( fig .
the
amounts of rpt3 immunoprecipitated in the endogenous complexes were probably
below the detection limit of the rpt3 antibody .
altogether , these findings
establish that endogenous and overexpressed rpt4 and rpt6 are engaged in two
complexes , the 26s proteasome and apis .
most of the overexpressed rpt6 and
rpt4 co - sedimented with apis and recruit other subunits of the complex .
we ,
thus , used this experimental setting to analyze whether rpts modulated htt
misfolding and aggregation .
figure 5.rpt6 , but not rpt6 lacking the coiled - coil region , binds to htt73 .
a , lysates of cells transfected with empty vector and htt73 alone or
together with rpt6 analyzed by immunoblot ( left panel ) or
immunoprecipitated ( ip ) with htt or gfp antibody followed by rpt6
immunoblot .
b , gst , gst - rpt6 , and gst - rpt6 fusion
proteins were expressed in e. coli , purified on glutathione - sepharose
beads , separated by sds - page , transferred onto nitrocellulose , and stained
with ponceau red .
c , htt73 containing cell lysates ( input ) were
incubated on beads containing gst , gst - rpt6 , or gst - rpt6 and
washed , and complexes bound to beads were revealed with htt antibody .
rpt6 , but not rpt6 lacking the coiled - coil region , binds to htt73 .
a , lysates of cells transfected with empty vector and htt73 alone or
together with rpt6 analyzed by immunoblot ( left panel ) or
immunoprecipitated ( ip ) with htt or gfp antibody followed by rpt6
immunoblot .
b , gst , gst - rpt6 , and gst - rpt6 fusion
proteins were expressed in e. coli , purified on glutathione - sepharose
beads , separated by sds - page , transferred onto nitrocellulose , and stained
with ponceau red .
c , htt73 containing cell lysates ( input ) were
incubated on beads containing gst , gst - rpt6 , or gst - rpt6 and
washed , and complexes bound to beads were revealed with htt antibody .
nucleation is the rate - limiting step of poly(q ) aggregation in
vitro ( 31 ) . to determine
whether similar features could be observed in cells , we expressed large
amounts of htt73 and performed biochemical fractionation .
we found that a
10 - 25-fold increase in the sds - soluble protein leads to a very modest increase
of nonidet p-40-insoluble , presumably misfolded htt73
( fig .
thus , cells
can accumulate a large amount of nonidet p-40-soluble htt73 , suggesting that
aggregation requires not only a high protein concentration but also additional
events . because the function of rpts is to unfold proteins and because they
co -
localize with poly(q ) inclusions , we asked whether these unfoldases might
assist the conversion from soluble to aggregated mutant htt . increasing the
levels of rpt6 or
rpt4 led to a remarkable and dose - dependent increase of
htt73 when low amounts of htt73 were co - transfected with rpt6 or rpt4
( fig .
3 , b - d ,
lanes 1 , 4 , and 8 , compared with
fig .
biochemical fractionation studies revealed that
nearly all the sds - soluble htt73 that accumulated upon rpt6 or rpt4
overexpression was nonidet p-40-insoluble , suggesting that it was in a
pre - aggregated misfolded state ( fig .
in contrast , when large amounts of htt73 were
transfected alone , cells contained very high levels of soluble htt73
( fig .
these
experiments revealed that rpt6 and rpt4 convert soluble htt into an insoluble
species . to further characterize the effect of rpt6 and rpt4 on htt73 ,
htt73
fused to gfp was overexpressed either alone or together with rpt6 or rpt4
( fig .
although
cells expressing htt73-gfp alone contained either diffuse fluorescence or
small fluorescent foci , cells co - expressing htt73-gfp and rpt6 or rpt4
contained large inclusions ( fig .
this was also observed in primary neuronal cells
expressing htt73-gfp alone or together with rpt6 , 3f ) .
northern blot analyses were carried out with cells co - expressing rpt6
with htt73 and revealed that the effects of rpt6 overexpression on htt
accumulation were not due to changes at the level of htt mrna ( supplemental
fig .
previous studies have indicated that rpt subunits of the 19s have
distinct functions ( 46 ) .
to
determine whether the effect on mutant htt aggregation was a general property
of all rpts or was restricted to rpt6 and rpt4 , htt 73 was overexpressed with
the other rpts .
increasing the levels of rpt6 or rpt4 led to a remarkable and
dose - dependent increase of aggregated htt73 , as revealed by immunoblots and
filter retardation assays ( supplemental fig .
2 ) . overexpression of rpt3
modestly enhanced aggregation of htt73 , in contrast to rpt2 , rpt1 , and rpt5 ,
which had no significant effect ( supplemental fig .
this indicated that
overexpression of an individual rpt was not sufficient to trigger htt
aggregation but , rather , that the effects of rpt6 and rpt4 on htt73 were
specific .
because cellular protein concentration varies with cell density and
equal cell numbers were seeded in each experiment , the fact that the levels of
the cellular protein vimentin did not vary in the different conditions
revealed that none of the transfected constructs elicited cell death
( supplemental fig .
this is in agreement with previously studies that
established that mutant htt is not toxic 2 days post - expression
( 47 ,
48 ) .
we next sought to
determine which domains were important for the activity of rpt6 .
the amino
terminus of rpt6 contains a coiled - coil domain , a region which was found
important for the chaperone function of other aaas
( 43 ) .
therefore , the
amino - terminal 79 amino acids of rpt6 containing the coiled - coil region were
deleted , generating a protein fragment , rpt6 .
overexpression
of increasing doses of rpt6 together with htt73 augmented the levels of htt73 ,
detected on immunoblots , by 1.3- , 3- , and 7-fold and the levels of aggregated
htt73 , detected on filter retardation assays by 2- , 5- , and 7-fold .
in contrast , rpt6 lacking the coiled - coil region , expressed at a level similar
to the full - length protein , was devoid of any activity on htt73
( fig .
4 ) . this result reveals
that the coiled - coil region of rpt6 is essential for its function on htt
aggregation .
figure 6.rpt6 and rpt4 enhance misfolding of proteins containing expanded poly(q )
independently of proteolysis . a , immunoblots and filter
retardation assays of cells overexpressing sca3 22q or 82q alone or together
with rpt6 and either left untreated or treated with 1 m
epoxomycin ( epoxo ) for 14 h. b , 293 t cells were transfected
with htt73 alone or together with rpt6 and rpt4 encoding plasmids and either
left untreated or treated with 0.5 or 1 m epoxomycin for 14 h
and analyzed by immunoblots and filter retardation assays .
rpt6 and rpt4 enhance misfolding of proteins containing expanded poly(q )
independently of proteolysis .
a , immunoblots and filter
retardation assays of cells overexpressing sca3 22q or 82q alone or together
with rpt6 and either left untreated or treated with 1 m
epoxomycin ( epoxo ) for 14 h. b , 293 t cells were transfected
with htt73 alone or together with rpt6 and rpt4 encoding plasmids and either
left untreated or treated with 0.5 or 1 m epoxomycin for 14 h
and analyzed by immunoblots and filter retardation assays .
the results presented so far indicate that rpt6 and rpt4 assist mutant htt
misfolding and aggregation .
we next tested whether this effect involved a
direct interaction between rpt6 and htt73 , as suggested by co - localization
studies ( fig .
coimmunoprecipitation experiments were carried out using cells expressing
htt73 alone or together with rpt6 .
5a ) . to determine whether rpt6 and htt73 interact
directly , recombinant rpt6 , either wild type or lacking the coiled - coil
region , was produced in e. coli as a fusion with gst and immobilized
on glutathione beads .
nonidet p-40 lysates containing htt73 produced in 293 t
cells were then incubated with gst and gst - rpt6 .
5b ) . deleting rpt6 coiled - coil region abolished its
ability to bind htt .
these interaction data suggest that the effect of rpt6 on
htt aggregation is mediated through a direct interaction and requires the
coiled - coil region .
because a fraction of rpts were found independently of the proteasome in
sca-3 ( 45 ) , we tested whether
overexpressing rpt6 affected accumulation of ataxin-3 , a protein unrelated to
htt , which contains a poly(q ) expansion in sca-3 .
rpt6 overexpression
dramatically enhanced mutant ataxin-3 aggregation but had no effect on
wild - type ataxin ( fig .
previous studies have reported that proteasome
inhibition increases accumulation and aggregation of htt
( 6 - 8 ) .
therefore , we next analyzed whether rpt6 or rpt4 overexpression generally
impaired proteasomal degradation , thereby enhancing accumulation of expanded
poly(q ) .
epoxomycin , which blocks the catalytic activity of the 20s
proteasome , provoked accumulation of both sds - soluble and aggregated htt73 or
aggregated mutant ataxin-3 ( fig .
a and b ) . similarly to epoxomycin treatment ,
rpt6 or rpt4 overexpression enhanced htt73 accumulation and aggregation
( fig .
6b ) . in
contrast , we found that overexpressing these 19s subunits did not stabilize
the short - lived proteins e2f-1 and p21
( fig .
6b ) , whereas
inhibition of the proteolytic activity of the proteasome markedly stabilize
these two short - lived proteins ( fig .
these results indicate that rpt6 or rpt4
overexpression does not impair proteasomal degradation . having shown that increasing the levels of rpt4 and rpt6 promotes mutant
htt misfolding , we next tested whether we could interfere with the endogenous
proteins in primary neurons .
two sirnas targeting rpt6 were found to
significantly reduce the levels of rpt6 by 25 and 40% , respectively .
similarly , two sirnas designed against rpt4 reduced the levels of the protein
by 50% ( fig .
7a ) .
we found that reducing the levels of rpt6 or rpt4 substantially reduced the
number of cells containing poly(q ) inclusions when we examined cells
expressing mutant htt together with sirna targeted against rpt6 or rpt4
( fig .
70% of neurons expressing mutant htt contained
inclusions , and 30% of cells exhibited diffuse fluorescence .
this ratio was
inverted in cells transfected with rpt4 or rpt6 sirnas
( fig .
the number
of cells expressing mutant htt was very similar when the levels of rpt4 and
rpt6 were reduced and in control cells , indicating that the sirnas were not
toxic under our experimental conditions ( data not shown ) .
this analysis
demonstrates that reducing the levels of rpt4 or rpt6 in primary neurons
significantly decrease inclusion formation .
a , immunoblots showing rpt6 , rpt4 , and vimentin in
extracts of primary neurons transfected with control , rpt6 , or rpt4 sirnas .
ratio of rpt6 and rpt4 proteins , normalized to vimentin , in protein extracts
of primary neurons transfected with control , rpt6 , or rpt4 sirnas .
quantifications were performed on three independent experiments .
sirna_2
together with htt73-gfp , fixed 24 h post - transfection , and labeled with tau-1
antibody .
c , ratio of primary neurons transfected with control ,
rpt6 , or rpt4 sirnas together with htt73-gfp , expressed as percentage of
transfected cells .
data were scored from 10 independent experiments
corresponding to more than 4000 cells in total . , p <
0.01 ; , p < 0.02 .
a , immunoblots showing rpt6 , rpt4 , and vimentin in
extracts of primary neurons transfected with control , rpt6 , or rpt4 sirnas .
ratio of rpt6 and rpt4 proteins , normalized to vimentin , in protein extracts
of primary neurons transfected with control , rpt6 , or rpt4 sirnas .
sirna_2
together with htt73-gfp , fixed 24 h post - transfection , and labeled with tau-1
antibody .
c , ratio of primary neurons transfected with control ,
rpt6 , or rpt4 sirnas together with htt73-gfp , expressed as percentage of
transfected cells .
data were scored from 10 independent experiments
corresponding to more than 4000 cells in total . , p <
0.01 ; , p < 0.02 . overexpressed rpt6 and rpt4 recruited other rpts
( fig .
2d ) , suggesting
that their stimulatory activity on htt misfolding was mediated by a complex
containing rpts . to directly examine this hypothesis , we next tested whether
purified 19s proteasome could modulate htt aggregation in vitro .
aggregation of mutant htt was monitored by filter retardation assay as
described in scherzinger et al .
cleavage of gst - htt
released the gst moiety and initiated htt aggregation
( fig .
the addition of substoichiometric amounts of purified
19s proteasome and atp strongly enhanced aggregation of mutant htt
( fig .
the
stimulatory effect of 19s proteasome on mutant htt aggregation in
vitro was significantly decreased by atp-s , a non - hydrolysable atp
analogue ( fig .
the addition of 19s proteasome with or without atps did not affect the
cleavage of gst - htt ( fig .
this suggests that the stimulatory activity of the
19s proteasome on htt aggregation is mediated by its unfoldase function .
altogether , the results presented here reveal that atpases of the 19s
proteasome function non - proteolytically in facilitating misfolding and
aggregation of proteins with a poly(q ) expansion . because htt inclusions are
ubiquitinated , htt is likely to be addressed to the proteasome by a
ubiquitin - dependent pathway ( 3 ,
4 ) .
we next investigated what
would be its fate if targeted to the proteasome by the degradation pathway
utilized by the evanescent protein odc
( 49 ) .
the 37 carboxyl - terminal
amino acids of odc have been found to be an efficient and autonomous
destabilizing sequence when fused to heterologous proteins
( 49 ) .
htt73 and
htt73odc were expressed in 293 t cells . although both soluble and aggregated
htt73 were readily detected by immunoblots and filter retardation assays ,
htt73odc was hardly detectable , even when large amounts of dna encoding this
protein were transfected , suggesting that the protein was very rapidly and
efficiently degraded ( fig .
we confirmed that htt73odc was fully degraded and
did not escape our detection ( supplemental fig .
3 ) . when the proteolytic
capacity of the proteasome was compromised , htt73odc was stabilized ,
confirming that htt73odc is a proteasome substrate
( fig .
the fact that htt73odc is insensitive to rpt6 overexpression but
is stabilized by inhibition of the catalytic activity of the proteasome
further supports the conclusion that rpt6 overexpression does not perturb
proteasomal degradation .
when the ability of the proteasome to degrade
proteins is compromised , small but detectable amounts of htt73odc aggregates
were found by means of their retention on a cellulose acetate filter
( fig .
these
observations suggest that , when the catalytic activity of the proteasome is
compromised , a small amount of htt73odc recovers its ability to aggregate . to
obtain further insight into the mechanism by which the odc carboxyl terminus
destabilizes mutant htt , we mutated cysteine 441 to alanine , as this point
mutation has been shown to stabilize odc
( 49 ) .
in contrast to htt73odc ,
htt73odcc441a was readily detectable , and its aggregation increased
upon rpt6 overexpression ( fig . 9 ,
c and d ) .
the finding that the odc destabilizing
sequence converts htt73 into an evanescent protein indicates that the
proteolytic capacity of the proteasome is not the limiting factor for mutant
htt degradation .
figure 8.the 19s enhances mutant htt aggregation in vitro .
a , time course of proteolysis of gst - htt50c with or without
19s and atps .
0.5 m gst - htt50 was cleaved with 10
units of thrombin in the absence or presence of 70 nm 19s and 15
mm atps .
aliquots of the reaction were analyzed after 3.5 ,
6 , or 8 h at 37 c by sds - page stained with coomassie brilliant blue .
b , samples shown in a
were analyzed by filter retardation assays and revealed with htt
antibodies .
the 19s enhances mutant htt aggregation in vitro .
a , time course of proteolysis of gst - htt50c with or without
19s and atps .
0.5 m gst - htt50 was cleaved with 10
units of thrombin in the absence or presence of 70 nm 19s and 15
mm atps .
aliquots of the reaction were analyzed after 3.5 ,
6 , or 8 h at 37 c by sds - page stained with coomassie brilliant blue .
b , samples shown in a
were analyzed by filter retardation assays and revealed with htt
antibodies .
in this study we report on the finding that in cells aggregation of htt
amino - terminal fragments with a pathogenic poly(q ) expansion is dependent on a
remodeling event mediated by rpt6 and rpt4 .
proteasomal chaperones are
subunits of 2 complexes , the 26s proteasome and the previously reported apis
or 19s - like complex ( this study and refs .
22 and
25 ) .
although the function of
the proteasome in protein degradation is well recognized , there is also
growing evidence indicating that 19s atpases , in particular rpt6 , function
independently of degradation
( 50 ) . in hd patients ,
inclusions
are often found in the nucleus , where a non - proteolytic function of
the 19s atpases has been extensively reported .
a remodeling activity of rpts
independently of degradation provides a unifying mechanism to reconcile their
diverse functions .
conditions provoking the dissociation of the 19s from the
20s have been reported previously .
binding
of alternative rp such as pa28 to the cp also provokes dissociation of the
atpases ( 17 ) .
intriguingly ,
pharmacological inhibition of the catalytic function of the proteasome
stimulates binding of pa28 to the proteasome
( 17 ) , suggesting that free 19s
particles may be released by this treatment .
what precisely modulates the
relative abundance of 26s proteasome and apis ( or free 19s - like complex )
remains to be determined .
however , it is noteworthy that aging was reported to
promote proteasome disassembly in flies
( 51 ) . in neurons of hd ( this study ) and sca-3
( 45 ) brains as well as in
primary neurons expressing mutant htt and
rpts , but not 20s subunits ,
co - localize with poly(q ) inclusions , indicating that rpts are not associated
with the catalytic particles in poly(q ) inclusions .
this suggests that rpts
may be involved in inclusion formation independently of the proteolytic
function of the proteasome . in support of this hypothesis
, we found that
overexpression of rpt6 , rpt4 , and more modestly rpt3 enhance mutant htt and
sca-3 aggregation .
because the majority of overexpressed rpt4 and rpt6
co - sediment with apis , we propose that rpt4 and rpt6 remodel proteins with a
poly(q ) expansion and thereby elicit their aggregation independently of
degradation .
overexpressing rpt6 or rpt4 has no effect on the levels of
vimentin or the short - lived e2f-1 , p21 , and htt7odc , showing unambiguously
that rpt6 or rpt4 overexpression does not impair proteasome degradation .
more
importantly , reducing the levels of rpt6 or rpt4 in primary neurons decreases
inclusion formation .
together , these results indicate that endogenous rpt6 and
rpt4 enhance htt misfolding , and this effect is likely mediated through an
unfoldase function of these proteins . in vitro reconstitution
experiments
support this model ; substoichiometric amounts of 19s complex
promote mutant htt aggregation , and this activity is markedly attenuated by
atps .
htt supplied in the in vitro aggregation assay is not
ubiquitinated , suggesting that the 19s does not required ubiquitination of htt
to recognize and remodel htt .
thus , ubiquitination does not seem to be
required for the aggregation process per se .
however , it is possible
that in cells ubiquitination of htt plays a role in targeting htt to the
proteasome .
this is in agreement with previous in vitro studies on
the chaperone function of the proteasome
( 52 ) . when the domain
composition of rpts was carefully examined , we found that the three atpases
enhancing poly(q ) aggregation , rpt6 , rpt4 , and rpt3 , possess a coiled - coil
domain of nearly 50 amino acids .
in contrast , the 19 s atpases unable to
enhance poly(q ) aggregation have either no coiled - coil region ( rpt5 ) or only a
short one ( rpt1 , rpt2 ) according to domain predictions with the web - based tool
smart .
the importance of the equivalent m - region of the bacterial
atpase clpb and the yeast hsp104 suggested that this finding might not be
fortuitous .
we , thus , deleted the coiled - coil region in rpt6 and found that
although rpt6 was expressed at levels similar to the
full - length protein , this mutant lacking the coiled - coil domain was inactive
toward poly(q ) aggregation ( fig .
4 ) . in agreement with this result ,
rpt6 lacking the coiled - coil
region was no longer able to bind mutant htt
( fig .
, our findings suggest that in the disease
process , the formation of misfolded and presumably toxic species may be
assisted by rpt6 and rpt4 . a recent study has revealed that tau inclusions ,
the pathological hallmark of alzheimer disease , pick disease , and other
tauopathies , are immunoreactive for rpt3 , raising the question of whether the
findings reported here for htt and sca-3 may be a generic feature of many
neurodegenerative diseases
( 53 ) .
a , lysates of cells transfected with increasing amounts
of htt73 or htt73odc encoding plasmids analyzed by immunoblots .
b ,
cells were transfected with htt73 ( left panel ) or htt73odc ( right
panel ) alone or together with increasing amounts of rpt6 encoding plasmid
and left untreated or treated with 0.25 , 0.5 and 1 m epoxomycin
( epoxo ) for 14 h. lysates were analyzed by immunoblots or by filter
retardation assays .
c , cells transfected with increasing amounts of
htt73odc or htt73odcc441a were analyzed by immunoblots .
d ,
cells were transfected with htt73odcc441a alone or together with
increasing amounts of rpt6 encoding plasmid and left untreated or treated with
0.25 , 0.5 , and 1 m epoxomycin for 14 h. lysates were analyzed
by immunoblots or by filter retardation assays revealed with htt antibody .
e , possible routes followed by htt : degradation by a proteasome
complex ( this study and ref .
13 ) or misfolding and
aggregation by apis or 19 s - like complex .
a , lysates of cells transfected with increasing amounts
of htt73 or htt73odc encoding plasmids analyzed by immunoblots .
b ,
cells were transfected with htt73 ( left panel ) or htt73odc ( right
panel ) alone or together with increasing amounts of rpt6 encoding plasmid
and left untreated or treated with 0.25 , 0.5 and 1 m epoxomycin
( epoxo ) for 14 h. lysates were analyzed by immunoblots or by filter
retardation assays .
c , cells transfected with increasing amounts of
htt73odc or htt73odcc441a were analyzed by immunoblots .
d ,
cells were transfected with htt73odcc441a alone or together with
increasing amounts of rpt6 encoding plasmid and left untreated or treated with
0.25 , 0.5 , and 1 m epoxomycin for 14 h. lysates were analyzed
by immunoblots or by filter retardation assays revealed with htt antibody .
e , possible routes followed by htt : degradation by a proteasome
complex ( this study and ref .
13 ) or misfolding and
aggregation by apis or 19 s - like complex .
monomeric pure poly(q ) peptides are in a random coil conformation
( 54 ) , but the addition of the
polyproline domain present in the carboxyl terminus of the poly(q ) stretch in
htt imposes structural constrain to the protein , which adopts a proline type
ii helical conformation ( 36 ) .
this explains why the proline - rich region of htt antagonizes the toxicity of
the poly(q ) expansion ( 34 ,
35 ) .
thus , a remodeling event
ought to be required to alleviate the inhibitory function of the proline - rich
region and to elicit the conformational transition into the -sheet
conformation . in agreement with biophysical data
, we found that in cells large
amounts of htt fragments with expanded poly(q ) can be expressed in a soluble
state ( this study and ref .
this implies that
aggregation is not just a consequence of an increase in concentration but is
dependent in the cellular milieu on additional events .
the data presented here
indicate that proteasomal chaperones facilitate the transition from soluble to
aggregated proteins with a poly(q ) expansion .
because inhibition of the proteolytic activity of the proteasome increases
htt aggregation , it has been proposed that aggregates form as a consequence of
an impairment of the proteolytic function of the proteasome .
however , two
conflicting studies question whether purified proteasomes can degrade poly(q )
peptides in vitro ( 9 ,
13 ) . here
we show that the
addition of wild type but not mutant odc destabilizing sequence to htt with a
poly(q ) expansion profoundly destabilizes this aggregation - prone protein .
it
is noteworthy that under certain conditions odc can be degraded by the 20s
proteasome ( 56 ) .
although the
mechanism by which wild - type but not mutant odc carboxyl terminus destabilizes
htt remains to be determined , it is likely that htt - odc is degraded by the
same ubiquitin - independent pathway as odc .
thus , the route by which htt is
targeted to the proteasome determines its fate
( fig .
furthermore , the fact that the addition of the odc destabilizing sequence
converts mutant htt into a short - lived protein shows unambiguously that , in
cells , the proteolytic capacity of the 20s proteasome is not a factor limiting
mutant htt degradation .
we propose that misfolding and aggregation of proteins
with poly(q ) expansion arise as a consequence of a remodeling event elicited
by a non - proteolytic function of proteasomal chaperones . in conclusion
, the data presented here reveal that aggregation of proteins
with poly(q ) expansion requires a transition from a benign to an
aggregation - prone conformation , a process which in cells is facilitated by
proteasomal chaperones .
aggregation of proteins with a poly(q ) expansion is ,
thus , reminiscent of a typical folding reaction , requiring the assistance of
chaperones .
| deposition of misfolded proteins with a polyglutamine expansion is a
hallmark of huntington disease and other neurodegenerative disorders .
impairment of the proteolytic function of the proteasome has been reported to
be both a cause and a consequence of polyglutamine accumulation .
here we found
that the proteasomal chaperones that unfold proteins to be degraded by the
proteasome but also have non - proteolytic functions co - localized with
huntingtin inclusions both in primary neurons and in huntington disease
patients and formed a complex independently of the proteolytic particle .
overexpression of rpt4 or rpt6 facilitated aggregation of mutant huntingtin
and ataxin-3 without affecting proteasomal degradation .
conversely , reducing
rpt6 or rpt4 levels decreased the number of inclusions in primary neurons ,
indicating that endogenous rpt4 and rpt6 facilitate inclusion formation .
in vitro reconstitution experiments revealed that purified 19s
particles promote mutant huntingtin aggregation . when fused to the ornithine
decarboxylase destabilizing sequence , proteins with expanded polyglutamine
were efficiently degraded and did not aggregate .
we propose that aggregation
of proteins with expanded polyglutamine is not a consequence of a proteolytic
failure of the 20s proteasome .
rather , aggregation is elicited by chaperone
subunits of the 19s particle independently of proteolysis . |
cardioembolic stroke , which is an important subtype of ischemic stroke , involves a large infarct volume and multiple vascular territories .
the neurologic deficits are grave and develop abruptly.1,2 because cardioembolic stroke is generally severe and frequently recurs , its long - term mortality is high.3,4 in addition , hemorrhagic transformation occurs frequently because of early or delayed recanalization.5,6 cardioembolic stroke accounts for 14 - 30% of all ischemic stroke.7 - 12 in korea , it accounts for approximately 17% , and the proportion is increasing ( figure 1).13,14 cardioembolic stroke is associated with chronological age7 and is thought to be one of the most important subtypes of ischemic stroke in aged or aging populations .
non - valvular atrial fibrillation ( nvaf ) is the most important cause of cardioembolic stroke , and patients with nvaf can complain of palpitations , chest pain , breathing difficulty , dizziness , or fainting.15 however , many patients do not have any symptoms or complain of vague and non - specific symptoms.16,17 probability of stroke incidence in patients with atrial fibrillation is 3 - 4%,18 and the risk of stroke increased by five times in all age groups.19,20 the percentage of strokes attributable to atrial fibrillation increases steeply from 1.5% at age 50 to 59 years to 23.5% at age 80 to 89 years.21 the prevalence of atrial fibrillation is globally increasing over time . in the united states , the number of patients with atrial fibrillation was 2.1 million in 1997 , but it increased to 2.3 million in 2001 .
it is estimated to increase to 5.6 million in 2050.22 the increased prevalence of atrial fibrillation is caused by improved survival rates of patients with heart disease.23 the prevalence of atrial fibrillation significantly increases with an increase in age.24 in korea , 57% of individuals with atrial fibrillation are older than 65 years , and the prevalence of atrial fibrillation is the highest in those older than 80 years.25 the importance of atrial fibrillation in ischemic stroke will further increase in korean society , which is rapidly aging .
risk factors increasing the incidence of stroke in patients with nvaf are known to be female gender , old age , history of stroke or transient ischemic attack ( tia ) , hypertension , heart failure , diabetes , and vascular diseases.23,26,27 history of stroke or tia increases the risk of stroke in patients with nvaf by three times.26 the incidence of stroke in patients with nvaf in their 70s is seven times that of patients in their 40s.28 when patients with nvaf have hypertension , the risk of stroke is thrice as high.19 since a risk stratification scale for embolic events in patients with nvaf was developed based on integration of these risk factors , it can be used to assess the risk of stroke in patients with nvaf and to select adequate preventive drugs . to classify the risk of stroke in patients with nvaf ,
the representative stratification systems currently being used include the chads2 and cha2ds2-vasc scores ( table 1).29,30 the chads2 score is widely used as the risk stratification scale , but after considering additional risk factors such as vascular disease , gender , and age of 65 - 74 years , a more specific evaluation can be made .
if a patient is classified into the group of low risk ( 0 or 1 ) in the point system by using the chads2 score , the cha2ds2-vasc score can be helpful for a more comprehensive risk assessment.30 in addition , the has - bled score , which is a convenient bleeding risk scale , is prepared from risk factors and a systematic review of bleeding in patients with nvaf ( table 2).31 if the has - bled score is 3 , a patient is classified into the high - risk group for bleeding.32 when antithrombotic therapy is started , special care and regular observation are required in this group .
in addition , microbleeds predict intracranial bleeding associated with warfarin.33,34 for the past 60 years , when the need for anticoagulation therapy was determined based on the calculated risk of stroke in patients with nvaf , warfarin has been administered .
previous studies demonstrated that warfarin was superior to other antiplatelet drugs in preventing stroke in patients with nvaf.35,36 in addition , it has been reported that the stroke is less severe when it occurs in patients taking warfarin.37 - 39 some patients have a very high tendency of relapse stroke , and stroke may recur despite the use of warfarin after stroke associated with nvaf .
although the primary reason is that warfarin is not properly administered or the international normalized ratio ( inr ) does not enter the target , other risk factors for recurrence of stroke are old age , female gender , and a history of stroke.40 if large artery atherosclerosis such as carotid or vertebral atherosclerosis is associated with nvaf , the recurrence rate of stroke also increases.41 in addition , a meta - analysis reported that stroke may be more recurrent in asian individuals taking warfarin compared to other races.42 in this context , the chads2 score can be useful in predicting the recurrence of stroke in patients with stroke associated with nvaf.40,42 furthermore , it was recently reported that the chads2 score can be used to predict the prognosis of stroke associated with nvaf ( figure 2).43,44
warfarin , a vitamin k antagonist , was the only oral anticoagulant available for a long time . however , its narrow therapeutic window and numerous food and drug interactions affect its safety , efficacy , and patient compliance .
in addition , its slow onset of action and variable pharmacologic effects make it difficult to maintain the appropriate antithrombotic effect.45 a meta - analysis reported that 44% of bleeding complications with warfarin were associated with supratherapeutic inrs , and 48% of thromboembolic events occurred in the subtherapeutic range.46 these drawbacks have encouraged the development of new anticoagulants .
several new oral anticoagulants ( noacs ) have been developed in the past 10 years.47 three of them were approved for use in the prevention of stroke and systemic embolism in patients with nvaf : dabigatran , a direct thrombin inhibitor , and rivaroxaban and apixaban , activated factor x inhibitors.48 - 50 they have predictable anticoagulant effects and fewer food and drug interactions , which allow a fixed dosing regimen without the need for monitoring .
however , strict patient compliance is required to maintain the desired anticoagulation levels . when rapid reversal of the anticoagulant effect is needed in the event of a major bleeding or emergency surgery
, there are no specific antidotes and standardized tests to monitor the anticoagulant status , which is a weakness of noacs.51 in terms of efficacy , according to the three randomized clinical trials that compared the efficacy of three noacs and warfarin , noacs were not always superior to warfarin .
the rates of the primary efficacy outcomes of any type of stroke or systemic embolism were significantly lower in the dabigatran 150 mg and apixaban groups compared to the warfarin group .
those in the dabigatran 110 mg and rivaroxaban groups were not significantly lower than those in the warfarin group .
in addition , all three studies had poor or moderately controlled patients ( defined as inr in the target range < 65% of the time ) as a control group.48 - 50 follow - up studies in which the control groups were divided based on inr level did not show the superiority of the dabigatran 150 mg dose compared with the well - controlled group ( inr in the target range 65% of the time).52 the superiority of the primary efficacy outcome in apixaban use was mostly caused by a reduction in hemorrhagic stroke , and there was no statistically significant difference in ischemic stroke rate.49 therefore , if the patient is well - controlled with warfarin and has minimal complications , the use of noacs may be controversial ( table 3 ) . in terms of safety ,
the major bleeding complication rate in the dabigatran 110 mg and apixaban groups was significantly lower than that in the warfarin group .
however , there was no significant difference between the dabigatran 150 mg and rivaroxaban groups and the warfarin group ( table 3).48 - 50 considering subtypes of bleeding , the intracerebral hemorrhage ( ich ) rate was significantly lower with both doses of dabigatran , but the major gastrointestinal ( gi ) bleeding rate was significantly higher in the dabigatran 150 mg and rivaroxaban groups.50,53 the ich rate was significantly lower and the gi bleeding rate was not significantly different in the apixaban group.49 hemorrhagic stroke rates related to receiving warfarin were higher in asian patients than in non - asian patients , but hemorrhagic stroke rates were significantly reduced by dabigatran in both asian and non - asian patients compared with individuals receiving warfarin.54 in non - hemorrhagic adverse events , approximately 10% of the patients in the dabigatran group complained of severe dyspepsia and approximately 21% of them discontinued the drugs .
tartaric acid created an acidic environment and increased the absorption of the drug independent of gastric ph.55 approximately 0.8% of patients taking dabigatran in the re - ly trial experienced myocardial infarction .
it was not statistically significant compared with the group taking warfarin , but it showed greater tendencies.56 a meta - analysis of 7 studies including the re - ly trial showed that the risk of myocardial infarction and cardiac death or unstable angina was significantly increased in the dabigatran group compared with the warfarin group.57 the bioavailability of dabigatran is only 6.5% and is not influenced by the coadministration of food .
approximately 80% of the drug is excreted unchanged in the urine , while only 20% is excreted in the feces .
thus , it is substantially unaffected by mild to moderate hepatic failure.58 because of the low plasma protein binding , it may be dialyzable when rapid reversal of its anticoagulation effects is needed.59 the bioavailability of rivaroxaban is dose - dependent and influenced by the coadministration of food .
the bioavailability at 10 mg and 20 mg in the fasting state is 80%-100% and 66% , respectively . taken with meals , its absorption is delayed but increased .
thus , therapeutic doses of rivaroxaban should be taken with meals.60 approximately 66% is excreted in the urine , and only 28% is excreted in the feces .
the safety and efficacy of rivaroxaban were consistent with warfarin in patients with moderate renal impairment , and it was also observed in japanese patients.61 two - thirds of the drug is converted into inactive metabolites through different cyp450 isoenzymes ( cyp3a4/5 or cyp2j2 ) and cyp - independent mechanisms.62 in this context , rivaroxaban should be avoided in patients with severe renal failure or moderate to severe hepatic impairment .
the bioavailability of apixaban is approximately 50% and is not influenced by the coadministration of food.63 one - third of the drug is metabolized through the cytochrome p-450 isoenzyme system ( mainly cyp3a4/5 ) .
approximately 25% is excreted in the urine , and > 50% is excreted in the feces.64 it may be suitable for patients with mild to moderate renal impairment because of its low renal excretion .
as previously mentioned , patients stabilized on warfarin may prefer to continue the same . however , the convenience and efficacy of noacs in the patients with inadequate inr are cause to consider the transition to noacs .
transition to warfarin also may be needed in patients who are unable to continue on noacs . in this situation , safe transition between anticoagulants is an important issue in current practice . in previous trials including patients who had warfarin before starting on dabigatran , the highest inr permitted at the time of transition was 2.0 or 2.3,65,66 whereas in the rivaroxaban once - daily , oral , direct factor x inhibition compared with vitamin k antagonism for prevention of stroke and embolism trial in atrial fibrillation ( rocket af ) , it was 3.0.50 in these trials ,
the overlap was not associated with an increased risk for bleeding . in transition from noacs to warfarin
, it is necessary to take into account the expected onset of warfarin as well as the half - life of the noacs .
the mean time to achieve therapeutic range ( inr 2 - 3 ) was approximately 5 days .
the half - life of dabigatran varies from 14 - 17 hours with normal renal function to 18 - 27 hours in moderate to severe renal impairment.67,68 the recently recommended approaches based on these data are summarized in table 4.69 - 71 the longer overlap period of rivaroxaban and warfarin shown in this table is caused by the shorter half - life of rivaroxaban.72 when assessing the inr during transitions between rivaroxaban and warfarin , we need to be cautious because rivaroxaban may increase the prothrombin time.73 perioperative management of the noacs is based on the urgency of the procedure , bleeding risk , and current renal function .
preoperative discontinuation of the drug is based on pharmacokinetic data and considerations regarding the bleeding risk ( table 5 ) .
interruption of dabigatran for 48 hours should be sufficient to ensure adequate hemostasis in patients with normal renal function because of the half - life of 14 - 17 hours.74 for procedures with a low risk of bleeding such as cardiac catheterization , diagnostic endoscopy , and minor orthopedic surgery , for which an inr of 1.5 for patients on warfarin would be acceptable , it is reasonable to interrupt dabigatran for 24 hours . in patients with decreased renal function , the period of interruption should be longer .
this principle is also applied to rivaroxaban . in the previous trial , rocket - af ,
rivaroxaban was interrupted approximately 2 days before the procedure.60 the short period of interruption does not require bridging therapy with unfractionated heparin or low - molecular - weight heparin .
reinitiation of noacs after the procedure depends on the postoperative risk of bleeding . for procedures with good hemostasis ,
the suggestion is to reinitiate noacs at a minimum of 4 - 6 hours after the procedure.65 the first dose of dabigatran should be a half - dose ( 75 mg ) , and the next scheduled dose should be the usual maintenance dose .
a similar strategy using a 10-mg dose for the first dose can be applied to rivaroxaban .
patients with bowel paralysis may require bridging with parenteral anticoagulants because they can not take oral anticoagulants .
for strategies to prevent stroke associated with nvaf in addition to antithrombotic therapy , studies on procedures to surgically block the left atrial appendage ( laa ) , which is the most important position in which thrombus is formed by nvaf , have been conducted.75 - 77 after a device to block laa is inserted , warfarin is administered for 45 days , and then both aspirin and clopidogrel are administered for 4.5 months .
it is not inferior to the group of conventional warfarin use when aspirin monotherapy is administered.78 in addition , when long - term follow - up continues for up to 2.3 years , it is non - inferior in occurrence of cardiovascular diseases including stroke compared with the group using warfarin.79 other strategies to prevent stroke associated with nvaf include rhythm control and heart rate control .
previous studies and meta - analyses showed that the beneficial effect between rate control and rhythm control was not different for prevention of stroke.80 - 85 a post - hoc analysis from a recent study showed that dronedarone , a newly developed rhythm control drug , was beneficial in preventing stroke,86 but another study using dronedarone in addition to standard therapy for rhythm control showed that dronedarone increased the risk of stroke more than two - fold in comparison with placebo.87 recently , a large - sized population - based observational study to compare rhythm control and rate control in patients with atrial fibrillation was conducted in canada.88 risk for the occurrence of stroke was decreased by 20% in patients who underwent rhythm control compared with those who underwent rate control .
when patients were classified into low- , moderate- , and high - risk groups based on chads2 scores , rhythm control was superior to rate control in the moderate- and high - risk groups .
to date , a complete conclusion about the effect of rate control and rhythm control on occurrence of stroke associated with nvaf has not been made .
more delicate risk assessment systems make physicians choose adequate preventive strategies to reduce stroke related to nvaf , and noacs may be additional therapeutic options for these patients in primary and secondary prevention . in this context , there are some clinical practice guidelines for the management of atrial fibrillation patients from the united states and europe.26,89,90 recently , a clinical practice guideline for stroke prevention was published and revised in korea , and it includes these issues about ischemic stroke associated with nvaf.91 ischemic stroke related to nvaf has been more prevalent , as life expectancy is increasing worldwide , and disease burden is also increasing steeply . | stroke associated with non - valvular atrial fibrillation ( nvaf ) is one of the most important subtypes of ischemic stroke , and its importance is becoming even more apparent in an aging population . to assess the risk of stroke associated with nvaf , the chads2 and cha2ds2-vasc scores are mainly used .
such scores can be used to predict the recurrence and prognosis of ischemic stroke .
in addition , new oral anticoagulants ( noacs ) and devices are being evaluated in the prevention of stroke associated with nvaf in addition to treatment with the conventional oral anticoagulant , warfarin . since clinical experience with noacs is not globally sufficient , a cautious approach is needed . |
this occurs mostly through the action of dedicated transcription factors ( tfs ) , as in the well - known case of the lac operon ( jacob et al . , 1960 ) .
on the other hand , gene expression is continuously adjusted to adapt to varying environmental conditions , leading to quantitative relationships between the molecular content of cells and their growth rates ( scott and hwa , 2011 ) . physiological factors
( berthoumieux et al . , 2013 ) , such as the concentration of rna polymerases ( rnaps ) ( klumpp and hwa , 2008 ) , the regulation of rna degradation ( chen et al . , 2015 ) , and topological properties of dna ( dorman , 1995 , hatfield and benham , 2002 , travers and muskhelishvili , 2005 ) ,
are thus known to continuously affect , at a system level , the transcriptional activity of genes .
the specificity , if any , of each of these mechanisms with respect to the set of co - regulated genes , nevertheless remains to be understood . in bacteria , the coordination of transcription
is strongly related to the linear organization of genomes ( kps et al . , 2012 ) .
at the smallest scale , many co - regulated genes are thus found within operons so that they can be co - transcribed into the same mrnas . despite their apparent simplicity , the operons have nevertheless raised important questions , not only about their determination but also about their definition ( okuda et al . , 2007 , gell et al . , 2011 , mazin et al . , 2014 ) and utility ( de lorenzo and danchin , 2008 , junier , 2014 ) .
for instance , although certain operons are easily recognizable by their functional homogeneity ( as , e.g. , for the lac operon ) , many of them are composed of genes whose function appears unrelated ( de lorenzo and danchin , 2008 ) .
soon after the seminal work of jacob and monod , studies on operons such as trp also revealed the possibility to have specific internal regulation of termination ( yanofsky , 2000 ) : mrna - based intrinsic terminators may abort transcription midway , whereas competing mrna secondary structures ( anti - terminators ) may attenuate this effect ( merino and yanofsky , 2005 , santangelo and artsimovitch , 2011 ) . together with the observation that the majority of operons actually contain alternative transcription start sites ( tsss ) ( sharma et al .
, 2010 , cho et al . , 2014 ) , high - throughput data have thus revealed the presence , inside operons , of differential initiation and termination points ( okuda et al .
, 2007 , gell et al . , 2011 , nicolas et al . , 2012 , mazin et al . , 2014 ) .
yet the impact of these internal elements on the genome - wide coordination of transcription has remained unexplored .
model systems such as the bacteriophage further revealed that operons may be part of larger functional genomic units with , in particular , the possibility of having subsequent operons transcribed in one go ( gottesman et al .
trt has actually been shown to be frequent and regulated by dedicated proteins ( stlke , 2002 , nudler and gottesman , 2002 ) , with the so - called factor playing a major role in many bacteria ( richardson , 2002 ) .
transcriptional co - expression has thus been shown to extend beyond operons ( jeong et al . , 2004 , carpentier et al . , 2005 , nicolas et al . , 2012 ) .
yet the systematic identification of supra - operonic units and of their regulatory mechanisms remains an open problem .
a system - level understanding of transcriptional coordination thus requires abandoning , at least in the first stage , our preconception of the potential units that may come at play .
a promising avenue along this line consists in analyzing in detail the genomic properties of proximal genes as a function of their degree of co - expression and to question the structural and regulatory properties that might be associated to the observed patterns ( ma et al . , 2013 ) . here ,
we perform such analysis in m. pneumoniae , a model organism with a reduced genome ( 820 kb ) that offers ideal properties to address questions about the fundamental mechanisms that govern bacterial cell physiology ( gell et al . , 2009 ,
in particular , although m. pneumoniae has two sigma factors ( torres - puig et al . , 2015 ) , a tiny tf repertoire ( table s1 ) and no factor ( himmelreich et al . , 1996 ) , it shows genome - wide complex specific regulatory patterns in response to different external perturbations ( gell et al . , 2009 ) .
this suggests the existence of fundamental mechanisms ensuring coordination of transcription , different from tfs and from the factor . to test this hypothesis and to identify associated regulatory mechanisms
, we analyzed rna sequencing ( rna - seq ) data obtained from m. pneumoniae under 115 different conditions ( figure 1a ) . to this end
, we built a co - expression measure particularly well poised to highlight basal co - expression . using a hierarchical clustering framework that is constrained to respect the 1d organization of the genome
, we then reveal the existence of three qualitatively distinct levels of co - expression associated to different organizations of adjacent genes and to different properties of intergenic regions .
we next show that the degree of co - expression between co - directional genes and operons is tightly related to the capacity of the rnap to transcribe them as if they belonged to the same operon .
we then reveal that such trt is both ubiquitous and condition dependent and that it is repressed by dna - bound rnaps , strong intrinsic terminators , and large intergenic distances .
we measured the transcriptional activity of 869 m. pneumoniae genes , of which 701 encode proteins ( lluch - senar et al . , 2015 ) , across 141 conditions ( 282 samples ) . to this end ,
m. pneumoniae m129 ( passage 34 , nc_000912 reference genome in the national center for biotechnology information [ ncbi ] ) was grown in modified hayflick medium and transformed by electroporation ( yus et al . , 2009 ) .
rna - seq data were then collected at various stages of the cell growth , after various perturbations and overexpression of different regulators ( table s2 ) .
the resulting transcription profiles were generally highly similar , even after shuffling the expression values between the conditions for each gene separately ( light gray distribution in figure 1a ) , showing that genes have mostly stable expression . to focus specifically on basal co - expression
we identified a large set of 227 highly similar samples corresponding to 115 conditions ( 112 for which two technical replicates are present ) , the 29 remaining conditions being characterized by a particularly low level of transcription ( table s2 ) . to analyze the basal coordination of transcription between all pairs ( i , j ) of genes
, we built a specific measure of transcriptional co - expression , cij , hereafter referred to as basal correlation .
cij quantifies the tendency of the expression of the two genes to systematically vary in parallel ( figure s1 ) , here among the 227 working samples .
specifically , it is equal to the difference between the number of pairs of samples for which the two genes co - vary and the number for which they vary in the opposite direction , normalized by the total number of possible pairs of samples . compared with other correlation measures to which it is related , such as the pearson correlation , cij is well suited to highlight the basal coordination of genes ( figure s1 ) . in particular
, cij0 indicates that the expression of the two genes varies as many times in the same direction as in the opposite direction .
in contrast , cij1 indicates that they always vary in the same direction , whereas cij1 indicates a systematic tendency to vary in the opposite direction .
figure 1b shows the resulting heatmap of cij , for which the genes are sorted according to their genomic position , and each pixel indicates the co - expression level between two genes .
one can distinguish the presence of specific contiguous clusters of highly co - expressed genes with , on average , a genomic extension that typically extends up to 10 kbp ( figure 1b , bottom ) .
this 10 kbp length scale is larger than the typical length scale of operons , corroborating that the co - expression of proximal genes extend beyond operons .
it is actually similar to that found in e. coli and b. subtilis when performing a similar analysis of co - expression ( jeong et al . , 2004 , carpentier et al . , 2005 , junier and rivoire , 2016 ) . to delineate in a more precise way the relationship between basal coordination of transcription and the established organization of m. pneumoniae into operons
( see experimental procedures for their definition ) , we analyzed in detail the genomic organization of co - expression . to this end
, we developed a hierarchical description of co - expression constrained to respect the linear organization of the genome .
briefly , we built a dendrogram in which pairs of adjacent genes were hierarchically fused on the basis of their co - expression ( figure 1c ) . using this dendrogram , we defined domains of the genome , which we call -domains , as the contiguous domains of genes for which all the adjacent genes have a co - expression larger than ( figure 1c ) .
an analysis of -domains for all possible values of reveals that although these domains may coincide with operons at certain values of , they are generally different .
this can be qualitatively appreciated for specific clusters of genes , such as that of the f - atpase machinery ( figure 1c ) .
more quantitatively , we evaluated the capacity of -domains to predict operons using our most recent manual annotation of operons as the ground truth ( table s1 ) . to this end
, we made vary from 1 to 1 and we assessed both the specificity and the sensitivity of predictions .
the resulting area under the receiver operating curve ( auc ) , which summarizes the balance between specificity and sensitivity by a single value , was equal to 0.76 ( figure 1d ) .
-domains are thus not perfect predictors ( in which case the auc would have been equal to 1 ) , corroborating the necessity to analyze co - expression properties independently of our knowledge of operons , at least in the first stage . to better understand the hierarchical properties of co - expression , we analyzed the relative orientations of adjacent genes as a function of their co - expression ( 846 pairs analyzed for which expression values were available for the two genes ) ( figure 2a ) .
we also analyzed the tendency of co - directional genes to overlap ( figure 2b ) , as well as the intergenic distances between the non - overlapping ones ( figure 2c ) .
notably , the three properties ( relative orientation , overlapping , and distance ) suggest a similar three - level organization of co - expression . specifically , for co - expression > 0.6 ( strong co - expression ) , 227 of 234 pairs of genes are co - directional , with a high proportion ( 52% ) of overlapping cases ( p4.1011 , hypergeometric test ) . for co - expression between 0.3 and 0.6 ( moderate co - expression , 375 pairs ) ,
genes significantly tend to be co - directional ( p7.107 ) and to overlap ( p7.105 ) , all the more that co - expression is large . at this level , although intergenic distances between non - overlapping genes are larger than those with strong co - expression , they remain relatively small . plotting the co - expression level of all pairs of non - overlapping co - directional genes
as a function of their intergenic distance actually reveals the existence of a 100 bp length scale below which typical co - expression is larger than 0.3 and above which co - expression is low and statistically insensitive to distances ( figure 2d ) . finally , below 0.3 ( low co - expression , 237 pairs ) , there is no enrichment for a specific relative orientation of genes ( p 1 , binomial test of the hypothesis that the probability of co - directionality is equal to 0.5 ) .
moreover , intergenic distances between co - directional genes are large , exceeding 100 bp and reaching typically 400 bp at very low co - expression ( figure 2c ) . performing the same analyses using pearson correlation led qualitatively to the same findings ( figure s2 ) .
the sharp delineation of the three different regimes as well as their correspondence between the different properties ( relative orientation , overlapping , and distance ) is less clear , though , than those obtained using the basal correlation .
because a significant number of operons have moderate co - expression levels and because trt pervades the transcriptomes of bacteria ( wade and grainger , 2014 ) , we wondered whether trt could explain the co - expression levels of co - directional genes belonging to distinct operons .
we thus investigated the tendency of all adjacent co - directional genes belonging to two different operons to be transcribed as a single transcript ( 268 pairs analyzed ) .
we examined the variation of expression in their intergenic region as a function of the variation of expression of the downstream gene ( figure 3a ) .
our rationale was that trt , if present , should leave a trace on the expression of the intergenic region that precedes the downstream gene .
we thus compared the co - expression between the downstream gene and the sense ( 5 3 ) intergenic region ( co - expression cs in figure 3a ) with that between the two genes ( co - expression c ) ; as a control , we considered the anti - sense ( 3 5 ) region ( figure 3a , right ) ( co - expression ca ) .
to prevent any bias arising from the transcription of the utrs inside operons , for this analysis , we defined intergenic regions as the sequences that separate the transcription termination site ( tts ) of the upstream gene and the tss of the downstream gene . for co - expression levels larger than 0.3 , we observed that the degree of co - expression between co - directional adjacent operons was higher when there was co - expression with the sense intergenic region ( figure 3a ) ; in contrast , it did not show any dependency with the anti - sense expression ( figure 3a , right ) .
the same analyses , but considering genes that are separated by more than 100 bp ( figure 3b ) , or using the pearson correlation ( figure s2e ) , led to the same conclusions .
next , to explicitly demonstrate the role of trt in basal coordination of transcription , we first studied the efficiency , , of trt extending between two co - directional adjacent operons , say , x and y with x preceding y ( figure s3a ) .
was defined as the ratio between the rna - seq expression levels measured at the tss of y and at the stop codon of the last cistron of x ( independently whether this was associated to a well - defined terminator ) .
we thus assumed that the rna - seq level just preceding y would result from the trt of x and would be representative of the basal level of y. according to this model , for which we provide below an experimental validation , the overall expression of y is thus equal to the sum of its basal level coming from trt , plus some contribution from its own tss ( figure s3a ) .
we analyzed seven pairs of genes with various degrees of correlations and distances : two pairs with strong correlation , including one overlapping case ( mpn155a - mpn155 ) ; four pairs with moderate correlation and distances larger than 100 bp , including two pairs with an intermediate gene located on the opposite strand of their intergenic region ; and one pair with low co - expression .
as shown in figure s3b , was close to 1 and varied little for pairs with strong correlation , but also for the pair mpn160-mpn161 ( highest moderate correlation , c=0.5 ) except during heat shock . for the other pairs ,
in particular , for the pair mpn161-mpn162 ( low correlation ) , we observed a 2-fold variation during cold shock that poorly correlated to the expression variation of the downstream gene .
we then tested the validity of our trt - based model of transcriptional coordination by confronting predictions of the model ( using rna - seq data ) to direct measurements of transcripts using real - time quantitative pcr ( qpcr ) .
specifically , for the aforementioned seven pairs , we measured the level of transcripts extending between the genes during cold shock , heat shock , and exponential growth ( control ) .
as shown in figure s3c , the variations of extended trt measured by real - time qpcr were qualitatively ( quantitatively in most cases ) similar to those predicted by the model .
notably , this was true for the cases with an intermediate gene on the opposite strand .
we thus conclude that trt is ubiquitous and can explain , in principle , many of the significant co - expression levels of co - directional adjacent operons .
these results also suggest that large pieces of genomes that extend beyond operons may be transcribed en bloc .
an instructive example concerns the ribosome - encoding genes : these genes are surrounded by transcription - related genes and other biological pathways , apparently forming altogether a large domain containing more than 50 genes ( corresponding to 15 manually annotated operons ) with a high level of background expression ( figure 3c ) . although some of this background is expected to result from the strong tendency of these promoters to initiate transcription , as demonstrated by our real - time qpcr analysis ( figure s3 ) it also results from trt extending between operons . in this context , it is important to recognize that large domains of coordinated expression , in which several genes and operons may be transcribed in a row , remain compatible with the very presence of operons .
this can be seen by the decrease of expression at the end of certain operons or by the presence of steep fold changes at their promoter ( vertical gray lines in figure 3c ) .
the pair mpn155a - mpn155 ( strong basal co - expression ) provides a good example of this effect as it shows extended trt between the two corresponding operons ( figures s3b and s3c ) but also a sharp tss at the downstream gene at late exponential phase ( in red in figure 3c ) and at stationary phase ( figure s3b ) .
rna - seq data and real - time qpcr show that trt may vary not only along the genome but also among conditions ( figure s3 ) .
the ten - gene ( four - operon ) domain containing the heat shock gene ( grpe ) provides an insightful example of such variations ( figure 5c ) , with the operon containing grpe differentiating into two sub - operons during heat shock and distinct operons becoming transcribed as a single operon during cold shock ( figures s3b and s3c ) .
notably , both our rna - seq analysis and our real - time qpcr measurements further suggest that trt is globally enhanced during cold shock ( table s3 ; figure s3 ) , in accord with reports in e. coli and b. subtilis of the anti - terminator role of cspa cold - shock proteins ( bae et al . , 2000 , stlke , 2002 ) .
to systematically quantify trt variations among conditions , we analyzed the behavior of the ttss internal to pairs of co - directional genes belonging to different operons ( 233 ttss analyzed ) , independently of whether a well - defined terminator was associated to the tts .
for each tts , we computed the variation of its downstream expression ( down ) as a function of the variation of its upstream expression ( up ) in response to perturbations ( 96 perturbations tested with respect to 19 controls ; figure 4a ) . using this approach
, we could identify at least six types of ttss ( figure 4b ) .
the three first types concern ttss for which a statistically significant positive correlation exists between the values of down and up that are computed over the different perturbations ( see legend of figure 4 for details of the statistical analyses ) .
they are respectively defined by downup ( stable trt , in red in figures 4b and 4c ) , downup ( stable trt plus some activation , in orange ) , and downup ( stable trt plus some repression , in yellow ) . for these tsss
, trt thus tends to be maintained at a similar level , irrespective of the conditions .
notably , these correspond to 85% of the total amount of ttss ( 50% if only considering downup ) and appear to account for all strong co - expression levels ( figure 4c ) .
the two next types correspond to activation only , with a majority of down0 ( in blue ) , and to repression only , with a majority of down0 ( in cyan ) , irrespective of the value of up . together with the ttss having independent or no apparent statistically significant variations of down ( in green ) , these three last types contribute mainly to low co - expression levels .
altogether , these results thus corroborate both the ubiquity of trt and its major role in basal co - expression .
the observation of pairs having both low co - expression and stable trts also suggest that trt does not systematically extend to the next operon . finally , to apprehend whether trt is stochastic or specifically regulated , we analyzed the behavior of the ttss upon each perturbation .
first , in accord with our real - time qpcr experiments ( figure s3 ) , we observed that the variations of trt ( activation or repression ) for a given perturbation tend to be the same for all ttss , suggesting that trt is specifically regulated at a genome - wide level .
these include cold shock , osmotic shock , and novobiocin treatments , whereas low ph and heat stresses tend to repress trt .
finally , by identifying the ttss and the corresponding perturbations for which the variation of trt was extreme ( supplemental experimental procedures ) , we found that conditions for which a large number ( 12 ) of ttss had an extreme behavior were strongly enriched in novobiocin ( gyrase inhibitor ) perturbations ( p8107 , hypergeometric test ) and strongly depleted in single - gene perturbations ( p2104 ) ( table s3 ) . because novobiocin targets topoisomerases and , hence , modify dna supercoiling , these results suggest that the mechanical properties of dna and its interaction with rnaps might play a crucial role in trt variations ( see the following discussion for further details ) .
our observations of a trt that depends strongly on conditions , with operons that can be transcribed uniformly , en bloc ( super - operons ) , or differentially ( sub - operons ) , raise at least two fundamental questions : what mechanisms are responsible ( 1 ) for promoting an operon - like transcription of adjacent genes and ( 2 ) for preventing it ?
in answer to the first issue , using co - expression levels as a proxy of transcription en bloc , the results in figures 2c and 2d suggest that compactness , with a distance between open reading frames smaller than 100 bp , may be required for efficient operon - like co - expression .
notably this length scale corresponds to the typical distance that is usually considered for operon prediction ( mcclure et al . , 2013 ) .
we note , nevertheless , that pairs of genes with intergenic regions larger than 100 bp can have a high level of trt ( figures 3 and s3 ) .
our analysis also shows that compactness alone is not sufficient , because a substantial number ( 52 ) of pairs of co - directional genes with low co - expression levels are separated by less than 100 bp ( among which 17 pairs concern overlapping genes ) . in answer to the second question ,
let us first mention that although compactness properties call for an important role of distances on the capacity of the rnap to transcribe multiple genes in a row , co - expression does not depend primarily on distances when these exceed 100 bp ( figure 2d ) .
to better understand the differences between intermediate co - expression levels and low co - expression levels , we thus investigated the possible impact of -independent intrinsic terminators found within mrna sequences ( -dependent termination is absent in m. pneumonia ) .
canonical intrinsic terminators consist of an rna hairpin followed by a u tract , a combination that is believed to favor the disruption of the mrna - dna template hybridization necessary for the rnap to process transcription ( peters et al . , 2011 ) .
we thus evaluated the presence , in the intergenic regions of all pairs of co - directional genes , of rna hairpins that were immediately followed by u tracts of various lengths ( nu2 ) ; as a control , we considered intergenic regions that were translated by an arbitrary amount of base pairs , which allowed us handling distance effects of intergenic regions , a longer sequence being more likely to contain an rna hairpin ( figures s4a and s4b ) , independently whether the latter plays a functional role .
we found that more than 15% of the gene pairs with low co - expression contained an rna hairpin with nu4 ( figure 5a ) , a proportion that was highly significant with respect to the control ( p4.103 , two - sided t test with unequal variances ) .
note here that the absence of an enrichment of terminators with shorter u tracts ( nu<4 ) corroborates previous observations that long u tracts are needed to have efficient termination ( chen et al . , 2013 ) .
similar trends were observed for intermediate co - expression levels , although involving a lower fraction ( typically half ) of gene pairs , in accord with the fact that at this level , trt is expected to occur in a larger subset of conditions . according to the above analysis , more than 80% of the co - directional gene pairs with low co - expression do not contain any strong intrinsic terminator in their intergenic region .
non - perfect u tracts or more complex termination signals that are yet to be identified might explain part of this low co - expression
. the action of nucleoid - associated proteins ( naps ) such as h - ns in e. coli ( singh et al . , 2014 ) could also be invoked .
data from our lab nevertheless show that m. pneumoniae contains only one nap , ihf ( gene mpn529 ) , with a low copy number ( < 100 ) . to better apprehend the mechanisms related to the repression of trt , we thus performed chromatin immunoprecipitation sequencing ( chip - seq ) analysis of the -subunit of the rnap .
consistent with results obtained in e. coli ( mooney et al . , 2009 ) ,
chip - seq profiles from cells in the stationary phase revealed the presence of well - defined peaks corresponding to preferentially rnap occupancy domains ( rpod ) ( figures 5b and 5c ; table s4 ) .
notably , we found that the majority of gene pairs with low co - expression contain rpods in their intergenic regions ( figure 5b ) .
for intermediate co - expression , rpods are less present but remain over - compared to strong co - expression .
note that in contrast to hairpins , the presence of rpods does not depend on the intergenic distances ( figure s4c ) , meaning that larger intergenic distances can not simply explain these results . because the average level of transcription strongly depends on rna degradation , we eventually compared the half - lives of transcripts between adjacent genes .
we found a remarkable correlation between the degree of transcriptional coordination and the similarity of half - lives ( figure 6 ) .
using a correlation measure well poised to quantify basal co - expression and applying it to rna - seq data obtained in more than 100 different conditions , we have revealed the existence in m. pneumoniae of three distinct levels of basal coordination of transcription ( strong , moderate , and low ) , corresponding to three qualitatively different properties for the relative orientations and intergenic regions of adjacent genes . in accord with the major role of operons in the coordination of gene expression ,
we have found that strong basal co - expression requires adjacent genes to be co - directional .
we have also found the existence of a 100 bp length scale , below which an operon - like behavior appears to be quasi - systematic and above which co - expression depends strongly on the sequence and structural properties of the intergenic region . in particular , although pairs of adjacent genes with low basal co - expression do not show any preferential relative orientations , 70% of the intergenic region of the co - directional pairs either contain a domain preferentially occupied by rnaps ( rpods ) or strong terminators ( 55% and 15% of the cases , respectively ) . by focusing specifically on co - directional adjacent genes , we have further revealed that the coordination of transcription is tightly related to the tendency of proximal genes to be transcribed en bloc , even though these genes may have not been categorized to belong to the same operon .
three extreme scenarios can then be considered ( figure s5a ) , which is in accord with our observation of the three qualitatively distinct co - expression levels .
the transcription en bloc may be systematic ( i.e. , it occurs with probability close to 1 in all conditions ) , in which case the genes behave as canonical operons ( green light in figure s5a ) . or it occurs from time to time , meaning that it can take place in specific conditions and be absent in others . variations may also occur in a given condition , because transcription may terminate with a certain probability due , for example , to the presence of an intrinsic terminator . in this case ,
gene expression may present staircase - like patterns ( gell et al . , 2009 ) ( orange light ) .
finally , transcription en bloc can never occur , in which case genes must be considered to belong to different transcriptional units ( red light ) . in accord with the rich zoology of operon - related structures that have been described over the past decade ( okuda et al .
, 2007 , gell et al . , 2009 , cho et al . , 2009 , nicolas et al . , 2012 ,
mazin et al . , 2014 ) and with the ubiquitous presence of pervasive transcription ( wade and grainger , 2014 )
, our findings thus indicate that operon - like behaviors are often stochastic and condition dependent , with frequencies of occurrence that depend on intergenic sequences .
in particular , transcriptional initiation may often occur on top of a background level of continuous expression . in this context
, we surmise that one of the most fundamental mechanism for the coordination of transcription relies on a high probability to have specific large domains of genes that are transcribed in a row , independently of the fact that these domains may contain several internal entry points and exit points for the rnaps ( see figure s5b for a schematic representation of this model ) .
these internal landmarks might then be used by the bacterium to adapt to a wide range of conditions ( see , e.g. , figure 5c ) .
they might also contribute to the activation of a given domain ( see the following discussion ) .
our scenario implies , on one hand , the existence of two mechanisms internal to the domains , which are a priori necessary to maintain a proper balance between transcripts .
first , there should exist a mechanism that enhances the transcription of upstream genes whenever transcription is initiated within the domain , in order to avoid a gradient of transcripts along the domain ( with downstream genes in larger quantity than upstream genes ) .
although at this stage we have no direct evidence of such phenomenon , this prediction suits the proposal , in bacteria , of a control of gene expression by dna supercoiling ( dorman , 1995 , hatfield and benham , 2002 , travers and muskhelishvili , 2005 ) .
it is also in accord with our observation of a strong impact of novobiocin ( a gyrase inhibitor ) treatment on trt properties ( table s3 ) .
the negative supercoiling that is generated upstream of the transcribing rnaps might indeed enhance the initiation of the upstream genes ( meyer and beslon , 2014 ) . considering that these effects can propagate all the way up to the borders of the domain because of the long - range nature of the transmission of supercoiling constraints ( krasilnikov et al . , 1999 )
, an internal initiation event should in principle be able to activate the expression of the whole domain ( figure s5b ) , in particular without the additional action of tfs .
second , produced transcripts should have similar degradation rates , which we confirmed by analyzing rna half - lives ( figure 6 ) .
well - defined domains of basal co - expression require , on the other hand , the ability , upstream , to prevent the activation of genes and , downstream , to terminate the transcription process .
supposing that the upstream activation is mainly the result of supercoiling transmission , stalled rnaps , as suggested by the presence of rpods ( reppas et al .
, 2006 , mooney et al . , 2009 ) , could act as topological barriers ( higgins , 2014 ) ( see figure 5c for a suggestive example ) .
downstream , in addition to the possibility of rpod roadblocks , strong intrinsic terminators are expected to play an important role in terminating transcription ( figure 5 ) .
other mechanisms can be contemplated , such as anti - sense transcription ( lybecker et al . , 2014 ) or the action of small rnas , although recent work from our lab shows that the latter have little impact on gene expression ( llorns - rico et al . , 2016 ) . here , and more particularly in the absence of the factor and of naps , which have been shown to prevent pervasive transcription ( singh et al . , 2014
) , the mechanisms at the core of the basal coordination of transcription in m. pneumoniae thus appear to rely solely on the physical entities ( rnap and mrna ) and mechanical properties of the transcription process itself .
although a strong terminator can efficiently prevent trt , it may prevent co - expression only partially
. this can be seen for instance by the adjacent genes 5s rrna ( mpnr03 ) and mpn095 , which is the unique pair showing both strong co - expression ( c=0.68 ) and the presence of a strong intergenic terminator .
although some specific processing of rrna might occur , overriding of the terminal signal , as suggested by the high level of co - expression with the sense intergenic region ( cs=0.55 ) , might explain the strong co - expression .
local concentration effects of rnaps might also contribute , more particularly because of the high expression level of the 5s rrna .
in such situations , intergenic distances might play a crucial role in the isolation of adjacent genes . specifically ,
compared to the 2030 nm size of the rnap , the 130 bp that separate the tts of the 5s rrna from the tss of mpn095 correspond to a maximal spatial distance of 45 nm ; 400 bp , the typical distance for pairs of genes with co - expression close to 0 ( figure 2c ) , correspond to 135 nm .
our scenario reckons with the intrinsic stochastic nature of transcriptional initiation , with the capacity of the rnap to transcribe multiple operons in one go ( santangelo and artsimovitch , 2011 ) , and with the possible role of supercoiling to transmit regulatory properties , especially in a bacterium that is depleted in tfs ( zhang and baseman , 2011 , dorman , 2011 ) .
it also opens new roads to understand the existence of preferential regions and promoters for the binding of rnaps ( reppas et al . , 2006 , mooney et al . , 2009 ) and suggests that a large part of the specific basal coordination of transcription might rely exclusively on the interplay among rnap , dna , and mrna .
importantly , our findings appear to hold in a wide range of bacterial species . a similar three - level organization of co - expression , with the same properties of relative orientations and of intergenic distances ( including the existence of a 100 bp length scale ) ,
is indeed observed both in e. coli and in b. subtilis , ( figure s6 ) .
domains of proximal genes that are conserved in phylogenetically distant bacteria have also been shown to correspond , both in e. coli and in b. subtilis , to domains of highly co - expressed genes and operons where trt is particularly enhanced ( junier and rivoire , 2016 ) .
finally , we note that -independent terminators , as well as attenuators of these terminators through , for example , the action of riboswitches , are often conserved among distant bacteria ( vitreschak et al . , 2004 , merino and yanofsky , 2005 ) . together with the dynamical interplay between dna and rnaps
, they may thus correspond to ancestral mechanisms upon which the basal functioning of bacteria has been tinkered . in particular , tfs and other types of gene control such as the invertible dna switches of bacteroides ( kuwahara et al . , 2004 )
may represent evolutionary solutions dedicated to specific needs related to the lifestyle of each bacterium .
rna isolation was performed using mirneasy kits from qiagen , and an in - column dnase treatment was included .
rna was measured using a nanodrop ( thermo ) , and integrity was confirmed in a 6000 nano chip bioanalyzer ( agilent ) .
we then used the truseq stranded mrna sample prep kit v2 ( illumina ) to obtain a paired - end strand - specific rna - seq library . see table s2 for further details of conditions .
chip - seq of rnap ( tap - tagged ; see khner et al . , 2009 ) was performed as previously described ( yus et al . , 2012 ) .
we identified all mrna tsss from their associated tssrnas ( yus et al . , 2012 ) .
we distinguished productive promoters from short tssrnas as explained previously ( llorns - rico et al . ,
regarding 3 sites , we used strand - specific deep sequencing and tiling array data to define approximately their positions ( gell et al . , 2009 ) .
we then used these data to refine our previously published operon map ( gell et al . , 2009 ) ( updated map in table s1 ) .
cells were collected in the indicated conditions , and rna was purified as described above .
retrotranscription and real - time qpcr of 800 base long regions were done in one step with the gotaq 1-step rt - qpcr system ( promega ) .
oligos ( table s5 ) were used at 0.15 m , and 25 ng total rna was used as a template .
potential intrinsic terminators were defined as a rna hairpin immediately followed by a u tract .
rna hairpins were identified as described previously ( mathews et al . , 1999 ) .
rpods were identified by the presence of significant peaks ( see supplemental experimental procedures ) in the chip - seq data of the rnap -subunit ( gene mpn191 ) at 6 and 96 hr .
rna half - lives were determined using a dna gyrase inhibitor ( novobiocin ) , which alters the chromosomal supercoiling releasing the rnap , thus stopping transcription ( dorman , 2011 ) .
after novobiocin treatment , rna was extracted at different time points , and rna - seq was performed to determine transcript levels .
| summarycoordination of transcription in bacteria occurs at supra - operonic scales , but the extent , specificity , and mechanisms of such regulation are poorly understood . here
, we tackle this problem by profiling the transcriptome of the model organism mycoplasma pneumoniae across 115 growth conditions .
we identify three qualitatively different levels of co - expression corresponding to distinct relative orientations and intergenic properties of adjacent genes .
we reveal that the degree of co - expression between co - directional adjacent operons , and more generally between genes , is tightly related to their capacity to be transcribed en bloc into the same mrna .
we further show that this genome - wide pervasive transcription of adjacent genes and operons is specifically repressed by dna regions preferentially bound by rna polymerases , by intrinsic terminators , and by large intergenic distances . taken together
, our findings suggest that the basal coordination of transcription is mediated by the physical entities and mechanical properties of the transcription process itself , and that operon - like behaviors may strongly vary from condition to condition . |
the face is widely regarded as a symbol of self and a smile as a window into one 's personality . in this , the teeth play important roles in the maintenance of a positive self - image , and loss of teeth may result in significant disabilities that can profoundly disrupt social activities .
tooth loss may be traumatic and upsetting , and it is regarded as a serious life event that requires significant social and psychological readjustment .
the nonacceptance of edentulousness and the individual 's feelings about dentures , which have been the traditional way of replacing missing teeth , are important for the acceptance of new dentures .
traditionally , missing teeth are replaced by removable partial dentures , fixed partial dentures ( bridges ) and complete dentures in cases of complete edentulousness . the need
to replace lost teeth with a near - natural successor has encouraged rapid research and advancement in the field of dental implants , especially in advanced economies .
dental implant is an artificial root that is surgically inserted into the jawbone to support a single tooth replacement ( crown ) , fixed partial or complete denture or maxillofacial prosthesis .
it is an ideal option for people with good oral health who have lost a tooth or teeth due to injuries , periodontal diseases , failure of endodontics , etc .
it is also used for the treatment of edentulousness and is associated with improved denture retention , stability and functional efficiency and , thus , improving the quality of life of the patient .
currently , dental implants are widely accepted as a prosthetic treatment of completely or partially edentulous patients , and studies have shown significant improvement in patients attitudes toward their dental health after treatment with implant prostheses .
unfortunately , the same can not be said of developing countries with poorer access to dental care , where different authors have reported wide variability in the acceptance of this newer option of tooth replacement . in our setting , a resource - challenged environment
, conservative dentistry is relatively young and there is dearth of information regarding patients knowledge of tooth replacement as a whole and the use of implant - retained prostheses as an option of replacement of missing tooth . with recent improvement in socioeconomic infrastructures in this environment and expansion of the middle class , access to oral health care
hence , in order to identify goals in the promotion of oral health and improvement of the overall quality of life of patients with tooth loss , the study was conducted to evaluate the knowledge of patients about tooth replacement as a whole and assess their awareness of implant - retained prosthesis as an option for tooth replacement .
a descriptive cross - sectional study of patients attending the dental centre of the university college hospital , ibadan , nigeria was conducted over a period of 6 months . the university college hospital , ibadan , is a major referral tertiary hospital in the south - western part of the country . in the conduct of this survey ,
the guidelines of ethical consideration were strictly adhered to and participants filled the questionnaire after signing informed consent .
information was obtained with the use of structured , self - administered questionnaires given to patients aged 18 years or older , who consented to participate in the study at the dental clinics during the study period between september 2012 and february 2013 .
data collected included the sociodemographic characteristics of the study participants , knowledge about implant - retained tooth replacement as an option of replacement , the cost , source of information and knowledge about other options of tooth replacement as a whole .
tests of association between variables were conducted using chi square statistics , with the level of statistical significance set at p < 0.05 .
a total of 220 participants took part in the study and 199 ( 90.5% ) properly filled questionnaires were analyzed .
the ages of the study participants ranged from 18 to 84 years , with a mean of 37.6 ( 16.5 ) years and a male to female ratio of 1:1.1 .
one hundred and two participants were married , 86 were single , 10 were widowed and one was divorced
. majority of the participants ( 141 , 70.9% ) had tertiary education , while only eight ( 4.0% ) had no formal education [ table 1 ] .
demographic characteristics the majority of the participants ( 184 , 92.5% ) were aware of the possibility of replacing lost teeth . a total of 102 ( 51.3% ) participants knew of different ways to replace missing teeth , while 94 ( 47.2% ) had no idea of such methods .
the awareness of the different possible options of tooth replacement as perceived by the participants is shown in table 2 .
a significantly higher proportion of those who were aware of the possibility of replacing missing teeth knew that removable partial dentures were an option ( p < 0.000 ) .
also , a significantly higher proportion of those that were aware of the possibility of replacement did not know about implant as a replacement option for missing teeth compared with removable partial dentures ( p < 0.01 ) [ table 3 ] .
knowledge on ways of replacing missing teeth awareness of tooth replacement and knowledge about options of replacement one hundred and forty ( 71.1% ) participants had not heard of dental implant while only 57 ( 28.9% ) of them had heard about it as an option for replacing missing teeth . of those who had heard about this option
, 22 ( 38.6% ) believed that they had received reasonable amount of information on the option , 26 ( 45.6% ) perceived that the information received had been inadequate and nine ( 15.8% ) were unsure of the quantity and quality of information they had on it .
only 21 of those participants who have heard about implant and claimed it was well explained could explain what they understood by dental implants , of whom 15 ( 71.4% ) gave the correct information about the implant - retained dental prosthesis . the majority ( 68% ) of the participants
got informed about implant - retained prosthesis through the dentist , 23% got it through the media while 6% of the participants got to know through the internet [ figure 1 ] .
source of information on dental implant while 45 ( 22.6% ) participants would like to have implant - retained prosthesis as an option for replacement of missing teeth , 24 ( 13.3% ) said they would not want it , while the majority , 112 ( 61.9% ) , were not sure if they would like to have it or not .
of the 78 participants who gave reasons why they would not like to have their missing tooth / teeth replaced with dental implant - retained prosthesis , majority ( 46.2% ) claimed that they would not consider it because they do not have any idea about what it is , while seven ( 9.0% ) thought it was expensive [ figure 2 ] .
reasons for not considering dental implant one hundred and fifty - four ( 84.6% ) of the participants would like to be educated on dental implant as an option for replacement of missing teeth , while just nine ( 4.9% ) did not want to know about it and 19 ( 10.4% ) cared less about the education on dental implant .
one hundred and ten ( 61.8% ) of the participants claimed they would consider implant - retained prosthesis as an option for tooth / teeth replacement if they had good knowledge about it .
while 15 ( 8.4% ) would not consider it , 53 ( 29.8% ) of the participants are not sure of considering it even if it is well explained to them .
a statistically significant relationship was found when education on implant and if participant would consider implant is well explained ( p = 0.000 ) . among the 57 participants who have heard about implant , only 21 ( 36.8% ) have the knowledge about the cost implication , of which 15 ( 71.4% ) have the right information on cost implication and the remaining six ( 28.6% ) have wrong information on the cost .
the study also showed a statistically significant relationship between those who have heard about implant - retained prosthesis and their knowledge of cost implication ( p = 0.000 ) .
the ages of the study participants ranged from 18 to 84 years , with a mean of 37.6 ( 16.5 ) years and a male to female ratio of 1:1.1 .
one hundred and two participants were married , 86 were single , 10 were widowed and one was divorced
. majority of the participants ( 141 , 70.9% ) had tertiary education , while only eight ( 4.0% ) had no formal education [ table 1 ]
the majority of the participants ( 184 , 92.5% ) were aware of the possibility of replacing lost teeth .
a total of 102 ( 51.3% ) participants knew of different ways to replace missing teeth , while 94 ( 47.2% ) had no idea of such methods .
the awareness of the different possible options of tooth replacement as perceived by the participants is shown in table 2 .
a significantly higher proportion of those who were aware of the possibility of replacing missing teeth knew that removable partial dentures were an option ( p < 0.000 ) . also , a significantly higher proportion of those that were aware of the possibility of replacement did not know about implant as a replacement option for missing teeth compared with removable partial dentures ( p < 0.01 ) [ table 3 ] .
knowledge on ways of replacing missing teeth awareness of tooth replacement and knowledge about options of replacement
one hundred and forty ( 71.1% ) participants had not heard of dental implant while only 57 ( 28.9% ) of them had heard about it as an option for replacing missing teeth . of those who had heard about this option , 22 ( 38.6% ) believed that they had received reasonable amount of information on the option , 26 ( 45.6% ) perceived that the information received had been inadequate and nine ( 15.8% ) were unsure of the quantity and quality of information they had on it .
only 21 of those participants who have heard about implant and claimed it was well explained could explain what they understood by dental implants , of whom 15 ( 71.4% ) gave the correct information about the implant - retained dental prosthesis . the majority ( 68% ) of the participants got informed about implant - retained prosthesis through the dentist , 23% got it through the media while 6% of the participants got to know through the internet [ figure 1 ] .
source of information on dental implant while 45 ( 22.6% ) participants would like to have implant - retained prosthesis as an option for replacement of missing teeth , 24 ( 13.3% ) said they would not want it , while the majority , 112 ( 61.9% ) , were not sure if they would like to have it or not .
of the 78 participants who gave reasons why they would not like to have their missing tooth / teeth replaced with dental implant - retained prosthesis , majority ( 46.2% ) claimed that they would not consider it because they do not have any idea about what it is , while seven ( 9.0% ) thought it was expensive [ figure 2 ] .
reasons for not considering dental implant one hundred and fifty - four ( 84.6% ) of the participants would like to be educated on dental implant as an option for replacement of missing teeth , while just nine ( 4.9% ) did not want to know about it and 19 ( 10.4% ) cared less about the education on dental implant .
one hundred and ten ( 61.8% ) of the participants claimed they would consider implant - retained prosthesis as an option for tooth / teeth replacement if they had good knowledge about it . while 15 ( 8.4% ) would not consider it , 53 ( 29.8% ) of the participants are not sure of considering it even if it is well explained to them .
a statistically significant relationship was found when education on implant and if participant would consider implant is well explained ( p = 0.000 ) .
among the 57 participants who have heard about implant , only 21 ( 36.8% ) have the knowledge about the cost implication , of which 15 ( 71.4% ) have the right information on cost implication and the remaining six ( 28.6% ) have wrong information on the cost .
the study also showed a statistically significant relationship between those who have heard about implant - retained prosthesis and their knowledge of cost implication ( p = 0.000 ) .
the present study showed a high rate of awareness of replacement of missing teeth in this environment , with 92.5% of the participants being aware that missing teeth can be replaced .
furthermore , the option of removable partial dentures for teeth replacement was the most recognized by the participants .
this may be because removable partial dentures have been the most widely available and traditional way of replacement in this country ; it is relatively cheap and affordable for most socioeconomic classes in resource - poor settings and has been found to be satisfactory to patients in terms of appearance , retention and stability .
while 28.9% of the participants have heard about dental implant , only 22.6% of them were aware that it is an option of replacement of missing teeth .
there was a significant relationship ( p = 0.01 ) when those who have heard of dental implant were compared with those who were aware of possible replacement of missing teeth , with a higher proportion of those who have knowledge of replacement of missing teeth showing low knowledge about dental implant as an option of tooth replacement .
the level of awareness of implant as an option for replacement of missing teeth recorded in this study is higher than what was recorded by kumar et al .
, who had an awareness of 4.83% . this may be due to the fact that the present study was conducted in a tertiary hospital situated in an urban environment , with the majority of the participants ( 84.8% ) having at least secondary school education as against the population studied by kumar et al .
, who were people of lower socioeconomic and education levels of the area ( khammam , andhra pradesh in india ) and were also unskilled workers educated only to the secondary level or lower .
however , the level of awareness recorded in this study is lower when compared with that reported by al johanny et al .
berge and best , who recorded the level of awareness in their different studies to be from 66.4% to as high as 77% .
this may be due to the relatively low level of practice of implant dentistry in this environment and sensitization of patients by dentists toward the use of implant - retained prosthesis as option of tooth replacement .
dentists were the major source of information ( 68% ) to those who have heard about dental implants followed by the media , internet and other sources such as friends .
this is similar to the study by kumar et al . , which recorded the dentist as being the main source of information ( 38.25% ) .
however , this result is contrary to some other studies that have media and internet as the major source of information on dental implant .
this could be due to the fact that majority of the people in this environment depend on professionals such as dentists , on their source of information and the fact that accessibility to the internet is relatively expensive and still limited to those of middle and high socioeconomic classes .
however , of the 21 participants who had this option of tooth replacement explained to them by dentists , 15 ( 71.4% ) had the right information while six ( 28.6% ) were either wrongly informed or did not understand what was explained about this option of tooth replacement .
although this misconception about dental implant has been recorded in a study , it was more through information from the internet and media . of the majority of the participants who claimed that they were not sure if they would like to have dental implants as an option to replace missing teeth , the major reason given by 46.2% of these participants was the fact that they do not have any idea about it , while 39.7% had no particular reason .
high cost of implant as perceived by seven ( 9% ) of the participants was another reason given for not considering dental implants .
this further buttresses the need for adequate education , as was confirmed in this study by the response of the participants to education on dental implants , with majority of the participants claiming that they would appreciate it if they could be better informed about the advantages and disadvantages that might make them consider it as a better option .
, satpathy et al . and al johany , who also recorded a higher percentage of their participants requesting to have more knowledge on dental implants .
a statistically significant relationship was also found when education on implant and consideration of dental implant for use by participant was compared ( p = 0.000 ) .
thus , if the procedure of implant - retained prosthesis is well explained to patients , they will consider having it as an option of replacing missing teeth .
there was misconception and low level of knowledge about the cost implication of dental implants among the 57 participants who have heard about it , with just 21 ( 36.8% ) claiming that they know the cost , of whom 15 ( 71.4% ) were rightly informed .
this low awareness and misconception about the cost is similar to that reported by rustemeyer and bremerich and tepper .
there was also a significant relationship about the knowledge of cost implication when those who have heard about the implant prosthesis were compared with their knowledge of the cost implication ( p = 0.000 ) .
thus , there is need for patients adequate information vis a vis the cost , advantages , disadvantages and possible complications of dental implant prostheses as a better option for teeth replacement .
the study has shown that there is a low level of awareness about dental implant in this environment even though people are aware of the possibility of tooth replacement .
adequate awareness and rich , right and detailed information are the necessary tools that project dental implant - retained prostheses as the best option for the tooth and lost maxillofacial tissue replacement .
the dentist as a professional has the major role to play in this regard , and this can be fulfilled by implementing patient education programmes and counseling centers on dental implant use , advantages and possible complications in order to prepare a patient 's mind . | aim : a survey was set out to evaluate the knowledge of patients about tooth replacement as a whole , and assess their awareness of implant - retained prosthesis as an option of tooth replacement.materials and methods : information on sociodemographic characteristics , knowledge about implant - retained tooth as an option for missing tooth replacement , cost implication , source of information and knowledge about other options of tooth replacement were obtained from patients attending the dental clinics of the university college hospital , ibadan , using structured self - administered questionnaires .
data were analyzed using spss version 20.result:a total of 220 patients aged 18 - 84 years with a mean age of 37.6 ( 16.5 ) years participated in the study , with a male to female ratio of 1:1.1 .
the majority ( 92.5% ) knew that missing teeth can be replaced , while a significantly lower proportion ( 28.9% ) knew about dental implants as an option ( p < 0.01 ) .
dentists were the major source of information on dental implants ( 68% ) . only 21 ( 36.8% ) of those who had heard about dental implant had knowledge about the cost ( p < 0.000).conclusion : a low level of awareness about dental implant as tooth replacement option exist in this environment , although most of the study participants were aware that missing teeth can be replaced . |
cronkhite - canada syndrome ( ccs ) was first described in 1955 by cronkhite and canada .
the main symptoms of ccs are diarrhea , weight loss , abdominal pain , and other gastrointestinal complications , such as protein - losing enteropathy and malnutrition with diffuse polyposis in the digestive tract .
ccs is a rare non - genetic disease of unknown cause characterized by alopecia , atrophic fingernails , and skin hyperpigmentation . in the past
, ccs was regarded clinically as a malignancy because of its poor prognosis , but recent advances such as high - calorie infusion and steroid therapy have achieved long - term survival in some cases .
ccs was considered a benign condition , however , there are several case reports of ccs associated with gastrointestinal carcinoma [ 2 , 3 , 4 ] and , since the etiology of this syndrome remains unknown , many cases are refractory to treatment and there are still fatalities . therefore , new therapies are urgently needed .
our patient initially presented with diarrhea , and sessile polyposis was revealed in the colon , intestine and stomach by colonoscopy and upper gastrointestinal endoscopy .
after successful eradication of h. pylori , she underwent ileostomy closure and complete polyposis resolution has since been observed .
we describe this rare case with complete regression of ccs lesions and consider novel treatments for ccs .
a 52-year - old woman , previously in good health without symptoms , developed diarrhea with lower abdominal pain during bowel movements and hematochezia , which continued for more than 2 weeks , after ingesting oysters and goat meat . at our hospital , colonoscopy demonstrated diffuse edema and a markedly erythematous elevated lesion from the end of the ileum to the rectum ( fig .
after hospitalization , infectious enteritis was suspected , but stool culture showed no obvious pathogens and there were no parasites in stool specimens .
upper gastrointestinal endoscopy showed salmon roe - like multiple elevated lesions with marked erythema involving almost the entire stomach ( fig .
2b ) , similar to that in the colon , as well as similar mild polypoid lesions in the duodenum .
initial pathological findings showed non - specific chronic inflammatory cell infiltration , edema and thickening of the colonic mucosa and crypts with cystic dilatation in the intestine ( fig .
however , neither foveolar gland hyperplasia nor interstitial inflammatory cell infiltration was found in the stomach .
her symptoms persisted after starting 5-asa and she also showed alopecia and glossitis 1 month later . at the second colonoscopy ,
an elevated tumor 30 35 mm was observed in the sigmoid colon ( fig .
1b ) . since the biopsy specimen was consistent with group 5 , a well - differentiated adenocarcinoma , high anterior resection of the sigmoid colon and ileostomy were performed .
even when these treatments were stopped , the diarrhea , alopecia and glossitis continued to gradually improve .
detailed examination of the surrounding mucosa revealed cystic dilatation of glands and edema , infiltration of eosinophils with plasma cells and lymphocytes , and spread of lymphangitis .
ccs associated with sigmoid colon cancer ( 01 , pm n0 m0 ) with tubular adenoma was thus diagnosed ( fig .
colonoscopic observation via the ileostomy , after surgery , revealed amelioration of the edema and erythema in the ileum , ileocecal region and ascending colon . on colonoscopy 6 months later , the findings in the ileum had almost disappeared and erythematous polypoid lesions and edematous mucosa showed regression in the colon .
although spread of cystic crypts was observed in some portions of the intestine , colonic histology , i.e. mucosal thickening , showed improvement 1 year later .
gastric foveolar hyperplasia and edema disappeared , and mucosal thickness was normal in the stomach . on the other hand , on colonoscopy 2 years later , while polyps were no longer visible , erythema and petechiae persisted , reflecting diversion colitis ( fig .
the thickness of the colonic mucosa was histologically normal with no edema , but extended cystic crypts remained in portions of the intestine .
upper gastrointestinal endoscopy confirmed the absence of gastric polyps or other abnormal findings in the duodenum ( fig . 2c ) .
histopathological features of the stomach indicated normal thickness without mucosal edema , and chronic gastritis with positive h. pylori histology and serology .
after 4 months , the endoscopic and histological findings of gastritis had also improved and active inflammation had disappeared ( fig .
surgical closure of an ileal fistula was carried out and , after 4 months , upper gastrointestinal endoscopy and colonoscopy showed that not only the ccs lesions but also the diversion colitis findings had completely disappeared .
as the patient remained asymptomatic during observation , she has since been followed at our outpatient clinic without medication .
after 2 further years , colonoscopy and upper gastrointestinal endoscopy showed normal findings ( fig .
the number of ccs cases in asia , including japan , is reportedly high . although ccs has been recognized as a benign non - genetic disease
, it manifests clinically as protein - losing gastroenteropathy , and relapse or exacerbation is common despite temporary improvements with medical therapy , such as steroids .
ccs was formerly considered to have a poor prognosis and a high mortality rate due to the complication of carcinoma , and many patients present with malignancy .
although the characteristics of ccs polyps are considered to be non - neoplastic in nature , a relatively high frequency of gastrointestinal carcinoma in ccs patients has been reported [ 2 , 3 , 4 , 5 ] .
the course in our case was also complicated by early - onset carcinoma necessitating partial resection of the sigmoid colon and ileostomy .
subsequently , symptoms and endoscopic and histological findings showed amelioration , and the patient was followed without further intervention .
she underwent h. pylori eradication 2 years after surgery and the ileostomy was closed the following year , after h. pylori eradication .
three months after this closure , ccs lesions of the colon and stomach were no longer visible macroscopically or histologically .
, there are no prior reports of ccs cases such as our present patient undergoing surgical treatment and eradication of h. pylori .
steroids have commonly been administered for ccs and cases achieving temporary improvement or even long - term remission have been reported .
however , there were numerous recurrences and effectiveness , as compared to other treatments , has not been established . among reports supporting surgical treatment of ccs
therapeutic effects would presumably have been obtained if the site in the digestive tract , harboring polyposis and causing protein leakage , had been resected along with the carcinoma .
reported in their literature review that symptomatic remissions were observed in 2 of 6 cases who underwent subtotal gastrectomy alone .
in addition , symptomatic remissions were attributed to the operations alone in 3 of 7 patients receiving large bowel resection , among 55 ccs cases . in our case , only partial resection of the sigmoid colon was performed for tumor removal from the sigmoid colon and polyposis remained at other sites .
therefore , it is possible that changes in bacterial dynamics within the intestinal flora are related to the improvement achieved by ileostomy in our patient . furthermore , clear evidence implicating h. pylori infection in the pathogenesis of ccs is lacking .
recently reported that lesions such as diffuse polyposis showed progressive remission after h. pylori eradication using antibiotics in patients with ccs .
h. pylori usually inhabits the gastric mucosa , but this microorganism has reportedly been cultured from stool specimens of h. pylori carriers .
h. pylori might injure the intestinal mucosa when passing through the intestinal tract , and/or activation of lymphocytes in the gastric mucosa by h. pylori may induce an immune response against this microorganism in the intestinal mucosa . as with maltoma of the stomach ,
based on these reports and our evidence , we can not rule out the possibility that h. pylori infection is involved in the development or exacerbation of ccs polyps , but the underlying mechanism remains uncertain .
accumulation of such cases and further research are necessary to explain the pathogenesis of ccs and to devise novel treatments for this disease .
| the etiology of cronkhite - canada syndrome ( ccs ) remains unknown and many cases are refractory to treatment .
therefore , new therapies are urgently needed .
furthermore , a number of ccs cases with gastrointestinal carcinoma have been reported .
our patient had rapid onset of ccs and early development of colon carcinoma associated with adenomas .
high anterior resection of the sigmoid colon and ileostomy were performed , and her symptoms and endoscopic and histological findings improved .
helicobacter pylori eradication was carried out 2 years later , surgical closure of an ileal fistula the following year .
after 4 months , upper gastrointestinal endoscopy and colonoscopy showed that the ccs lesions had completely disappeared , and biopsies confirmed a normal stomach , duodenum , ileum and colon histologically .
the patient has maintained remission for 2 years .
the clinical course of this case , showing complete regression of ccs lesions following abdominal colectomy and h. pylori eradication , suggests the significance of h. pylori infection in the treatment of ccs . |
we used data from the 2006 brazos valley community health assessment ( bvha ) , which was developed by a collaboration of local and regional academic and community - based organizations in the brazos valley of central texas .
participants were recruited from adult community residents who resided in one of six rural and one urban county by a professional independent survey research firm that identified 9,940 valid telephone numbers through random digit dialing . of these telephone numbers ,
more than 2,500 adults ( 19.4% minority , 71% female , and 61% rural residents ) who resided in the seven counties returned the mailed survey ; the response rate was 73.8% ( 25 ) .
this study used data from 1,878 adult participants in the bvha who had complete responses for demographic characteristics , eating behaviors , and household food - related hardship ( experience of running out of food , without money to obtain more ) ( 26 , 27 ) ; 649 participants ( 25.7% ) were excluded due to missing data . there were no statistically significant differences between included and excluded participants with regards to demographic characteristics or rural residence .
the texas a&m university institutional review board approved the study protocol and all participants provided informed consent . demographic characteristics included age ( 1844 years , 4564 years , and 65 years ) , race / ethnicity ( non - hispanic white vs. all others ) , household income ( poverty : 100% fpl [ federal poverty level ] , low income : 101199% fpl , and above low income : 200% fpl ) , employment status ( employed full - time outside the home for wages vs. not employed full - time outside the home ) , marital status ( married vs. not married ) , 1 child under the age of 18 years living in the household , and body mass index ( bmi ) , which was calculated from self - reported height and weight ( kg / m ) .
the bmi was categorized as normal ( bmi < 25 kg / m ) , overweight ( bmi 2529.9 kg / m ) , and obese ( bmi 30 kg / m ) .
eating behaviors were selected based on prior community - based work in north carolina and included prevalence and consumption of ssbs , frequency of fast food meals , frequency of eating a regular breakfast meal , and daily intake of fruit and vegetables ( 25 , 28 , 29 ) .
ssb consumption was assessed with the following question : how many cans of regular soda ( not diet ) or glasses of sweet tea do you drink on an average day? six response categories included 0 , 1 , 2 , 3 , 4 , 5 , or 6 ; and more than 6 .
the prevalence of ssb consumption was defined as the proportion of adults who reported any consumption of ssb ( 1 can or glass per day ) .
based on a distribution of responses , a dichotomized variable for a high level of ssb consumption was defined as 3 cans or glasses per day versus <3 cans or glasses .
frequency of fast food meals was determined from the question : how many times a week do you eat fast food meals? the same six response categories were provided as above ; and a similar approach for a dichotomized variable for frequent fast food meal consumption was defined ( 3 times / week vs. <3 times / week )
. the following question was used to describe breakfast meals frequency : how many days a week do you eat a regular breakfast meal? from the six possible responses , a dichotomized breakfast meal variable was constructed as <3 days / week versus 3 days / week .
two questions from a validated , self - reported two - item screener were combined to describe fruit and vegetable intake : ( 1 ) how many servings of fruit do you usually eat each day ( a serving= cup of fruit or cup of fruit juice ) ? and ( 2 ) how many servings of vegetables do you usually eat each day ( a serving= cup of cooked or one cup raw vegetables ) ? ( 30 , 31 ) .
a three - category variable was constructed for total daily intake of fruit and vegetables : 02 servings , 34 servings , and 5 servings .
the first quantitative food depletion item in the household hunger dimension of the radimer - cornell measure of hunger and food insecurity was used to determine the presence of household food insecurity in the past 30 days ( 27 , 3235 ) .
respondents were asked to choose the frequency ( often true , sometimes true , or never true ) that the following occurred for their household in the past 30 days : the food that we bought did n't last and we did n't have enough money to buy more. responses of often true and sometimes true were combined to indicate food - related hardship ( often true or sometimes true ) versus no food - related hardship ( never true ) .
this measure describes the household experience of running out of food without money to obtain more ( 26 , 27 ) .
release 11 of stata statistical software was used for all statistical analyses ; p<0.05 was considered statistically significant .
descriptive statistics were estimated for demographic characteristics , eating behaviors , and food - related hardship .
the difference between rural and urban adults was assessed with contingency tables by using the statistic .
bivariate correlations between theoretically selected variables ( demographic characteristics , eating behaviors , and food - related hardship shown in table 1 ) and ssb intake were estimated .
correlations at p<0.10 were retained for inclusion in the logistic regression model that included rural and urban respondents ; excluded variables included sex , overweight , ages 4564 years , and employment status . using backward elimination of all variables with p>0.05 , a combined multivariable logistic regression model ( n=1,878 ) was estimated for high level of ssb consumption ( 3 cans / glasses per day vs. <3 cans / glasses ) . using the final model for the combined sample , separate multivariable logistic regression models were estimated for the 734 urban respondents and the 1,144 rural respondents .
difference in demographic characteristics , eating behaviors , and household food - related hardship between urban and rural adults ( n=1,878)a comparisons were performed using test . statistically significant after using bonferroni correction for multiple comparison ( bonferroni - corrected p=0.002 ) .
1 can or glass of regular soda or sweet tea on an average day , compared with < 1 on an average day .
3 cans or glasses of regular soda or sweet tea on an average day , compared with <3 on an average day .
we used data from the 2006 brazos valley community health assessment ( bvha ) , which was developed by a collaboration of local and regional academic and community - based organizations in the brazos valley of central texas .
participants were recruited from adult community residents who resided in one of six rural and one urban county by a professional independent survey research firm that identified 9,940 valid telephone numbers through random digit dialing . of these telephone numbers ,
more than 2,500 adults ( 19.4% minority , 71% female , and 61% rural residents ) who resided in the seven counties returned the mailed survey ; the response rate was 73.8% ( 25 ) .
this study used data from 1,878 adult participants in the bvha who had complete responses for demographic characteristics , eating behaviors , and household food - related hardship ( experience of running out of food , without money to obtain more ) ( 26 , 27 ) ; 649 participants ( 25.7% ) were excluded due to missing data . there were no statistically significant differences between included and excluded participants with regards to demographic characteristics or rural residence .
the texas a&m university institutional review board approved the study protocol and all participants provided informed consent .
demographic characteristics included age ( 1844 years , 4564 years , and 65 years ) , race / ethnicity ( non - hispanic white vs. all others ) , household income ( poverty : 100% fpl [ federal poverty level ] , low income : 101199% fpl , and above low income : 200% fpl ) , employment status ( employed full - time outside the home for wages vs. not employed full - time outside the home ) , marital status ( married vs. not married ) , 1 child under the age of 18 years living in the household , and body mass index ( bmi ) , which was calculated from self - reported height and weight ( kg / m ) .
the bmi was categorized as normal ( bmi < 25 kg / m ) , overweight ( bmi 2529.9 kg / m ) , and obese ( bmi 30 kg / m ) .
eating behaviors were selected based on prior community - based work in north carolina and included prevalence and consumption of ssbs , frequency of fast food meals , frequency of eating a regular breakfast meal , and daily intake of fruit and vegetables ( 25 , 28 , 29 ) .
ssb consumption was assessed with the following question : how many cans of regular soda ( not diet ) or glasses of sweet tea do you drink on an average day? six response categories included 0 , 1 , 2 , 3 , 4 , 5 , or 6 ; and more than 6 .
the prevalence of ssb consumption was defined as the proportion of adults who reported any consumption of ssb ( 1 can or glass per day ) .
based on a distribution of responses , a dichotomized variable for a high level of ssb consumption was defined as 3 cans or glasses per day versus <3 cans or glasses .
frequency of fast food meals was determined from the question : how many times a week do you eat fast food meals? the same six response categories were provided as above ; and a similar approach for a dichotomized variable for frequent fast food meal consumption was defined ( 3 times / week vs. <3 times / week ) .
the following question was used to describe breakfast meals frequency : how many days a week do you eat a regular breakfast meal? from the six possible responses , a dichotomized breakfast meal variable was constructed as <3 days / week versus 3 days / week . two questions from a validated
, self - reported two - item screener were combined to describe fruit and vegetable intake : ( 1 ) how many servings of fruit do you usually eat each day ( a serving= cup of fruit or cup of fruit juice ) ? and ( 2 ) how many servings of vegetables do you usually eat each day ( a serving= cup of cooked or one cup raw vegetables ) ? ( 30 , 31 ) .
a three - category variable was constructed for total daily intake of fruit and vegetables : 02 servings , 34 servings , and 5 servings .
the first quantitative food depletion item in the household hunger dimension of the radimer - cornell measure of hunger and food insecurity was used to determine the presence of household food insecurity in the past 30 days ( 27 , 3235 ) .
respondents were asked to choose the frequency ( often true , sometimes true , or never true ) that the following occurred for their household in the past 30 days : the food that we bought did n't last and we did n't have enough money to buy more. responses of often true and sometimes true were combined to indicate food - related hardship ( often true or sometimes true ) versus no food - related hardship ( never true ) .
this measure describes the household experience of running out of food without money to obtain more ( 26 , 27 ) .
demographic characteristics included age ( 1844 years , 4564 years , and 65 years ) , race / ethnicity ( non - hispanic white vs. all others ) , household income ( poverty : 100% fpl [ federal poverty level ] , low income : 101199% fpl , and above low income : 200% fpl ) , employment status ( employed full - time outside the home for wages vs. not employed full - time outside the home ) , marital status ( married vs. not married ) , 1 child under the age of 18 years living in the household , and body mass index ( bmi ) , which was calculated from self - reported height and weight ( kg / m ) .
the bmi was categorized as normal ( bmi < 25 kg / m ) , overweight ( bmi 2529.9 kg / m ) , and obese ( bmi 30 kg / m ) .
eating behaviors were selected based on prior community - based work in north carolina and included prevalence and consumption of ssbs , frequency of fast food meals , frequency of eating a regular breakfast meal , and daily intake of fruit and vegetables ( 25 , 28 , 29 ) .
ssb consumption was assessed with the following question : how many cans of regular soda ( not diet ) or glasses of sweet tea do you drink on an average day? six response categories included 0 , 1 , 2 , 3 , 4 , 5 , or 6 ; and more than 6 .
the prevalence of ssb consumption was defined as the proportion of adults who reported any consumption of ssb ( 1 can or glass per day ) .
based on a distribution of responses , a dichotomized variable for a high level of ssb consumption was defined as 3 cans or glasses per day versus <3 cans or glasses .
frequency of fast food meals was determined from the question : how many times a week do you eat fast food meals? the same six response categories were provided as above ; and a similar approach for a dichotomized variable for frequent fast food meal consumption was defined ( 3 times / week vs. <3 times / week ) .
the following question was used to describe breakfast meals frequency : how many days a week do you eat a regular breakfast meal? from the six possible responses , a dichotomized breakfast meal variable was constructed as <3 days / week versus 3 days / week . two questions from a validated , self - reported two - item screener were combined to describe fruit and vegetable intake : ( 1 ) how many servings of fruit do you usually eat each day ( a serving= cup of fruit or cup of fruit juice ) ? and ( 2 ) how many servings of vegetables do you usually eat each day ( a serving= cup of cooked or one cup raw vegetables ) ? ( 30 , 31 ) .
a three - category variable was constructed for total daily intake of fruit and vegetables : 02 servings , 34 servings , and 5 servings .
the first quantitative food depletion item in the household hunger dimension of the radimer - cornell measure of hunger and food insecurity was used to determine the presence of household food insecurity in the past 30 days ( 27 , 3235 ) .
respondents were asked to choose the frequency ( often true , sometimes true , or never true ) that the following occurred for their household in the past 30 days : the food that we bought did n't last and we did n't have enough money to buy more. responses of often true and sometimes true were combined to indicate food - related hardship ( often true or sometimes true ) versus no food - related hardship ( never true ) .
this measure describes the household experience of running out of food without money to obtain more ( 26 , 27 ) .
release 11 of stata statistical software was used for all statistical analyses ; p<0.05 was considered statistically significant .
descriptive statistics were estimated for demographic characteristics , eating behaviors , and food - related hardship .
the difference between rural and urban adults was assessed with contingency tables by using the statistic .
bivariate correlations between theoretically selected variables ( demographic characteristics , eating behaviors , and food - related hardship shown in table 1 ) and ssb intake were estimated .
correlations at p<0.10 were retained for inclusion in the logistic regression model that included rural and urban respondents ; excluded variables included sex , overweight , ages 4564 years , and employment status . using backward elimination of all variables with p>0.05 , a combined multivariable logistic regression model ( n=1,878 ) was estimated for high level of ssb consumption ( 3 cans / glasses per day vs. <3 cans / glasses ) . using the final model for the combined sample , separate multivariable logistic regression models were estimated for the 734 urban respondents and the 1,144 rural respondents .
difference in demographic characteristics , eating behaviors , and household food - related hardship between urban and rural adults ( n=1,878)a comparisons were performed using test . statistically significant after using bonferroni correction for multiple comparison ( bonferroni - corrected p=0.002 ) .
servings of fruit and vegetables usually eaten each day . 1 can or glass of regular soda or sweet tea on an average day , compared with < 1 on an average day .
3 cans or glasses of regular soda or sweet tea on an average day , compared with <3 on an average day .
rural respondents were older than urban counterparts ; a larger proportion were women , reported a household income 101199% fpl , and were obese ; and a smaller proportion were employed full - time outside the home for wages or had at least one child under the age of 18 years living in the household . compared with urban respondents , the prevalence and high level of ssb consumption ( 3 cans or glasses of ssb / day ) was greater among rural adults .
a greater proportion of rural adults ate a regular breakfast meal less than three times a week and consumed fewer servings of fruit and vegetables . on the other hand
, a larger proportion of urban adults ate fast food meals at least three times a week . finally , a larger proportion of rural adults reported household food - related hardship than urban counterparts ( 23.7% vs. 17.2% ) .
several differences between urban and rural adults remained significant after correcting for multiple comparisons with a bonferroni - corrected level of statistical significance .
several demographic variables were not correlated with ssb consumption ; namely sex , overweight status ( bmi 2529.9 kg / m ) , age category of participants 4564 years , and employment status .
although statistically significant , the strength of individual correlations was weak ( r 0.15 )
. age category of participants 1844 years ( r=0.11 , p<0.001 ) , minority status ( r=0.10 , p<0.001 ) , poverty - level household income ( r=0.15 , p<0.001 ) , presence of 1 child in the household ( r=0.14 , p<0.001 ) , and obesity ( r=0.07 , p=0.005 ) were positively correlated with ssb consumption ; older age category ( 65 years ) was negatively correlated . among the variables for eating behaviors , frequency of fast food meals ( r=0.09 , p<0.001 ) , low fruit and vegetable intake ( r=0.15 , p<0.001 ) , and consuming <3 breakfast meals / week ( r=0.17 , p<0.001 ) were positively correlated with ssb consumption ; high fruit and vegetable intake of 5 servings / day was negatively correlated with ssb consumption ( r=0.12 , p<0.001 ) .
food - related hardship was positively associated with ssb consumption ( r=0.21 , p<0.001 ) .
minority status ( p=0.64 ) , employment status ( p=0.57 ) , age ( p=0.19 ) , and obesity ( p=0.17 ) were sequentially removed from the final model for the combined rural and urban sample , which adjusted for demographic characteristics , eating behavior , and household food - related hardship .
independent of demographic characteristics , eating behaviors , and food - related hardship , rural residence was associated with greater odds for reporting a high level consumption of ssbs ( or 1.8 ; 95% ci 1.3 , 2.4 ; p<0.001 ) than urban residence . among all adults having a poverty - level household income ( or 2.2 ; 95% ci 1.6 , 3.1 ) , children in the household ( 1.8 ; 95% ci 1.3 , 2.3 ) , frequent consumption of fast - food meals ( 1.6 ; 95% ci 1.2 , 2.2 ) , infrequent breakfast meals ( 1.7 ; 95% ci 1.3 , 2.3 ) , low fruit and vegetable intake ( or 2.1 ; 95% ci 1.4 , 3.3 ) , and food - related hardship ( or 1.9 ; 95% ci 1.4 , 2.6 )
table 2 shows the results from the multivariable regression model for rural adults . among rural adults , a higher level of ssb consumption
was associated with greater odds for respondents with poverty - level household income , presence of child in the household , frequent consumption of fast - food meals , infrequent consumption of regular breakfast , low fruit and vegetable intake , and food - related hardship . among urban adults ( table 3 ) ,
one eating behavior ( infrequent consumption of a regular breakfast meal ) , household food - related hardship , and one demographic characteristic ( children in the home ) were associated with ssb consumption .
interestingly , frequency of fast - food meals and low fruit and vegetable intake were not associated with a high level of ssb consumption among urban adults .
odds ratios and 95% ci from multiple variable logistic regression models correlating demographic characteristics , eating behaviors , and household food - related hardship with consumption of sugar - sweetened beverages among 1,144 rural adultsa dependent variable is consumption of 3 cans / glasses of regular soda or sweet tea on an average day compared with <3 cans / glasses .
1 child under 18 years living in the household with the adult respondent compared with no children .
eat a regular breakfast meal <3 days a week compared with 3 days a week . in the last month , food bought did n't last and there was not enough money to buy more compared with food did last .
odds ratios and 95% ci from multiple variable logistic regression models correlating demographic characteristics , eating behaviors , and household food - related hardship with consumption of sugar - sweetened beverages among 734 urban adultsa
dependent variable is consumption of 3 cans / glasses of regular soda or sweet tea on an average day compared with <3 cans / glasses .
1 child under 18 years living in the household with the adult respondent compared with no children .
eat a regular breakfast meal <3 days a week compared with 3 days a week . in the last month , food bought did n't last and there was not enough money to buy more compared with food did last .
although research findings suggest a link between consumption of ssbs and health outcomes ( 1 , 2 , 12 ) , there are few studies that have examined the influence of less - healthy eating behaviors and food - related hardship on the consumption of high levels of ssb , especially among rural adults .
this is critical considering the dramatic increase in prevalence of overweight and obesity ( 36 , 37 ) , ssb consumption ( 3 , 9 , 3840 ) , frequency of fast - food meal consumption ( 41 ) , and nutrition and health disparities associated with rural residence ( 25 , 4246 ) .
however , studies of ssb consumption rarely have considered eating behaviors and adequacy of household food supplies as contributing factors .
findings from this study of 1,878 rural and urban adults extend our understanding of the influence of less - healthy eating behaviors and household food - related hardship on higher levels of ssb consumption .
first , the prevalence and high level of consumption of ssb were significantly greater among rural adults compared with urban counterparts .
second , a high level of ssb consumption was associated with less - healthy eating behaviors , especially among rural adults . to our knowledge , this is apparently the first study that simultaneously evaluated the association of multiple eating behaviors and household food - related hardship among a large sample of rural adults .
unlike primarily urban studies that used a single definition of ssb consumption such as once or more a week ( 17 ) , one 12-ounce serving of sugar - sweetened soda per day ( 6 ) , 1 ssb / day ( 47 ) , and > 1 bottle / day ( 48 ) , this study considered prevalence ( 1 can or glass of ssb / day ) and a high level of ssb consumption ( 3 cans or glasses of ssb / day ) . more than 52% of rural adults , compared with 43.7% of urban adults , consumed at least one ssb per day .
this appears to be higher than a similar size study of rural adults ( n=1,817 ) in wyoming , montana , and idaho that defined ssb consumption as less than once / week versus once or more per week ( 17 ) or the large , primarily urban nurses health study ii that found that 9.5% of the sample consumed 1 ssb / day ( 47 ) .
compared with previous studies of ssb consumption , our finding that rural adults consumed higher levels of ssb than urban adults is apparently new .
one possible explanation may be that previous studies did not attempt to examine high levels of ssb consumption ; but chose lower levels of consumption , such as at least one ssb per day or week ( 6 , 17 , 47 , 48 ) .
another explanation may be that rural residents have greater access to convenience and non - traditional food stores and fast - food opportunities where ssb are more available and affordable ( 43 , 45 , 4951 ) .
preference and greater household availability for ssb such as regular soft drinks or sugar - sweet tea , which has been identified through household food inventories , may provide another explanation for high levels of ssb consumption ( 52 , 53 ) .
in addition to consumption of ssb , three additional less - healthy eating behaviors that are associated with poor diet quality were examined ; namely , infrequent breakfast meals ( 28 , 54 , 55 ) , frequent consumption of fast - food meals ( 56 ) , and fewer portions of fruit and vegetables ( 57 , 58 ) .
rural adults compared less favorably with urban adults in two of these three eating behaviors .
a greater proportion of rural adults infrequently consumed a regular breakfast meal and ate less than three daily servings of fruit and vegetables .
lower fruit and vegetable intake among rural adults may be the result of limited access to food stores that market fruit and vegetables store availability and transportation infrastructure ( 46 , 59 , 60 ) . in the united states , there has been an overall decline in breakfast consumption ( 61 ) .
one explanation for less frequent breakfast meal consumption among rural adults may be that rural adults travel a greater distance in the morning to work and do not have the time for a regular breakfast . in both urban and rural areas , there are increased opportunities for fast food through traditional fast - food restaurants and marketing of fast food through convenience and other retail stores , often referred to as
an explanation for greater utilization of fast - food meals by urban adults may be greater accessibility and availability .
inadequate household food supplies or household food - related hardship are known to influence food choice and dietary intake ( 46 , 51 , 63 ) .
we identified great nutritional disparity between rural and urban adults , which has been absent from the literature .
more than 23% of rural adults compared with 17.2% of urban adults reported that in the past 30 days purchased food did not last and there was no money to buy more , which is supported by secondary analysis of national surveys ( 64 ) .
one explanation for the higher prevalence of both household food - related hardship and ssb consumption for rural adults may be related to the coping strategies food - insecure individuals employ to mitigate the consequences of food - related hardship ( 6568 ) such as consuming inexpensive and inflationary - resistant energy - dense foods ( 69 ) .
findings from multiple variable regression models confirmed geographic differences and similarities in the association of demographic characteristics , eating behaviors , and food - related hardship with high levels of ssb consumption .
although poverty - level household income increased the odds for ssb consumption among rural adults and not urban adults , in both geographic groups the presence of a child in the household was associated with a high level of ssb consumption .
all three eating behaviors frequent fast - food meals , infrequent breakfast , and low intakes of fruit and vegetables were associated with ssb consumption among rural adults , but only infrequent consumption was significant among urban residents .
food - related hardship was associated with ssb consumption among both rural and urban adults ; the effect size was greater among the urban sample .
thus , multiple less - healthy eating behaviors have a greater association with ssb consumption among rural adults than among urban adults .
interestingly , two less - healthy eating behaviors were not independently associated with ssb consumption in our urban subsample .
a prior rural study found an increased likelihood of overweight or obesity associated with greater frequency of ssb and fast food ( 17 ) .
thus , it is critically important to understand individual and household contextual influences on high levels of ssb consumption .
our findings revealed linkages among multiple less - healthy eating behaviors , which enhance results from a similar study of rural adults ( 25 ) .
adults , especially rural adults who frequently ate fast - food meals , infrequently consumed a breakfast meal , or had fewer daily servings of fruit and vegetables were also more likely to consume high levels of ssb .
just as healthier food patterns are associated with healthier beverage patterns ( 70 ) , the present study shows that consumption of ssb appears to be closely linked to less - healthy eating patterns ( 71 , 72 ) . as such , ssb consumption may serve as a marker of other less - healthy eating behaviors and overall poor nutrition .
first , the self - reported measure of ssb consumption may understate actual frequency and amount of ssb consumed on a usual day .
future work will include specific prompts for calorically sweetened beverages to include carbonated and non - carbonated soft drinks , fruit punch , fruit drinks , lemonade , sweetened powder drinks , bottled coffees , and coffees or teas with added sugar ( 73 ) .
third , data were not available on the type and amount of fast - food items consumed or the source of fast - food meals or ssb .
finally , measures on sedentary behaviors ( e.g. television viewing , computer use , video gaming ) should be included ( 74 ) . despite these limitations ,
this study advances our knowledge about less - healthy eating behaviors and household food - related hardship .
results from this study provide impetus for understanding interactions among multiple eating behaviors especially among economically and geographically disadvantaged adults . considering that americans are consuming more total calories per day , with much coming from ssb and fast food ( 75 ) , new strategies are needed for educating consumers not only about how to moderate their ssb intake , but also how to simultaneously disrupt the co - occurrence of undesirable eating behaviors ( e.g. fast - food consumption and skipping breakfast ) and promote healthful behaviors ( e.g. eating a regular breakfast and increasing fruit and vegetable intake ) .
challenges include the perception and observation that ssb are priced and promoted preferentially with meal deals at fast - food outlets and other venues that market fast - food items ( 49 , 76 ) , and that energy - dense foods are not only least expensive but also most resistant to inflation ( 69 ) .
given the economic disincentive for consumers to make healthier selections at fast - food restaurants and other venues ( 49 , 76 , 77 ) and the reality of low - cost accessible energy - dense foods , strategies must consider convenience and cost ( 69 ) especially for low - income and/or rural families ( 51 ) .
supported in part by the national institutes of health ( nih)/national center on minority health and health disparities ( # 5p20md002295 ) and by cooperative agreement # 1u48dp001924 from the centers for disease control and prevention , prevention research centers program through core research project and special interest project nutrition and obesity policy research and evaluation network .
the content is solely the responsibility of the authors and does not necessarily represent the official views of the nih and cdc . | backgroundsugar - sweetened beverage ( ssb ) consumption is associated with the increasing prevalence of overweight and obesity in the united states ; however , little is known about how less - healthy eating behaviors influence high levels of ssb consumption among rural adults.objectivewe assessed the frequency of ssb consumption among rural and urban adults , examined the correlates of frequent ssb consumption , and determined difference in correlates between rural and urban adults in a large region of texas.designa cross - sectional study using data on 1,878 adult participants ( urban=734 and rural=1,144 ) , who were recruited by random digit dialing to participate in the seven - county 2006 brazos valley community health assessment .
data included demographic characteristics , eating behaviors ( ssb consumption , frequency of fast - food meals , frequency of breakfast meals , and daily fruit and vegetable intake ) , and household food insecurity.resultsthe prevalence of any consumption of ssb and the prevalence of high consumption of ssb were significantly higher among rural adults compared with urban counterparts .
the multivariable logistic regression models indicated that a high level of ssb consumption ( 3 cans or glasses ssb / day ) was associated with demographic characteristics ( poverty - level income and children in the home ) , frequent consumption of fast - food meals , infrequent breakfast meals , low fruit and vegetable intake , and household food insecurity especially among rural adults.conclusionsthis study provides impetus for understanding associations among multiple eating behaviors , especially among economically and geographically disadvantaged adults .
new strategies are needed for educating consumers , not only about how to moderate their ssb intake , but also how to simultaneously disrupt the co - occurrence of undesirable eating and promote healthful eating . |
thunderclap headache attributed to reversible cerebral vasoconstriction ( tharcv ) is a clinical - radiological entity observed in a number of cases reported since 1978 .
the tharcv syndrome may include several of the patients experiencing thunderclap headache [ 28 ] , headache associated with sexual activity [ 2 , 812 ] , isolated benign cerebral vasculitis [ 13 , 14 ] , or migranous vasospasm or stroke [ 1517 ] .
it is widely accepted that the aura of a migraine crisis is associated with a vasoconstriction or vasospasm in the arterial territory of the aura - related cerebral cortex , which seems to be correlated with spreading depression , oligemia , or both [ 1821 ] . in rare situations
other conditions may induce much longer and intense cerebral artery narrowing [ 2244 ] , such as : subarachnoid hemorrhage ( sah ; largely the leading cause of cerebral vasospasm ) , drug abuse or sympathomimetic drug administration ( methamphetamine , heroin , ephedrine , and cocaine ) [ 2325 ] , intracranial surgical manipulation , head injury , herpes zoster , pheochromocytoma , eclampsia , ergot derivatives , angiographic contrast material , lyme disease , systemic lupus erythematosus ( sle ) , periarteritis nodosa , primary granulomatous angiitis of the central nervous system ( gacns ) , and other types of cerebral arteritis [ 3436 ] .
intracranial aneurysm rupture is a relative frequent event ( 10/100,000/year ) and it is very important to identify those high - intensity headaches of abrupt onset mimicking that of ruptured cerebral aneurysm .
primary thunderclap headache ( 4.6 ) and reversible cerebral vasoconstriction syndrome ( 6.7.3 ) are two clinical entities that may simulate a headache associated with subarachnoid hemorrhage .
the combination of both thunderclap headache and reversible cerebral vasoconstriction is being increasingly identified in a number of patients assisted in the emergency unit . according to the ihs 2004 classification
, the diagnostic criteria of primary thunderclap headache are the following : ( a ) severe headache fulfilling criteria b and c ; ( b ) both of the following characteristics : ( 1 ) sudden onset , reaching maximum intensity in < 1 min ; ( 2 ) lasting from 1 h to 10 days ; ( c ) irregular occurrence over subsequent weeks or months and ( d ) not attributed to another disorder .
the headache may recur within the first week after onset and a normal cerebrospinal fluid ( csf ) and normal brain imaging are required .
on the other hand , the ihs 2004 criteria for headache attributed to benign ( or reversible ) angiopathy of the central nervous system are : ( a ) diffuse , severe headache of abrupt or progressive onset , with or without focal neurological deficits and/or seizures and fulfilling criteria c and d ; ( b ) strings and beads appearance on angiography and subarachnoid hemorrhage ruled out by appropriate investigations ; ( c ) one or both of the following : ( 1 ) headache develops simultaneously with neurological deficits and/or seizures , ( 2 ) headache leads to angiography and discovery of strings and beads appearance , ( d ) headache ( and neurologic deficits , if present ) resolves spontaneously within 2 months . in the present article , we review the concept of this new headache syndrome ( i.e. , tharcv ) using the clinical - radiological findings of 25 patients . considering the management of patients with the tharcv syndrome , we shall discuss the diagnostic criteria and the differential diagnosis .
a medline search was performed using the english keywords : thunderclap headache , explosive headache , sudden headache , headache and vasospasm .
patients with a clear history of thunderclap headache and angiograph confirmation of cerebral vasoconstriction were selected to be studied as a group . in order to achieve a more uniform diagnosis of the tharcv syndrome we propose the diagnostic criteria listed in tables 1 and 2 . since we consider the tharcv as a secondary form of headache attributed to the reversible cerebral segmental vasoconstriction it is further divided into ( i ) idiopathic , or ( ii ) symptomatic headaches.table 1proposed classification of reversible cerebral vasoconstriction syndrome as idiopathic or symptomaticiidiopathiciisymptomaticiiaassociated with intracranial abnormalities other than cerebral vasoconstriction ( i.e. , aneurysm , vascular malformation , brain tumor , hemorrhage , venous thrombosis , etc.)iibassociated with systemic diseasesiicassociated with use of vasoactive drugsiidassociated with pregnancytable 2proposed diagnostic criteria for thunderclap headache attributed to idiopathic reversible cerebral vasoconstriction ( tharcv ) syndromea .
pregnancy , post - partum or eclampsiaobs . : the following characteristics when present strongly suggest tharcv syndrome : ( a ) female sex , ( b ) pulsating quality , ( c ) recurrence of headache , ( d ) precipitation or aggravation of the headache attack by physical or sexual activities , and ( e ) past history of migraine proposed classification of reversible cerebral vasoconstriction syndrome as idiopathic or symptomatic proposed diagnostic criteria for thunderclap headache attributed to idiopathic reversible cerebral vasoconstriction ( tharcv ) syndrome obs . : the following characteristics when present strongly suggest tharcv syndrome : ( a ) female sex , ( b ) pulsating quality , ( c ) recurrence of headache , ( d ) precipitation or aggravation of the headache attack by physical or sexual activities , and ( e ) past history of migraine the medical literature until 2003 was reviewed , and 25 reported cases of patients ( table 3 ) , including the one described by our own group , were selected according to the proposed diagnostic criteria for idiopathic tharcv syndrome ( table 2 ) .
although several other cases were published in the recent years , we believe those 25 individuals represent a sample of the increasing number of tharcv cases described in the literature.table 3clinical and radiological characteristic of 25 reported cases of patients with thunderclap headache attributed to idiopathic reversible cerebral vasoconstriction syndromepatient # ref .
# age / genderhistory of migraineheadache and other characteristicsdurationprecipitating factorrecurrencearteriography ( time after headache)arterial narrowingsideischemic strokeseizureothers1127 fnr bifrontal , generalized , throbbing , nauseas , vomitingseveral hourssneeze12 daysdiffuse , ac , pcbil.yesnopenicillin hypersensitivity21339 fyespulsatile , nausea7 days nrno11 daysmca , aca , stabil.yesyes 33730 fyesoccipital , nausea , vomiting , abdominal cramps , passage of several diarrheal stools , photophobia13 days nr33 weeksdiffuse , ac , pcbil.nr worsed with propranolol43851 f nrthrobbing occipital48 h nr19 daysdifuse?leftyesnodiabetic hypertensive , steroid therapy53825 myesthrobbing , forehead bilaterally extending down behind the eyes , photophobia , vomiting1 h to few dayssit ups , push - ups , skipping ropeseveral12 days parieto - occipital arteriesleftyesno
63948 fnosense of spinning , nausea , vomiting , blurred vision nr14 daysdiffuse , ac , pcbil.yesyes3 strokes , cortical cerebral artery biopsy normal73937 fyesleft - side , losing consciousness6 daysnono12 daysdiffuse , ac , pcbil.yesno 83919 m nrawoke with a frontal and bitemporal headache , nausea , vomiting , photophobiamore than 16 days worsened following angiogram15 daysdiffuse , ac , pcbil.tiano 91627 fyesthrobbing bitemporal , associated with acute onset of right hemiparesis and expressive dysphasia nr7few daysica at c1 vertebral levelleftyesnostroke 4 days after discharged with amitriptyline101230 m nrthrobbimg occipital ; generalized6 hsexual intercourse , exertion176 hdiffuse , ac , pcbil.nono 111055 fnrbifrontal , throbbing that later became a dull ache , vomitingfew hours to 10 daysorgasmseveral10 daysdiffuse , ac , pcbil.nonoimproved with clonidine124036 fyesthrobbing , occipito - nuchal , quichly extended over the entire head and face3 days nr18 daysva + aca + pcabil.nonoprophylactic therapy with propranolol134130 fyesabrupt exarcebation with nausea and vomiting , throbbing pain in the right temple>24 hget up to drank some lemonadeno nrcalcarine arteryrightyesno 141431 myes?associated with vomiting9 days nr1 nrsca , mcabil.nono 151437 f nrabrupt , severe headache few hours nr1 nraca , mcabil.noyesasthma .
hypothyroidism + levothyroxine164226 fyespounding , nausea , vomiting , that worsened by standing up associated with visual blurring and photophobiaseveral days nrnr few days?aca + mcabil.nono 17944 fyesexplosive , throbing , global3 hsexual intercourse1 nrdiffusebil.yesnofamiliar history of sex headache .
papilloedema with arteriolar narrowing18755 fyesnausea , vomiting , shooting pain in the right occipital , entire craniumfew hours to 2 daysstraining , bending4 nrdiffuse , ac , pcbil.yes?no 19762 fyesthrobbing , explosive , nausea , supraorbital110 daysexposure to cold , bending2>5 days aca + mcabil.nonoatrial fibrillation20760 fyesexcruciating , pulsatile , nausea , photophobia nr1 nrdiffuse , ac , pcbil.?yeslung carcinoma21738 m nracute exacerbation of a bifrontal throbbing headache , explosive arngiogram13 daysmca , acoma , pcabil.yesnoprophylactic therapy with propranolol .
c6 quadriplegia ( diving injury)22249 myesgeneralized , throbbing , nausea , photophobia , crushingat least 3 hswimming , snorkeling16 daysdiffuse , ac , pcbil.nono 23247 mnoholocephalicfew hours sexual intercourse17 daysdiffuse , ac , pcbil.nono 241138 fnopulsatile , vertex3 dayssexual intercourse1>6 days laterdiffuse , ac , pcbil.nonopast history of neurocysticercosis25844 fyesgeneralized , throbbing1015 minsexual intercourse , masturbation16 daysmcarightnonoposterior fossa arachnoid cystac anterior circulation , pc posterior circulation , mca middle cerebral artery , aca anterior cerebral artery , sta superficial temporal artery , ica internal carotid artery , va vertebral artery , pca posterior cerebral artery , sca superior cerebelar artery , pcoma posterior communicating artery , bil . bilateral clinical and radiological characteristic of 25 reported cases of patients with thunderclap headache attributed to idiopathic reversible cerebral vasoconstriction syndrome ac anterior circulation , pc posterior circulation , mca middle cerebral artery , aca anterior cerebral artery , sta superficial temporal artery , ica internal carotid artery , va vertebral artery , pca posterior cerebral artery , sca superior cerebelar artery , pcoma posterior communicating artery , bil . bilateral analysis of the data shown in the table 3 shows a profile of this particular headache subtype . in this series of patients , 72% were women , the mean age at the onset of first headache was 39.4 2.3 years , ranging from 19 to 62 years ( women , 2662 years of age , mean 41.4 2.7 , median 38.5 ; men , 1949 years of age , mean 34.1 4.2 , median 31.0 ) .
no significant difference in age was observed between women and men ( student t - test ; p = 0.1665 ) .
eight percent of the patients were 1625 years of age , 28% 2635 years of age , 32% 3645 years of age , 24% 4655 years of age , and 8% 5665 years of age .
the characteristics of the headache described , in addition to the sine qua non condition of being abrupt and severe at the onset , differed according to patient description .
the duration of the headache attack ranged from a short period of time , i.e. , 1015 min , to as much as 34 weeks ( table 4 ) . in approximately half of the cases
the headache persisted up to 3 days . in one - third of the patients the pain continued for a few hours ( less than 24 h ) .
other headache features were reported , such as throbbing , pulsatility , or pounding ( 68% ) , being accompanied by nausea ( 44% ) , vomiting ( 40% ) , and photophobia ( 24% ) .
the headache was described usually as generalized ( 32% ) and bilateral ; in three cases it was unilateral at least at the onset.table 4duration , location , characteristic , number of occurrence of the headache , and past - history of headache in the 25 patients reported with tharcv syndromen%duration < 24 h1040 24 h3 days520 4 days7 days312 > 7 days728location generalized832 unilateral312 frontal520 occipital416 fronto - temporal14 temporal14 vertex14characteristic abrupt onset25100 severe intensity25100 throbbing , pulsatile , or pounding1768 nausea1144 vomiting1040
photophobia624 explosive312 blurred vision28 dull ache14 excruciating14 crushing14 sense of spinning14 losing consciousness14number of recurrences none416 11560 214 314 414 714 > 728past history of headache15/2560 migraine1352 with aura416 without aura312 unclassified624 tension - type headache14 associated with ccd14 associated with pregnancy14ccd contraceptive drugsome of the patients presented recurrent headaches of different duration duration , location , characteristic , number of occurrence of the headache , and past - history of headache in the 25 patients reported with tharcv syndrome ccd contraceptive drug some of the patients presented recurrent headaches of different duration in 21 of the 25 patients ( 84% ) there was at least one recurrence or a sudden increase in headache intensity .
the time interval between the first headache and recurrent attacks varied from a few hours up to 3 years . in 15 of 21 patients , recurrence was observed within the first week , and in one patient five such recurrences took place within this short period of time . in seven patients ,
recurrence was observed on the first day after the onset of headache . in three it occurred from 8 to 30 days .
a past history of headache was present in 15 of the 25 reported cases ( table 4 ) , including migraine in 13 patients ( 52% ) , at least four of them with aura , one associated with hormonal contraceptives and another after headaches that had begun 8 years before during the first trimester of pregnancy .
precipitating factors were identified by 14 patients ( 56% ; table 5 ) , with sexual intercourse being the major precipitating factor , reported by six patients ( 24% ) . of the 25 patients with tharcv syndrome studied , 12 ( 48% ) developed focal neurological signs , transitory ischemic attack ( n = 1 ) , or ischemic stroke ( n = 11 , 44% ) , and two ( 8% ) of them manifested seizures .
stiffness of the neck was observed in one patient.table 5precipitating factors identified in 14 of the 25 reported cases of patients with tharcv syndromeprecipitating factorn%sexual intercourse624exertion520angiography28sneezing14exposure to cold14total number of patients14/2556activities defined as exertion were calisthenics ( i.e. , sit ups , push - ups , and skipping rope ) , getting up from bed , straining , bending , swimming , snorkeling , and chasing off and screaming at an attacking dog precipitating factors identified in 14 of the 25 reported cases of patients with tharcv syndrome activities defined as exertion were calisthenics ( i.e. , sit ups , push - ups , and skipping rope ) , getting up from bed , straining , bending , swimming , snorkeling , and chasing off and screaming at an attacking dog probably cerebral artery narrowing was present for several days after the onset of headache . in the different reports vasoconstriction was diagnosed by arteriography between two days and three weeks after the onset of headache in 18 of the reported cases . even though the cerebral vasoconstriction was confirmed by angiography in all 25 patients , in the other seven cases the interval between the headache and the observed
a control angiography , performed 324 weeks later , was normal . in seven other patients a second angiography performed 232 weeks later demonstrated that most of the previously identified focal narrowing had resolved . in one case ( patient # 8 in table 3 ) an arteriography done 16 days later still demonstrated diffuse arterial narrowing . in another patient ( # 9 in table 3 )
an arteriography was repeated 15 days later showing normalization of the previously stenotic left internal carotid , but disclosing another arterial narrowing in the right internal carotid .
any of the intracranial major arteries can be affected ; in one subject ( patient # 2 in table 3 ) the external carotid artery was examined , disclosing involvement of the superficial temporal artery in addition to the presence of intracranial artery narrowing .
no drastic abnormalities were observed in the csf of patient with tharcv syndrome , with the exception of an eventual increase in protein levels .
in this article we described the clinical and radiological features of 25 reported cases of patients with a neurological syndrome characterized by tharcv .
the management of patients with the tharcv syndrome can be subdivided into pathophysiology determination and diagnostic and therapeutic strategies .
the headache of the tharcv syndrome typically is of acute onset and increases abruptly in severity , reaching peak severity in less than 60 s ( thunderclap headache ) .
thus , we propose some minor changes in the thunderclap diagnostic criteria modified by dodick et al . : ( a ) time to reach peak severity up to 60 s ; ( b ) headache duration of 5 min or longer , since the explosive type of headache occurring during sexual activity is considered to be a thunderclap headache and may be of short duration . even though the international classification of headache disorders , 2nd edition ( ichd - ii ) criteria stipulate that the pain of primary thunderclap headache last from 1 h to 10 days ( code 4.6 ) .
we could subdivide the tharcv syndrome into idiopathic and symptomatic . the symptomatic tharcv syndrome should fulfill the a and b items of the proposed diagnostic criteria , and is categorized into four subgroups : ( iia ) associated with intracranial abnormality ( vascular malformation , aneurysm , subarachnoid hemorrhage , cns vasculitis or trauma ) ; ( iib ) associated with systemic disease ( e.g. , pheochromocytoma or systemic vasculitis ) ; ( iic ) associated with drug abuse and use of vasoactive drugs ( e.g. , ergotamine , cocaine , heroin , methamphetamine , ephedrine , tryptans , intravenous use of contrast substances ) ; or ( iid ) associated with pregnancy , post - partum or eclampsia . as exclusion criteria we consider the presence of systemic vasculitis or a central nervous system ( cns)-meningeal biopsy showing inflammatory features related to cns vasculitis . more specifically , during the management of a patient with a thunderclap headache , as a general rule a ct brain scan without contrast is required . if a diagnosis is not made by this ct , lumbar puncture and csf examination is mandatory during the acute phase to rule out any intracranial symptomatic lesion , particularly sah and its causes .
both the ct scan and csf are expected to be normal , with the exception of patients with stroke , cerebral edema , or intracranial hypertension , in which csf abnormality is the result of these complications .
conventional cerebral angiography should be avoided , since the contrast medium could enhance the vasospasm and this , in turn , would increase the chance of a stroke or even cause the deterioration of a previously critical neurological condition , particularly in migraineurs [ 16 , 47 ]
. the next step would be the request of a good - quality magnetic resonance ( mr ) angiography or angio - computed tomography ( ct ) of intracranial vessels as soon as possible , in such a way that any moderate to severe ( reversible ) vasoconstriction would be visualized .
the conventional arteriography should be performed later , 34 weeks after the last severe headache attack . during the arteriography a study of the external carotid artery and its branches
should be included in an attempt to find a segmental narrowing in a superficial artery .
patients with thunderclap headache in the absence of cerebral vasospasm have been reported [ 48 , 49 ] .
this probably is factual , but the possibility of a short - lived cerebral arterial spasm occurring only at the onset of or during headache still needs to be excluded .
discussed why delayed arteriography would miss a transient spasm / stenosis to confirm migrainous stroke , to justify the lack of abnormality found in angiograms of patients who developed stroke during a migraine attack .
hence , all the other possibilities that could provoke an intracranial arterial narrowing should be taken into account .
the lack of systemic features enhances the possibility of a given arterial luminal narrowing to be part of the more benign tharcv syndrome when compared with cns vasculitis , in which no resolution of the vascular disease is expected without adequate treatment .
although reversibility of intracranial arterial narrowing is anticipated in the tharcv syndrome it may take weeks to happen . even though the reversibility of the vasoconstriction is not important during the initial diagnostic criteria , the reversibility of the condition needs to be proved , as the syndrome is called reversible .
since this has not always been made in previous studies as well as in ihs 2004 classification , the confirmation of the reversibility of the vasoconstriction should be included in the final diagnostic criteria , thanks to the reliability of less expensive or invasive techniques ( e.g. , angio - rm or angio - ct ) . in the discussion of some of the particularities that should differentiate the tharcv syndrome from other identified causes of arterial narrowing
we may mention some features , particularly those concerning cns vasculitis , a rare inflammatory disease of vessel walls associated with many causes ( i.e. , infection , malignancy , ionizing radiation , drug abuse , and autoimmune disease ) .
since angiographic features in cerebral arteritis are not completely specific , the associated radiologic and clinical findings often are fundamental elements for a diagnosis .
narrowing of a vessel may be the result of spasm , edema , cellular infiltration , or proliferation of the vessel wall as well as compression by adjacent thickened meninges , exudates , or fibrosis .
cerebral arterial narrowing might be subdivided into four groups : ( 1 ) vasospasm ( contraction of arterial smooth muscle ) ; ( 2 ) thickening of the vessel wall ; ( 3 ) extrinsic compression ; and ( 4 ) developmental or congenital .
very probably the tharcv is a form of headache attributed to reversible cerebral vasoconstriction syndrome ( benign or reversible angiopathy of the cns , ichd - ii code 6.7.3 ) [ 39 , 45 ] . according to the ihs 2004 criteria for headache attributed to benign ( or reversible )
angiopathy of the central nervous system , a severe headache of progressive onset occurring during the clinical presentation of the syndrome is permitted . in the present article
furthermore , we do not agree with the ihs d criterion : headache ( and neurological deficits , if present ) resolves spontaneously within 2 months .
vasoactive substances may cause vasospasm , such as chemical substances ( ergotamine , amphetamine , cocaine ) , metabolite substances ( in eclampsia or pheochromocytoma ) , and toxins ( angiographic contrast material ) . in the group of autoimmune mediated diseases
we may mention primary gacns , sle , polyarteritis nodosa , giant cell arteritis , and sjgren syndrome .
the physician should also search for other conditions that are known to cause vasculitis , such as drug hypersensitivity , cancer , infection , rheumatoid arthritis , and inflammatory bowel disease .
noninfectious vasculitides can be classified on basis of the predominant type of vessel affected : large - vessel vasculitis ( giant - cell arteritis and takayasus arteritis ) , medium - sized - vessel vasculitis ( polyarteritis nodosa , kawasakis disease , and primary gacns vasculitis ) , and small - vessel vasculitis ( anca - associated small - vessel vasculitis , immune - complex small - vessel vasculitis , paraneoplastic small - vessel vasculitis , and inflammatory bowel disease vasculitis ) .
infectious arteritis includes bacterial , tuberculous , fungal , yeast , luetic ( meningovascular syphilis ) , cysticercosis , rickettsia , and viral disorders .
primary granulomatous angiitis of the cns is a process usually restricted to vessels of 0.5 mm diameter , an arteriopathy that frequently is not visualized by angiography .
the diagnosis of isolated angiitis of the cns should be taken into consideration if the patient is young , presenting focal neurological signs , seizures , or constant severe headache . without treatment (
i.e. , glucocorticoid ) the clinical course of the gacns , as may be expected , is often progressive . to establish a definitive diagnosis
a leptomeningeal biopsy is necessary [ 14 , 50 ] . to decide which patients are candidates for biopsy it does depend on the gravity of the neurological picture , the functional importance of the cerebral artery to be biopsied , and lack of response to corticoid therapy .
similar segmental narrowing was also reported in atypical cluster headache involving proximal portions of the middle and anterior cerebral arteries .
several cases of women with preeclampsia - eclampsia and a history of sudden , severe headache accompanied by cerebral artery narrowing have been reported previously [ 30 , 39 , 53 ] .
preeclampsia is a frequent disorder characterized by hypertension , abnormal peripheral edema , and proteinuria .
most of the patients experience neurological signs and symptoms , such as headache , visual changes , confusion , disturbances of consciousness , or seizures . such manifestations of the disease are identical to those of hypertensive encephalopathy .
clinical and radiographic signs of hypertensive encephalopathy probably are related to the consequences of an acute increase in systemic blood pressure , which , in turn , would affect the autoregulation of the cerebral vasculature .
call et al . reported four patients with reversible cerebral segmental vasoconstriction and concluded that their cases may represent a severe clustering
these authors identified 12 other cases reported in the literature with clinical and angiographic similarities to their four cases , six occurring in the postpartum period , five being idiopathic , and one associated with an unruptured aneurysm .
the cited authors also commented that the clinical feature of presentation resembles the rupture of an intracranial aneurysm , with a sudden , high - intensity headache associated with nausea , vomiting , and photophobia . in six cases biopsy or autopsy of one vasoconstricted artery disclosed no anatomic abnormalities . on the other hand
, the occurrence of vascular abnormality ( narrowing ) in young women and during the postpartum period , strongly suggests a hormonal influence exerted by sex steroids .
cerebral postpartum angiopathy was described by rascol et al . in four women with distal arterial occlusion .
evaluated the effect of preeclampsia on cerebral artery blood flow velocity and concluded that cerebral vasospasm of the smaller diameter vessels is a major component of preeclampsia .
they described the case of a 27-year - old woman , who 15 min postpartum complained of headache , nauseas , and epigastric pain and whose cerebral angiogram showed diffuse spasm .
reported the case of a 34-year - old woman that during delivery , 10 min after receiving an intravenous administration of methylergometrine , experienced violent headache , associated with vomiting and arterial blood hypertension .
the authors postulated that the ergot - derived drug caused vasoconstriction through noradrenergic and serotoninergic actions .
additionally , bromocriptine for lactation suppression in combination with a sympathomimetic agent caused hypertension , severe headache , seizures and cerebral vasospasm in a patient during the puerperium .
these facts may indicate that , during delivery or the postpartum period , patients are more susceptible to administration of vasoactive drugs and this , in turn , can precipitate arterial spasm .
sudden surges in blood pressure may trigger a cerebral vasospasm , as also observed after adrenergic stimulation by the use of cocaine , by pheochromocytoma , or eclampsia .
kaye and fainatat described the case of a cocaine addict , a 22-year - man , who developed right - sided headache associated with transient blurring of his vision .
four days later he presented weakness of the left side , and an arteriogram performed 5 days after the onset of the hemiparesis demonstrated the presence of narrowing of the supraclinoid portion of the right internal carotid and the occlusion of the proximal segment of the middle cerebral artery .
patients with pheochromocytoma usually experience bilateral paroxystic episodes of bifrontal headache , radiating to temporal regions or secondarily generalizing , described as pulsating , moderate to severe in intensity .
the above - mentioned features are similar to the headache described by patients with tharcv syndrome , which may suggest that sudden increases in arterial blood pressure in the presence of a cerebral arterial narrowing may trigger a headache attack .
. stated that during a migraine attack narrowing of the internal carotid artery usually involves either its extradural or intracavernous portion .
two important questions should be answered : what are the precipitants of vasoconstriction ? and is vasoconstriction the cause of pain ? considering the vasospasm due to sah and the biology of blood vessels , we could speculate about the pathophysiology of the supposed vasospasm found in tharcv syndrome .
the regulation of cerebral blood flow ( cbf ) is a fast and selective phenomenon and dramatic changes in blood flow or volume can be induced within a short period of time ( seconds or minutes ) regarding specific or regional brain areas either in normal or abnormal ( e.g. , epilepsy ) physiologic conditions .
the oscillations present in intracranial pressure are secondary to spontaneous changes in cbf velocity as a result of rhythmic changes in cerebral vessel diameter triggered by monoaminergic and serotonergic centers present in the brainstem . in addition ,
arterial constriction followed by dilatation is postulated to be part of the pathophysiology mechanism of migraine .
the vascular endothelium synthesizes vasorelaxant substances , e.g. , endothelium - derived relaxing factor ( edrf ) , acetylcholine ( ach ) , bradykinin , purines ( i.e. , atp ) , histamine , vasopressin , substance p , neurokinin a and b , and prostaglandin f2. on the contrary , endothelium - derived constricting factors may also be involved in the control of vascular tone , including serotonin , norepinephrine ( ne ) , prostaglandin e2 , thromboxane a2 , leukotriene c4 , endothelin ( et)-1 , and et-3 . in addition , ach releases edrf .
ne also induces release of edrf and substance p , which seem to attenuate the vasoconstrictor response to ne .
edrf was identified as being nitric oxide ( no ) , which is produced by neurons , glia , and endothelium .
sympathetic nerve varicosities release ne and other putative transmitters such as atp , neuropeptide y ( constrictor ) , vasoactive intestinal peptide ( dilators ) , and calcitonin gene - related peptide ( dilator ) .
blood - borne ne and stimulation of sympathetic nerves do not affect significantly brain circulation .
after chronic trigeminal ganglionectomy there was an increase in the constrictor response of pial arteries to ne . also , inhibition of edrf synthesis of or endothelial denudation enhances the vasoconstriction induced by ne .
likewise , acute hypertension allows the occurrence of important vasoconstrictor effects induced by sympathetic stimulation , indicating that , under certain circumstances , cerebral vessels may respond to noradrenergic stimuli .
interestingly , acute hypertension generates superoxide anion , which , in turn , inactivates edrf .
this may reverse the ach - induced cerebral arterial dilatation and augment cerebral vasoconstriction induced by ne , or sympathetic stimulation .
high levels of vasoactive and spasmogenic substances are present in the csf and plasma of patients with sah and vasospasm . in the presence of sah ,
endothelium - dependent relaxation is impaired by the production of potent endothelium - derived contracting factors ( cyclooxygenase products of arachnoid acid and et ) .
endothelin , a 21 amino acids peptide , has very potent and long - lasting constrictive effects .
many lines of evidence suggest that et may be intimately involved in the genesis of the cerebral vasospasm . the plasma concentration of et-1 is higher in patients with vasospasm than in patients without symptomatic vasospasm , with the peak concentration coinciding with the occurrence of the vasospasm . in isolated human cerebral artery segments , et produced intense and sustained vascular constriction , which was inhibited by sodium nitroprusside or verapamil . the enhanced vascular tone induced by et is resistant to ne antagonists , serotonin , isoproterenol , histamine , ach , and angiotensin ii . in canine basilar artery , calcium - channel blockers such as nicardipine and
papaverine reversed the contraction induced by et-1 . the arterial contraction induced by both ne and serotonin
the cerebral vessels are hyperreactive to et , indicating that in a given situation of higher reactivity of a particular segment of the cerebral arterial system ( by a reason not yet known ) sudden release of ne , serotonin , or any other vasoconstrictor into the circulation could precipitate a severe and long - lasting segmental arterial constriction . or yet again , the lack of action of endogenous substances with vasodilator properties would facilitate vasoconstriction .
a close interaction between et ( a vasoconstrictor ) and no ( a vasodilator ) appears to take place and to play a major physiologic role in the control of cbf and vessel caliber .
so , any disturbance that may occur in the equilibrium between constrictor and dilator factors could generate arterial spasm .
endothelin-1 levels increase during [ 6769 ] and between migraine attacks , suggesting that the peptide is implicated in the physiopathogenesis of migraine .
it was reported that a variant of the eta receptor gene modulates the risk for migraine .
this may imply that migraineurs with qualitatively or quantitatively altered eta receptor may present dysregulation of arterial tone , resulting in inadequate dilatation or constriction of cerebral vessels in response to different stimuli . supporting
the hypothesis that no might also participate in the genesis of pain , nitroglycerin , by provoking vasodilatation via no formation , is able to induce in healthy subjects an immediate , short - lasting , bilateral fronto - temporal and pulsating headache , that can be aggravated by routine physical activity .
intriguingly , nitroglycerin causes a more severe pain in migraine patients [ 72 , 73 ] .
alteration of intracranial vessel tone and regional instability of the cbf were documented in migrainous patients during the headache - free interval [ 7477 ] .
if so , this fact may justify the frequent occurrence of the syndrome in migranous subjects .
described the case of a 16-year - old girl experiencing an attack associated with headache , when she suddenly felt tired and faint ( not well characterized if it was abrupt or severe ) that was repeated several times within a period of four months .
such attacks were thought to be the expression of migraine events , in view of the slow progress of some episodes , accompanied by fatigue , drowsiness , and cold hands .
this case led to the assumption that the tharcv syndrome may eventually course with atypical headache episodes or even without any pain at all , including a number of young adult patients with stroke .
most but not all the headaches associated with reversible cerebral vasoconstriction have the characteristics of thunderclap headache according to most recent findings .
such reported cases were not included in our analyzed series to study better the profile of typical patients with tharcv syndrome .
dr . robert h. ackerman commented about the multiple areas of narrowing , beading , and sausage - shaped dilation of cerebral vessels observed during angiography of patient # 3 in table 3 as follows :
these segmental changes are nonspecific signs that can be found in a number of primary and secondary disorders affecting the pial vessels , which lie in the subarachnoid space and receive an investiture of pia as they enter the brain .
these data suggest the possibility of a local relationship between csf , arterial vessels , and brain parenchyma to explain segmental vasospasm .
interestingly , the pattern of the cerebral arterial spasm generated by a number of different etiologic causes has the particular feature of being segmental in nature .
in addition , it is a short lasting ( hours or days ) , reversible phenomenon .
this may suggest that the constriction of a major cerebral artery is segmental and does not involve the entire extension of the vessel , particularly during high - intensity stimulation .
are those localized vascular constrictions observed in intracranial arteries during the conditions discussed above anatomic - functional sphincters controlling local blood flow ? or is the vasospasm occurring in the tharcv syndrome primarily a sphincter disturbance ? precipitating factors , such as sexual activity , may induce vascular changes with profound hemodynamic fluctuations , which may trigger the pain if an arterial narrowing is present . in this regard , during continuous monitoring of intra - arterial blood pressure mann et al . documented that during coitus , peaks values of up to 300/175 mmhg were registered , with a mean of 237/138 mmhg for men and of 216/127 mmhg for women , in hypertensive subjects .
tricyclic antidepressants ( i.e. , amitriptyline ) , potent inhibitors of the neuronal uptake of ne , systemically administered to humans caused a reduction of the whole body ne spillover to plasma , due to the reduction in nerve firing rates .
propranolol , by blocking vascular -receptors , could impair the anticipated vasodilatation induced by activating -adrenergic receptors .
nonselective beta - blockers can also have the adverse effect of increasing platelet aggregability . in this regard
, the association of propranolol with stroke in migraineurs has been mentioned previously [ 82 , 83 ] .
lance and goadsby advise to avoid the use of beta - blockers in migraineurs with prolonged aura or severe focal neurological symptoms . in this respect ,
three of the patients presented in table 3 ( # 4 , 12 , and 21 ) were under prophylactic use of propranolol , and patient # 3 had aggravation of the neurological manifestation after introduction of propranolol in her prescription .
similar vascular changes in reactivity to endogenous substances can happen with the therapeutic use of amitriptyline . by the way , two patients of the 11 cases described as isolated angiitis of the cns were using either propranolol ( case 6 ) or tricyclics ( case 9 ) .
the diagnosis of a growing number of patients with the tharcv syndrome is anticipated nowadays , mainly with the broad use of the harmless ct or mr angiography and a greater knowledge of the syndrome .
this study was previously presented at the brazilian headache congress in 2002 as a new headache syndrome named
abrupt severe headache associated with segmental artery narrowing ( ashcan ) [ 8 , 86 ] and since then several other cases of the syndrome have been reported [ 44 , 79 , 8789 ] . in conclusion ,
the present article was written in an attempt to better characterize the profile of the patients with tharcv syndrome .
this syndrome is a neurological disturbance ; perhaps more frequent than expected , preferentially affecting migrainous middle - aged women , with an unpredictable prognosis , either having a benign course or leading to stroke . | thunderclap headache attributed to reversible cerebral vasoconstriction ( tharcv ) is a syndrome observed in a number of reported cases . in this article we reviewed this new headache entity ( idiopathic form ) using the clinical - radiological findings of 25 reported patients . in this series of patients 72% were women , the mean age at the onset of first headache episode was 39.4 2.3 years .
in addition to the sine qua non condition of being abrupt and severe ( thunderclap ) at the onset , the headache was usually described as being explosive , excruciating , or crushing . the feature of pulsatility , accompanied or not by nausea was described by 80% of the patients .
forty percent of the cases manifested vomiting and 24% photophobia .
usually the headache was generalized , and in three cases it was unilateral at least at the onset . in 21 of 25
patients ( 84% ) there was at least one recurrence or a sudden increase in the intensity of the headache .
a past history of migraine was present in 52% of the patients .
precipitating factors were identified in 56% of the patients .
sexual intercourse was described by six patients . of
the 25 patients with tharcv syndrome studied , 12 ( 48% ) developed focal neurological signs , transitory ischemic attack ( n = 1 ) , or ischemic stroke ( n = 11 , 44% ) , and two ( 8% ) of them manifested seizures . the tharcv syndrome is a neurological disturbance perhaps more frequent than expected , preferentially affecting middle aged female migraineurs , and having an unpredictable prognosis , either showing a benign course or leading to stroke . |
97% of the deaths related to childhood cancer occur in the developing world and with the poor resources and multiple other associated problems the survival rates of most childhood cancers do not approach the figures for the developed world .
nephroblastoma or wilms tumour is the most common renal tumour seen in children and accounts for 6 - 7% of cases of childhood cancer in the developed world . in the south african children 's tumour registry they account for 12% of childhood cancers .
the overall survival rate of nephroblastoma approaches 90% in the developed world but in developing countries the survival rates are much less and in some sub - saharan countries it is only 40% at 8 months after diagnosis [ 4 , 5 ] . the tumour is supposed to be more common in africa than in europe and the usa with a median age at diagnosis of 42 .
the peak age is between 24 and 36 months and 75% of patients will be less than 60 months of age at diagnosis and 95% of patients will be less than 120 months of age at diagnosis .
there is usually a slight female predominance [ 2 , 4 ] but this is not seen in all studies [ 58 ] .
there are two major treatment philosophies ; there is the siop approach of neoadjuvant chemotherapy followed by surgery and continuation of chemotherapy depending on the stage at surgery while the national wilms tumor study group ( nwtsg ) approach is primary surgery followed by appropriate chemotherapy depending on the stage of the tumour .
the primary goal of each is to treat patients with as little chemotherapy as possible and achieve the best possible survival rates .
there are very few studies from countries in africa ; one from johannesburg showed the survival of white patients in south africa as > 90% while the black african patients did significantly worse with a survival rate of approximately 65% .
this study also showed that the stage 4 patients had a survival rate of < 50% while stage 1 and 2 patients had rates that approximated 90% .
two further studies showed a male to female ratio that varied from 1.85 to 1 to 1.3 to 1 [ 7 , 8 ] . in tanzania
the average age of the patients seen in a dar es salaam study was almost 70 months , much older than the usual age for developed countries .
south africa is a developing country with a diverse population and a discrepant health system .
it consists of a diverse population in terms of ethnicity but this can broadly be classified into black , white or caucasian , coloured or mixed ethnicity , and indians .
the coloured population genetically consists of an ancestral mix between european and various southern african black tribes .
this ethnic group is widespread across south africa but there is a higher concentration of this population group in specifically the western cape region .
the indians are predominantly found in kwa zulu natal region situated in the eastern part of the country .
most of the patients are dependent on the public health services while there is a select group who can afford private medical care .
this is an audit of the experience of four paediatric oncology units at different sites within south africa .
each of the units functions independently and all followed the siop principles including neoadjuvant chemotherapy . in south africa
all treatment is free of charge for children under 6 years of age and for older children the cost of treatment is directly related to their income .
during the study period ( 20002010 ) 875 new patients with wilms tumour were seen in south africa . of this number 165
were treated in centres following nwtsg principles and were excluded . of the remaining 710 children 416
the other paediatric oncology units employing the siop protocol did not have complete data so they could not participate in this study .
the 4 units that were elected to form part of the study represent a fair reflection of the population distribution in south africa , tygerberg hospital , with larger coloured and white populations , while the other three units had a larger black population .
the 4 south african oncology units included in the study were the following.inkosi albert luthuli central hospital is a tertiary care public hospital in durban which is allied to the nelson r mandela school of medicine at the university of kwazulu - natal .
the hospital provides oncology care to the population of kwazulu - natal , currently 10.5 million ( 4.2 million children ) , as well as parts of the eastern cape.the universitas hospital academic complex ( uhac ) in bloemfontein provides cancer care to over 1.5 million children .
it is the referral pediatric oncology unit for the whole of the free state and northern cape provinces , as well as some areas of both the north west and eastern cape provinces and lesotho.chris hani baragwanath hospital is situated in johannesburg and is the largest hospital in the world with approximately 3 200 beds .
this unit serves the province of gauteng as well as north west with occasional patients from mpumalanga.tygerberg hospital unit is situated in cape town in the western cape .
tygerberg hospital is a tertiary hospital located in parow , cape town , serving the eastern metropolitan region of cape town and the north - eastern districts of the western cape province.the diagnosis of nephroblastoma was made on the clinical examination and ultrasound and/or ct abdomen and in all cases that proceeded to surgery on the resected specimens . before treatment patients had biopsies with pathological specimens which were processed and reviewed by the national health laboratory attached to the reporting institution .
inkosi albert luthuli central hospital is a tertiary care public hospital in durban which is allied to the nelson r mandela school of medicine at the university of kwazulu - natal .
the hospital provides oncology care to the population of kwazulu - natal , currently 10.5 million ( 4.2 million children ) , as well as parts of the eastern cape .
the universitas hospital academic complex ( uhac ) in bloemfontein provides cancer care to over 1.5 million children .
it is the referral pediatric oncology unit for the whole of the free state and northern cape provinces , as well as some areas of both the north west and eastern cape provinces and lesotho .
chris hani baragwanath hospital is situated in johannesburg and is the largest hospital in the world with approximately 3 200 beds .
this unit serves the province of gauteng as well as north west with occasional patients from mpumalanga .
tygerberg hospital is a tertiary hospital located in parow , cape town , serving the eastern metropolitan region of cape town and the north - eastern districts of the western cape province .
the following information was extracted and further analysed : date of birth and age at diagnosis , sex and race of all patients , date of diagnosis , stage at presentation and the outcome , and date on which the patient was last seen or died .
all patients were diagnosed between january 2000 and december 2010 and followed up till december 2011 .
there were 36 patient records in which there were certain elements missing from the data .
all patients ' gender , age , and race were documented . in 30 patients the stage of the disease
was not documented while in 6 the outcome or date on which the patient was last seen was not documented .
as the asian group was so small the figures for this group were not further analysed but they were included in the analysis of the whole group of patients .
all results were analysed using graphpad prism 4 programme and survival curves calculated using the same programme .
ethical approval was obtained by all 4 participating units and confidentiality of the patients was kept entirely during the study .
a total of 416 patients with the diagnosis of nephroblastoma were seen in these 4 units over a 10-year period ( table 1 ) .
this comprises 58.5% of all nephroblastoma patients notified to the sa childhood cancer tumour registry .
there were 214 male and 202 female patients , with a male to female ratio of 1.05 to 1 .
the median age of the female patients was 43 months , 3 months older than the male cohort .
our peak age was between 24 and 36 months and 73% of our patients were < 60 months at diagnosis : > 95% of our patients were < 120 months old at diagnosis .
the median age of the mixed ethnicity group was the youngest at 31 months while the african group had a median age of 42 months .
the stage of disease was documented in 386 patients . as all patients were treated on siop based protocols the staging was done after preoperative chemotherapy except for some of the stage 4 patients , who were staged at diagnosis , based on the imaging studies .
the patients who were not staged included those who died soon after admission or failed to proceed to surgery and thus no formal staging could be performed .
stage 4 was the most common stage seen in the four units and comprised 112 ( 27% ) of the patients ; stage 1 and 3 patients each contributed 22% while stage 2 made up 15% of the patients .
of all our patients 50% had advanced stage disease at presentation or after the neoadjuvant chemotherapy .
the survival rates according to the stages showed , as expected , that stage 4 patients and those not staged did the worst .
stage 1 patients , which should do the best , had a survival rate similar to stage 3 patients while stage 2 patients did the best at 85% ( figure 1 )
. amongst the african patients stage 4 was the most common ( 28% ) followed by stage 3 at 22% .
stage 1 and 2 patients contributed to the remaining 34% of this group . in 8% of cases the patients had bilateral nephroblastoma . in the mixed ethnicity group
46% of the patients were stage 1 while stage 3 and 4 patients made up 17.5% each of the remainder .
the incidence of stage 5 was 7.5% while stage 2 made up the remaining 10% of cases .
in the white population stage 1 was the most common at 53% of cases ; stage 4 comprised 28% and the remaining stages each made up 7% .
the percentage of advanced disease was 50% in the african group while in the white and mixed ethnicity cohorts it was lower at 32% and 34% , respectively .
the outcome of 406 patients was assessed by documenting the date on which the patient was last seen or the date of death . in 10 patients the outcome or date on which the patient was last seen was not documented .
a total of 281 patients were alive when last seen and their survival ranged from 0 months to 139 months with a median of 25-month survival after diagnosis .
there were 130 patients who had confirmatory evidence of death ; their survival ranged from 0 months to 132 months with a median survival of 28 months after diagnosis .
the median survival rates for each of the ethnic groups were also analysed : the african patients had a median survival of 26 months compared to the mixed ethnicity group of 39 months while the white patients had a survival of 21 months .
our projected survival of all patients at 60 months after diagnosis was estimated to be 66% .
this is the largest cohort of patients with nephroblastoma reported from south africa and the largest from africa [ 611 ] .
nephroblastoma is the commonest renal tumour that occurs in children and in developed countries it has an overall survival rate that approaches 90% . in developing countries , for a variety of reasons the survival rates seldom approach these figures [ 1 , 10 ] .
there were slightly more males than female patients in our study as it is described in the literature [ 2 , 10 ] although other reports have shown that there is a varying male to female ratio that ranges from 2 : 3 to 1 : 1 [ 5 , 6 , 8 ] .
the age range in patients with nephroblastoma is reported from under 3 months to 180 months of age [ 6 , 10 , 11 ] .
our youngest patient was less than 1 month of age while the oldest was 178 months of age which fits in with the age distribution as described in the literature .
other studies have reported different age ranges of patients that range from under 24 months to 180 months [ 6 , 7 , 10 , 11 ] .
the median age of children with nephroblastoma is reported to be 42 months of age [ 2 , 8 ] which is the same as our median age of 42 months .
there are reports of older median ages that approach 5070 months [ 5 , 7 ] .
a study from south africa showed a median age of 39 months and , although a somewhat different population group , it is very close to our median age of 42 months . there is a study from the gfaop group who report a median age of only 36 months in their cohort of patients .
the nwtsg studies report a median age for male of 36.5 months while females are somewhat older at 42.5 months .
our females had a median age similar to the nwtsg study but our male cohort was about 3 months older .
it is evident from this study that the median ages of the patients who died , whether they are female or male , are about 4 - 5 months older than those who survived .
the median age of the different racial groups was also very different with the black cohort being the eldest at 42 months and the mixed ethnicity cohort the youngest at 34 months : both very dissimilar to the studies from cape town and the nwtsg .
the peak age range for this study ( between 24 and 60 months ) was similar to previous reports , and more than 95% of our patients were diagnosed before 120 months of age .
the racial distribution of our patients reflects the population of south africa which estimates that 80% of the population is black , 9% each of white and mixed ethnicity and 2.5% asian origin .
each unit has a different racial mix with two units seeing predominantly black patients , one mainly saw patients of mixed ethnicity while the last unit saw patients of all race groups .
in another published study from south africa , in an area of high prevalence of mixed ethnicity , the racial mix was 42% black patients compared to our figure of 85% while the mixed ethnicity was 45% compared to our 10% incidence .
they also reported a higher proportion of white patients compared to our study ( 12% versus 3.6% ) .
our incidence of white patients was extremely low reflecting the locations of the units involved in the study and the communities they serve .
these differing demographics impact the presentation and outcome of all patients , including those with wilms tumour .
the study of the groupe franco africain d'oncologie pediatrique ( gfaop ) of the treatment of nephroblastoma in 8 african pilot units showed an incidence of stage 1 disease of 35% while stage iv disease accounted only for 18% of their patients compared to our incidence of advance disease that approaches 50% .
the siop figures from europe also show a different stage incidence with almost 60% of cases being stage 1 after preoperative chemotherapy .
figures from other african countries quote that over 70% of their patients had advanced stage disease at presentation ; our figure , although lower , is still significant at just under 50% .
our stage 4 patient ratios were very similar except for the mixed ethnicity patients who had the lowest incidence at 17% .
our 5-year overall survival rate was 66% with a difference in the different stage survivals as well as a race difference with the white patients faring worst and the mixed ethnicity patients doing the best .
the survival rates for our patients according to stage showed that our stage 4 survival rate was lower than 50% .
. this apparent paradox may be due to understaging of large tumours as stage 1 or due to the dominance of ethnic african patients in stage 2 .
our stage 5 patients also appeared to do relatively well and had a survival rate of 63% .
this is a retrospective study and limitations are related to the missing information or incomplete data . despite the fact that the histology was confirmed in all cases by a pathologist after surgery and all cases were discussed in a tumor board meeting further complete information about the pathological staging was difficult to obtain in all cases .
this study is limited to the description of epidemiology , demographics , characteristics related to ethnicity , and overall survival of the south african patients .
no further information was collected regarding the nutritional status , socioeconomic implications , and associated morbidities which will be analysed and discussed in a future study .
this south african cohort is similar to reports from the developed world in terms of the gender ratio and age range .
our patients of mixed ethnicity were younger than our other two major population groups and we could not demonstrate the stage distribution reported by european groups using siop protocols where more than 60% of patients are stage 1 after neoadjuvant chemotherapy .
our survival rate at just 66% after 5 years is significantly worse than that described in the literature for developed countries using siop protocol .
awareness programs are essential as the diagnosis and treatment of early stages could increase the overall survival in south african children with nephroblastoma . | background .
nephroblastoma is the commonest renal tumour seen in children .
it has a good prognosis in developed countries with survival rates estimated to be between 80% and 90% , while in africa it remains low
. method .
retrospective study of patients diagnosed with nephroblastoma who are seen at 4 paediatric oncology units , representing 58.5% of all south african children with nephroblastoma and treated following siop protocol between january 2000 and december 2010 .
results .
a total of 416 patients were seen at the 4 units . over 80% of our patients were african and almost 10% of mixed ethnicity .
the most common stage was stage 4 .
the median survival was 28 months after diagnosis with the mixed ethnicity patients recording the longest duration ( 39 months ) and the white patients had the shortest median survival .
the overall 5-year survival rate was estimated to be 66% .
stage 2 patients did significantly better ( 85% ) .
conclusions .
our patients are similar with regard to gender ratio , median age , and age distribution as described in the literature , but in south africa the more advanced stage disease seen than in other developed countries is translated into low overall survival rate . |
chip - seq , gro - seq , or rna - seq libraries were also generated from imr90 , huvec , mcf7 or lncap cells and sequenced on the illumina hi - seq2000 platform .
more information about the experiments and detailed descriptions of hi - c data analysis pipeline , including data filtering , normalization , statistical modeling and interaction calling can be found in supplementary methods .
| a large number of cis - regulatory sequences have been annotated in the human genome1,2 , but defining their target genes remains a challenge3 .
one strategy is to identify the long - range looping interactions at these elements with the use of chromosome conformation capture ( 3c ) based techniques4 .
however , previous studies lack either the resolution or coverage to permit a whole - genome , unbiased view of chromatin interactions . here
, we report a comprehensive chromatin interaction map generated in human fibroblasts using a genome - wide 3c analysis method ( hi - c)5 . we determined over one million long - range chromatin interactions at 510 kb resolution , and uncovered general principles of chromatin organization at different types of genomic features .
we also characterized the dynamics of promoter - enhancer contacts upon tnf- signaling in these cells .
unexpectedly , we found that tnf- responsive enhancers are already in contact with their target promoters prior to signaling .
such pre - existing chromatin looping , which also exists in other cell types with different extra - cellular signaling , is a strong predictor of gene induction .
our observations suggest that the three - dimensional chromatin landscape , once established in a particular cell type , is rather stable and could influence the selection or activation of target genes by a ubiquitous transcription activator in a cell - specific manner . |
thromboembolic complications have been described in hiv infected patients with higher incidence seen in patients with active opportunistic infections ( oi ) or malignancy , and in patients with acquired immunodeficiency syndrome ( aids ) .
various abnormalities predisposing to hypercoagulable state have been reported in aids patients including the presence of antiphospholipid antibodies , lupus anticoagulant , deficiencies of proteins c and s , heparin cofactor ii , and antithrombin and use of protease inhibitors . though more than one thrombotic episode has been reported in adults , it has rarely been reported in children .
we report an hiv infected boy who had three thromboembolic episodes in a matter of 6 months and finally succumbed to the disease .
a six - year- old hiv infected boy on 2 drug antiretroviral therapy ( art ) consisting of zidovudine ( azt ) and lamivudine ( 3tc ) for the past 3 years presented with tuberculous meningitis and pulmonary tuberculosis ( tb ) in december 2004 . on investigation
, he was also found to have cardiomyopathy ( left ventricular dilation with hypertrophy ) on echocardiography , elevated liver transaminases , anemia , elevated , basal ganglia calcifications on ct of brain and normal kidney sizes on ultrasound of abdomen ( right kidney = 7.9 4.1 cm , left kidney = 7.7 3.7 cm ) and normal renal function tests .
his birth history was uneventful . on examination , he was malnourished ( weight = 12 kg ) , had oral thrush , clubbing and hypertension .
his urine examination showed no proteinuria and ultrasound colour doppler of renal vessels was normal .
he was started on antituberculous therapy ( att ) ; art was shifted to azt , didanosine ( ddi ) and efavirenz ( efv ) .
after 3 months in march 2005 , he was again hospitalized with convulsion and hypertension .
his csf was normal , sputum for acid fast bacilli was negative and chest x - ray had normalized .
hemoglobin had also increased to 16.7 g / dl , and esr was 15 mm at end of 1 h. liver transaminases were decreasing ( sgot = 97 iu / l , sgpt = 39
iu / l ) . however , ultrasound of kidneys showed small left kidney ( left kidney = 5.2 1.8 cm , right kidney = 9.9 4.2 cm ) .
renal function tests were normal and urine examination was also normal and serum toxoplasma igg was positive .
he was treated with sulfadiazine- pyrimethamine for 6 weeks and hypertension was controlled with increasing dose of nifedipine .
thus , he was determined to have renal hypertension most likely due to renal artery stenosis .
the child was discharged and after 2 months he presented with right - sided hemiplegia and uncontrolled hypertension in may 2005 .
mri of brain showed old and new infarcts with angiography showing no flow beyond left middle cerebral artery .
the child was advised left - sided nephrectomy in view of non functioning kidney and uncontrolled hypertension , which parents refused .
thrombotic workup in form of anticardiolipin antibodies ( acla ) , antiphospholipid antibodies ( apla ) , lupus anticoagulant were negative in this child .
protein c , protein s and antithrombin iii levels could not be done due to nonaffordability .
decrease in natural anticoagulants such as protein c and protein s has been found in hiv infected patients without a thrombotic episode .
it appears that cd4 counts < 200/ mm , presence of opportunistic infections , aids - related neoplasms or autoimmune disorder associated with hiv such as autoimmune hemolytic anemia ( aiha ) may predispose to thrombosis . in our patient , though cd4 counts were normal , the child did have advanced hiv disease , had ois in form of tuberculosis and suspected cns toxoplasmosis which could have predisposed him to thrombosis .
though his apla , acla were negative , we could not do his protein c and protein s level which would have helped to determine the cause of his recurrent thrombosis .
earlier we had reported a child with aps and cerebral infarct , but recurrent thrombosis inspite of anticoagulation is rarely seen as was seen in our patient . in adults on art with protease inhibitor ,
found that out of 28 adult male homosexuals with aids , who had thrombotic episodes , six patients had two thrombotic events .
these patients had either deep vein thrombosis , pulmonary thromboembolism or renal vein thrombosis . however , our patient had renal artery thrombosis , followed by middle cerebral artery thrombosis and subsequently hepatic vein thrombosis which has never been reported earlier . though anticoagulants and/or aspirin have been used to prevent recurrent thrombosis , in our patient inspite of warfarin , the child had a recurrent thrombosis that proved to be fatal .
our patient initially presented with hypertension , subsequently developed small non - functioning kidney due to renal artery stenosis and proteinuria though he had normal renal function tests on blood examination .
these include hiv - associated nephropathy ( hivan ) , hiv - related immune complex disease , nephropathy secondary to antiretroviral therapy ( art ) or antibiotics , thrombotic microangiopathy , and diseases related to common comorbidities such as opportunistic infections .
the broad spectrum of clinical presentation includes acute renal failure ( arf ) , nephrotic syndrome , progressive chronic renal dysfunction , proteinuria , tubular function abnormalities and electrolyte disturbances .
proteinuria is believed to be the earliest and most consistent clinical finding for the diagnosis of hiv - associated nephropathy .
however , our patient presented with hypertension and only later in the disease developed proteinuria suggestive that renal involvement was due to vasculopathy rather than renal involvement of hiv per se . in adults , hypertension in hiv infected patients has been found to be related to increasing age , longer duration of hiv , higher body mass index , and diabetes , with a trend for african american ethnicity .
we did not get any information on renal artery stenosis in hiv infected children , but postulate that it may be related to thrombotic tendency with hiv infection as the child subsequently had thrombosis in cerebral vessels and also the hepatic veins .
further studies to elucidate the mechanisms underlying this abnormal hemostatic phenomenon , factors predisposing to recurrent thrombosis are required .
thus , clinicians caring for hiv infected children should be aware of thromboembolic disease as a possible complication of aids . | though thromboembolic complications in hiv infected patients have been described in literature , recurrent thrombosis is very rare .
we present a six - year - old hiv infected boy who presented with recurrent thrombosis .
he initially had renal artery thrombosis , then middle cerebral artery thrombosis and finally hepatic vein thrombosis that was fatal . |
mitochondria are highly dynamic organelles that frequently fuse and divide in disease , aging , and development . in vertebrates ,
mitofusin-1 and -2 ( mfn1 and mfn2 ) are involved in mitochondrial fusion , whereas drp1 and mitochondrial fission factor ( mff ) control mitochondrial fission .
recently , it was reported that the er plays an active role in defining the sites of mitochondrial division .
in fact , mitochondria and the er physically interact by close structural juxtaposition , via the mitochondrial - associated er membrane ( mam ) . to clarify the role of mitochondrial fission in this communication
, we generated mice lacking the mitochondrial fission protein drp1 in the liver ( drp1liko ) .
when the mice were fed with a high - fat diet ( hfd ) , analysis of gene expression in the liver demonstrated marked elevation of er stress markers .
we also found a second subset of genes , discussed here , that are involved in the immune response .
mice were fed ad libitum with a normal chow diet ( 5.4% fat , crf-1 , orient yeast co. , tokyo , japan ) and kept under a light dark cycle of 12 h. for the hfd study , 4-week - old mice were put on high - fat diet ( 24% fat , lard fat , 45 kcal % fat , d12451 ; research diets , new brunswick , nj ) for 24 weeks . all mouse procedures and protocols were in accordance with the guide for the care and use of laboratory animals and were approved by the ethics committees on animal experimentation ( kyushu university , graduate school of medicine ) .
after 24 weeks high fat diet , mice were fasted for 17 h and then sacrificed .
total rna was isolated from cells using trizol reagent ( nitrogen ) and purified using sv total rna isolation system ( promega ) .
crna was amplified and labeled using a low input quick amp labeling kit ( agilent technologies ) .
crna was hybridized to a 44k 60-mer oligomicroarray ( whole mouse genome microarray 4 44k v2 ; agilent technologies ) according to the manufacturer 's instructions .
the relative hybridization intensities and background hybridization values were calculated using feature extraction software version 9.5.1.1 ( agilent technologies ) .
the scanned images were analyzed with feature extraction software 9.5.1.1 ( agilent ) using default parameters to obtain background subtracted and spatially detrended processed signal intensities .
the raw signal intensities and flags for each probe were calculated from the hybridization intensities and spot information according to the procedures recommended by agilent technologies using the flag criteria in the genespring software .
in addition , the raw signal intensities of two samples were log2-transformed and normalized by the quantile algorithm with the bioconductor .
mice were fed ad libitum with a normal chow diet ( 5.4% fat , crf-1 , orient yeast co. , tokyo , japan ) and kept under a light dark cycle of 12 h. for the hfd study , 4-week - old mice were put on high - fat diet ( 24% fat , lard fat , 45 kcal % fat , d12451 ; research diets , new brunswick , nj ) for 24 weeks . all mouse procedures and protocols were in accordance with the guide for the care and use of laboratory animals and were approved by the ethics committees on animal experimentation ( kyushu university , graduate school of medicine ) .
after 24 weeks high fat diet , mice were fasted for 17 h and then sacrificed .
total rna was isolated from cells using trizol reagent ( nitrogen ) and purified using sv total rna isolation system ( promega ) .
crna was amplified and labeled using a low input quick amp labeling kit ( agilent technologies ) .
crna was hybridized to a 44k 60-mer oligomicroarray ( whole mouse genome microarray 4 44k v2 ; agilent technologies ) according to the manufacturer 's instructions .
the relative hybridization intensities and background hybridization values were calculated using feature extraction software version 9.5.1.1 ( agilent technologies ) .
the scanned images were analyzed with feature extraction software 9.5.1.1 ( agilent ) using default parameters to obtain background subtracted and spatially detrended processed signal intensities .
the raw signal intensities and flags for each probe were calculated from the hybridization intensities and spot information according to the procedures recommended by agilent technologies using the flag criteria in the genespring software .
in addition , the raw signal intensities of two samples were log2-transformed and normalized by the quantile algorithm with the bioconductor .
genes were selected using the criterion of a z score of 2 , which identified 526 up - regulated and 640 down - regulated genes in drp1liko mice .
the top 5 up - regulated genes have been reported in other publications , of these , 3 genes are known to be er stress response genes , which was the topic of our previously published article . to further investigate these data
, we use the david tool to functionally cluster up - regulated and down - regulated genes by similarly annotated gene ontology ( go ) biological process terms , respectively .
the top ten significantly enriched annotation clusters of up - regulated genes were shown in fig .
1 . we found that seven of the ten clusters were related to the immune system ;
these clusters included terms such as immune response , phagocytosis , antigen processing and presentation , defense response and response to virus .
the remaining clusters were related to 2-deoxyribonucleotide biosynthetic process , amine biosynthetic process and cell death .
next , to further define connections between immune molecules that were regulated by mitochondrial fission , we list the 32 genes selected by using the term immune response in table 1 .
2 , and include lipid biosynthetic process , fatty acid metabolic process , acute inflammatory response , actin filament - based process , cell fate commitment , response to wounding , actomyosin structure organization , complement activation , alternative pathway , actin cytoskeleton organization and brown fat cell differentiation .
we described here that lack of drp1 increased the expression levels of a large number of genes involved in immune response . within the past several years
, a couple of studies have shed light on the connection between mitochondrial dynamics and antiviral innate immunity .
indeed , castanier and colleague have observed that knockdown of drp1 increased innate immune signaling and they have highlighted the importance of the interconnectedness and interdependence of mitochondria fission in antiviral innate immunity in vitro . in this study , we provided the compelling evidence that mitochondrial fission regulates the expression of the genes responsible for the immune system in the liver .
| dynamin - related protein 1 ( drp1 ) is a member of the dynamin family of large gtpase , which cycles between the cytosol and the mitochondrial outer membrane , and mediates mitochondrial fission . using microarray analysis of gene expression in the livers of wild - type and drp1 knockout mice
, we have previously identified that endoplasmic reticulum ( er ) stress marker genes are significantly increased by the absence of drp1 [ 1 ] . here , we provide methodological and analytical details of the microarray data , which have been deposited in the gene expression omnibus as data set gse64222 .
we have performed further gene ontology analysis of the data and found the differential expression of a subset of genes that are involved in the immune response in the livers of drp1 knockout mice versus wild - type controls . |
medication overuse headache ( moh ) is a chronic headache disorder due to overuse of analgesic medicines , taken to treat a primary headache , and remitted by drug discontinuation .
moh has long been considered as a biobehavioural disorder , in which the biological predisposition to develop a chronic headache interacts with an individual behaviour prone to develop a dependence disorder [ 2 , 3 ] .
the molecular basis of moh are largely unknown , though it has been hypothesized that this subtype of headache could be mediated by cognitive impulsivity , and shares some pathophysiological mechanisms with drug addiction , including dysfunction of the frontostriatal system and central pain sensitization induced by drug overuse .
based on this hypothesis , there seems to be a role in moh for bdnf ( brain - derived neurotrophic factor ) , because of its role in central pain sensitization and its relationship with substance - related disorders [ 5 , 6 ] .
bdnf is a member of neurotrophin family , a unique group of polypeptide growth factors that influence differentiation and survival of neurons in the developing nervous system .
bdnf is involved in long - term potentiation ( ltp ) , and therefore in learning and memory mechanisms .
also , a pathogenic role for bdnf has been shown in pain transduction and sensitization , in epilepsy , and in neuropsychiatric disorders .
bdnf gene , mapped to chromosome 11p , encodes for pro - bdnf that is subsequently cleaved in its mature form .
recently , it has been observed that a common 196 g > a single - nucleotide polymorphism ( snp ) of bdnf gene , resulting in a valine at residue 66 to methionine ( val66met ) substitution in pro - bdnf protein , exhibits a functional relevance in that it seems to reduce bdnf activity by altering neurotrophin trafficking .
the val66met variant affects anatomy , cognition and behaviour in adults and leads to memory impairment and susceptibility to obsessive - compulsive , eating , substance abuse and mood disorders [ 6 , 810 ] .
the rationale behind our study derives from two findings , which have been consistently reported in previous researches : ( 1 ) overuse of analgesic drugs is a defining feature of moh and , for this reason , moh has been related to behavioural syndromes associated with substance abuse [ 13 ] , ( 2 ) there is evidence that proneness to use / abuse substances is associated with the allele a of bdnf polymorphism .therefore , we tested the frequency of the val66met genotype in a sample of moh patients , and compared clinical features between individuals homozygous for g allele ( g / g ) and carriers of the allele a ( non - g / g ) .
aim of this study is to confirm our hypothesis that allele a of the examined bdnf polymorphism interfere with moh , seeking for clinical and psychiatric differences between the two groups , mainly in the monthly drug consumption , that is both the clinical hallmark of disease , and the feature closest to abuse disorder .
we would like to emphasize that , although such studies are necessary to get a deeper understanding of the association between moh and bdnf polymorphism , the present study did not intend to ascertain whether the bdnf polymorphism is a risk factor for the development of moh , but only an aggravating factor for subjects that have already developed the disorder .
unrelated consecutive moh patients were enrolled from two italian university headache clinics : the inpatient headache - unit of irccs c. mondino institute of neurology foundation ( pavia ) , and headache outpatient department of university of rome la sapienza , among those admitted with a diagnosis of probable moh , to undergo withdrawal from their overused medications .
although revised criteria were formulated for moh , patients enrolment was started before their statement , thus original ichd - ii version ( i.e. in the presence of headache improvement within 2 months after analgesics withdrawal ) , that is more conservative , was adopted to recruit all patients ; a retrospective analysis will also confirm that all enrolled patients fulfilled the revised criteria .
socio - demographic and clinical data were obtained by anamnesis ; consumption of analgesic drugs per month ( from here , monthly drug number ) , days with headache per month ( from here , monthly headache days ) and days with drug consumption per month ( from here , monthly drug days ) were self reported by patients through a headache diary , received at the time of first visit .
inclusion criteria were : ( a ) patient written informed consent , ( b ) absence of other major medical conditions , ( c ) absence of psychiatric disorders that could sustain a chronic pain condition ( psychotic and/or somatoform disorders ) , ( d ) to have accurately completed the last 3-month pre - withdrawal diary , ( e ) ability to complete the psycho - diagnostic assessment , ( f ) diagnostic confirmation of moh after withdrawal .
presence of categorical psychiatric diagnosis was investigated through four modules of the structured clinical interview for dsm - iv axis i disorders ( scid - i ) , clinician version : mood episodes , mood disorders , anxiety disorders , and eating disorders . moreover ,
a dimensional assessment was made with the beck depression inventory ( bdi ) , a self - reported questionnaire to measure the presence and severity of depressive symptoms . with the patients
written informed consent , 10 cc of peripheral blood were collected , total genomic dna purified , and genotyping for the bdnf val66met - bdnf variant was performed essentially as reported .
all data obtained in the three phases of the study ( socio - demographic and clinical recordings , psychiatric assessment , and genetic determinations ) were merged in a all - comprehensive database by an independent data - manager , which opened the blind and performed the sample size analysis on a preliminary group , to indicate the minimal number of patients needed in the study , and statistical analyses on definitive data . on the base of initial 36 patients ,
a sample size analysis was performed to obtain the required number of subjects to fulfil a desired power of 80% with significance level at 5% . because our primary endpoint was to detect a difference in number of analgesics consumed per month by patients between the two groups ( see introduction ) ,
we divided g / g from non - g / g subjects and calculated the monthly mean of drug consumption : 21 non - g / g subjects consumed meanly 45.57 doses versus 32.93 doses of 15 g / g subjects ( t = 1.887 , p = 0.07 , cohen s d = 0.65 ) .
sample size resulted to be 87 subjects , respectively , 51 for non - g / g and 36 for g / g group .
descriptive statistics to compare bdnf genotypes were performed using parametric or , when appropriate , non - parametric tests .
a stepwise multiple linear regression analysis was employed to identify significant predictors of the monthly drug number in the entire sample .
in addition , to well - know indexes of null hypothesis significance testing , effect size measures were also reported to recognize the value of the degree of association among variables , particularly between bdnf polymorphism group and monthly drug number [ 17 , 18 ] .
effect sizes were calculated with the phi ( ) coefficient [ or , when appropriate , cramr s v ( c ) ] for test , with cohen s d for student s t test , with partial eta squared \documentclass[12pt]{minimal }
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\begin{document}$$ ( \eta_{\text{p}}^{2 } ) $ $ \end{document } for the analysis of variance ( anova ) , and with cohen s f for multiple regression .
unrelated consecutive moh patients were enrolled from two italian university headache clinics : the inpatient headache - unit of irccs c. mondino institute of neurology foundation ( pavia ) , and headache outpatient department of university of rome la sapienza , among those admitted with a diagnosis of probable moh , to undergo withdrawal from their overused medications .
although revised criteria were formulated for moh , patients enrolment was started before their statement , thus original ichd - ii version ( i.e. in the presence of headache improvement within 2 months after analgesics withdrawal ) , that is more conservative , was adopted to recruit all patients ; a retrospective analysis will also confirm that all enrolled patients fulfilled the revised criteria .
socio - demographic and clinical data were obtained by anamnesis ; consumption of analgesic drugs per month ( from here , monthly drug number ) , days with headache per month ( from here , monthly headache days ) and days with drug consumption per month ( from here , monthly drug days ) were self reported by patients through a headache diary , received at the time of first visit .
inclusion criteria were : ( a ) patient written informed consent , ( b ) absence of other major medical conditions , ( c ) absence of psychiatric disorders that could sustain a chronic pain condition ( psychotic and/or somatoform disorders ) , ( d ) to have accurately completed the last 3-month pre - withdrawal diary , ( e ) ability to complete the psycho - diagnostic assessment , ( f ) diagnostic confirmation of moh after withdrawal .
presence of categorical psychiatric diagnosis was investigated through four modules of the structured clinical interview for dsm - iv axis i disorders ( scid - i ) , clinician version : mood episodes , mood disorders , anxiety disorders , and eating disorders . moreover ,
a dimensional assessment was made with the beck depression inventory ( bdi ) , a self - reported questionnaire to measure the presence and severity of depressive symptoms .
with the patients written informed consent , 10 cc of peripheral blood were collected , total genomic dna purified , and genotyping for the bdnf val66met - bdnf variant was performed essentially as reported .
all data obtained in the three phases of the study ( socio - demographic and clinical recordings , psychiatric assessment , and genetic determinations ) were merged in a all - comprehensive database by an independent data - manager , which opened the blind and performed the sample size analysis on a preliminary group , to indicate the minimal number of patients needed in the study , and statistical analyses on definitive data . on the base of initial 36 patients ,
a sample size analysis was performed to obtain the required number of subjects to fulfil a desired power of 80% with significance level at 5% . because our primary endpoint was to detect a difference in number of analgesics consumed per month by patients between the two groups ( see introduction ) ,
we divided g / g from non - g / g subjects and calculated the monthly mean of drug consumption : 21 non - g / g subjects consumed meanly 45.57 doses versus 32.93 doses of 15 g / g subjects ( t = 1.887 , p = 0.07 , cohen s d = 0.65 ) .
sample size resulted to be 87 subjects , respectively , 51 for non - g / g and 36 for g / g group .
descriptive statistics to compare bdnf genotypes were performed using parametric or , when appropriate , non - parametric tests .
a stepwise multiple linear regression analysis was employed to identify significant predictors of the monthly drug number in the entire sample .
in addition , to well - know indexes of null hypothesis significance testing , effect size measures were also reported to recognize the value of the degree of association among variables , particularly between bdnf polymorphism group and monthly drug number [ 17 , 18 ] .
effect sizes were calculated with the phi ( ) coefficient [ or , when appropriate , cramr s v ( c ) ] for test , with cohen s d for student s t test , with partial eta squared \documentclass[12pt]{minimal }
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\begin{document}$$ ( \eta_{\text{p}}^{2 } ) $ $ \end{document } for the analysis of variance ( anova ) , and with cohen s f for multiple regression .
a hundred and seven moh patients ( 84 women and 23 men ; age , mean sd : 46.741 11.36 years ) were enrolled in the study . depending on the type of overused drug ,
patients were divided into four groups : 29 patients ( 27.10% ) consumed exclusively triptans , 18 ( 16.82% ) exclusively non - steroidal antinflammatory drugs ( nsaids ) ; 27 ( 25.23% ) exclusively combination drugs ( combination ) , and 33 ( 30.84% ) associated different types of drugs ( association ) .
none of the patients consumed opioid drugs ; all types of combination drugs consumed by our patients resulted to contain caffeine . by genotyping entire moh population , we identified seven cases of harboured a / a genotype ( 6.54% ) , 53 were g / a ( 49.53% ) , and 47 were g / g ( 43.93% ) .
samples were in hardy - weinberg equilibrium ( = 2.460 , df = 1 , p = 0.12 for the null hypothesis ) .
a preliminary analysis was performed to investigate differences in monthly drug numbers between a / a ( mean sd 54.86 19.05 ) , a /
g ( 49.06 27.19 ) and g / g ( 33.83 14.04 ) group . due to small sample size in a / a group
whitney u test , and the monte carlo method , a bootstrapping technique with a resampling process that produces nonparametric significance estimates .
the three groups differed statistically ( among them ) in number of monthly used drug [ mean rank : a / a , 76.21 ; a / g , 61.43 ; g / g , 42.31 ; h = 13.317 , df = 2 , p < 0.0001 ( 99% confidence interval < 0.00010.001 ) ] .
pairwise comparisons ( significance threshold adjustment with bonferroni s correction , p = 0.0167 ) showed higher values in a / a [ mean rank : a / a , 43.00 ; g / g , 25.19 ; u = 56.000 , z = 2.794 , p2-tailed = 0.004 ( 99% ci 0.0030.006 ) ] and a / g [ mean rank : a / g , 58.82 ; g / g , 41.12 ; u = 804.500 , z = 3.046 , p2-tailed = 0.002 ( 99% ci 0.0010.003 ) ] when compared with g / g patients , but no difference between a / a and a / g [ mean rank : a / a , 37.21 ; a / g , 29.61 ; u = 138.500 , z = 1.082 , p2-tailed = 0.29 ( 99% ci 0.280.30 ) ] . till now obtained results confirmed our decision to consider together carriers of allele a in the non - g / g group : ( 1 ) above - mentioned explorative analysis showed that a / a and a / g groups were not different in terms of monthly drug consumption , ( 2 ) as expected , a little size of a / a subjects was found , and it makes difficult to perform complex , multivariate statistical analysis with an acceptable degree of reproducibility . as mentioned , to perform further statistical analyses , the population was divided into two groups : g / g genotype ( 47 subjects , 43.93% ) , homozygotes for valine ( allele g ) , and non - g / g genotype ( 60 subjects , 56.07% ) , carriers of metionine 66 ( allele a ) , either in heterozygosity or in homozygosity . clinical characteristics and descriptive statistics for the two groups are illustrated in detail in table 1 .
non - g / g showed a higher consumption of monthly drug number ( non - g / g vs. g / g , mean sd : 49.73 26.31 vs. 33.83 14.04 ; t = 4.011 , p < 0.0001 , d = 0.76 ) and longer moh duration ( 6.43 7.78 vs. 3.09 3.66 ; t = 2.944 , p = 0.004 , d = 0.56 ) when compared to g / g patients .
there were no significant differences between g / g and non - g / g patient groups in terms of other clinical and socio - demographic features .
moreover , no significant associations were found among the two groups and current occurrence of psychiatric disorders.table 1table shows descriptive socio - demographic , headache , and psychiatric data for the entire study samplenon - g / g ( n = 60 ; 56.07%)g / g ( n = 47 ; 43.93%)statisticsgender women47 ( 43.93%)37 ( 34.58%) = 0.002p = 0.96 = 0.005 men13 ( 12.15%)10 ( 9.35%)age ( years)47.57 11.4744.94 11.15 t = 1.192p = 0.24d = 0.23education ( years)12.34 4.4012.59 4.09
t = 0.262p = 0.79d = 0.06employment yes38 ( 35.51%)32 ( 29.91%) = 0.263p = 0.61 = 0.050 no22 ( 20.56%)15 ( 14.02% ) age of primary headache onset ( years)16.20 7.4215.32 7.14 t = 0.620p = 0.54d = 0.12 primary headache duration ( years)32.07 11.9630.43 14.28 t = 0.647p = 0.52d = 0.13 age of moh onset ( years)41.53 11.8242.66 12.64 t = 0.475p = 0.64d = 0.09 moh duration ( years)6.43 7.783.09 3.66 t = 2.944p = 0.004d = 0.56 monthly headache days24.82 6.0725.89 5.70 t = 0.935p = 0.35d = 0.18 monthly drug number49.73 26.3133.83 14.04 t = 4.011p < 0.0001d = 0.76 monthly drug days23.63 6.1422.17 6.46 t = 1.195p = 0.24d = 0.23drug type triptans18 ( 16.82%)11 ( 10.28%) = 0.707p = 0.87c = 0.081 nsaids10 ( 9.35%)8 ( 7.48% ) association17 ( 15.89%)16 ( 14.95% ) combination15 ( 14.02%)12 ( 11.21%)current psychiatric diagnosis present45 ( 42.06%)34 ( 31.78%) = 0.096p = 0.76 = 0.030 absent15 ( 14.02%)13 ( 12.15%)current mood diagnosis present27 ( 25.23%)22 ( 20.56%) = 0.035p = 0.85 = 0.018 absent33 ( 30.84%)25 ( 23.36%)current anxiety diagnosis present32 ( 29.91%)24 ( 22.43%) = 0.054p = 0.82 = 0.023 absent28 ( 26.17%)23 ( 21.50%)current eating diagnosis present6 ( 5.61%)2 ( 1.87%) = 1.257p = 0.26* = 0.108 absent54 ( 50.47%)45 ( 42.06%)current double psychiatric diagnosis present18 ( 16.82%)15 ( 14.02%) = 0.045p = 0.83 = 0.021 absent42 ( 39.25%)32 ( 29.91% ) bdi ( score)13.15 8.5312.09 8.73 t = 0.634p = 0.53d = 0.12continuous data are expressed as mean sd , whereas categorical data are frequencies and percentages of total subjects .
differences between non - g / g and g / g patients , with bivariate statistics results , are also illustrated .
bold entries represent the statistical significant differencesmoh medication overuse headache , bdi beck depression inventory , monthly drug days number of days per month in which patient assumed at least one dose of drug to treat headache , drug type triptans ( exclusively one , or more , types of triptan ) , nsaids ( exclusively one , or more , type of non steroidal antinflammatory drugs ) , association ( use of different types of drug in association ) ; combination ( drugs of combination , with more active ingredients ) * fisher s exact test , two - tailed : p = 0.46 table shows descriptive socio - demographic , headache , and psychiatric data for the entire study sample continuous data are expressed as mean sd , whereas categorical data are frequencies and percentages of total subjects .
differences between non - g / g and g / g patients , with bivariate statistics results , are also illustrated .
bold entries represent the statistical significant differences moh medication overuse headache , bdi beck depression inventory , monthly drug days number of days per month in which patient assumed at least one dose of drug to treat headache , drug type triptans ( exclusively one , or more , types of triptan ) , nsaids ( exclusively one , or more , type of non steroidal antinflammatory drugs ) , association ( use of different types of drug in association ) ; combination ( drugs of combination , with more active ingredients ) * fisher s exact test , two - tailed : p = 0.46 a two - way analysis of covariance ( ancova ) was employed to investigate the group effect ( two between - subject factors : genotype , non - g / g vs. g / g ; drug type , triptans vs. nsaids vs. association of drugs vs. drugs of combination ) on monthly drug number ( dependent variable ) ; monthly headache days and monthly drug days were used as covariate in this analysis to reduce the error variance .
the ancova model was highly significant ( f9,97 = 7.149 , p < 0.0001 ) and explained more than one - third of variance ( adjusted r = 0.343 ) . the drug type
effect was not significant ( f3,97 = 0.383 , p = 0.77 , \documentclass[12pt]{minimal }
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drug type was also significant ( f3,97 = 2.776 , p = 0.04 , \documentclass[12pt]{minimal }
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among the covariate , monthly drug days was highly significant ( f1,97 = 16.918 , p < 0.0001 , \documentclass[12pt]{minimal }
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but there was no effect of monthly headache days ( f1,97 = 0.043 , p = 0.84,\documentclass[12pt]{minimal }
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pairwise comparisons [ fisher s least square difference ( lsd ) ; significance threshold adjustment for multiple comparison test with bonferroni s correction ( 0.05/8 ) : p = 0.00625 ] showed no genotype difference in patients who consumed triptans ( p = 0.74 ) and nsaids ( p = 0.16 ) ; on the contrary , when compared with g / g , non - gg patients had a statistically higher consumption of drug both among those that used association of drugs ( p = 0.009 ; this value , however , did not reach the adjusted threshold of significance ) and among consumers of drugs of combination ( p < 0.0001).fig .
1interaction effect of genotype drug type on monthly drug number .
markers ( black circles and squares ) indicate mean , vertical bars denote 0.95 confidence intervals interaction effect of genotype drug type on monthly drug number . markers ( black circles and squares ) indicate mean , vertical bars denote 0.95 confidence intervals as showed in table 2 , in the multiple regression analysis , the val66met bdnf polymorphism ( beta = 0.33 , t = 3.669 , p < 0.0001 , f = 0.134 ) and the bdi score ( beta = 0.19 , t = 2.090 , p < 0.04 , f = 0.037 ) emerged as significant , independent predictors of the monthly drug consumption in moh patients .
no further variables , considered as possible predictors , entered the regression model ( see table 2).table 2results of stepwise multiple regression analysis : bdnf polymorphism ( g / g genotype ) and bdi score ( depressive symptoms ) are the predictors of monthly drug number in moh patientsvariablebetatpbdnf gene0.333.669<0.0001bdi0.192.090<0.04moh duration0.121.2420.22drug type0.080.9170.36gender0.080.9080.37employment0.070.7540.45current psychiatric diagnosis0.050.5320.60age of headache onset0.020.1670.87age0.010.1230.90significant predictors are in boldstepwise model summary : adjusted r = 0.137 , f2,104 = 9.424 , p < 0.0001 , f = 0.181stepwise model change statistics : first step , variable entered = bdnf gene , r change = 0.118 , f1,105 to enter / to remove = 14.030 , p < 0.0001 , f = 0.134 ; second step , variable entered = bdi , r change = 0.036 , f1,104 to enter / to remove = 4.368 , p < 0.04 , f = 0.037moh medication overuse headache ,
bdnf brain - derived neurotrophic factor , bdi beck depression inventory results of stepwise multiple regression analysis : bdnf polymorphism ( g / g genotype ) and bdi score ( depressive symptoms ) are the predictors of monthly drug number in moh patients significant predictors are in bold stepwise model summary : adjusted r = 0.137 , f2,104 = 9.424 , p
< 0.0001 , f = 0.181 stepwise model change statistics : first step , variable entered = bdnf gene , r change = 0.118 , f1,105 to enter / to remove = 14.030 , p < 0.0001 , f = 0.134 ; second step , variable entered = bdi , r change = 0.036 , f1,104 to enter / to remove = 4.368 , p < 0.04 , f = 0.037 moh medication overuse headache ,
bdnf brain - derived neurotrophic factor , bdi beck depression inventory
consistent with our working hypothesis , monthly drug consumption in moh patients resulted to be influenced by the examined bdnf polymorphism ( fig .
2 ) , both if univariate , and both if multiple regression analyses ( to control the association for such possible confounding factors ) are performed .
the relationship between monthly drug consumption and val66met polymorphism was also confirmed by effect size calculation ( medium effect at t test , small to medium effect at regression analysis ) .
the fact that a unique bdnf snp could account for these observed effects is noteworthy , because drug consumption is a multifactorial variable , surely influenced by many other genetic and environmental factors.fig .
g group , the black circles show a / a patients , the grey ones with black ring a / g patients .
vertical bars denote 0.95 confidence intervals monthly drug number in g / g and non - g / g patients .
g group , the black circles show a / a patients , the grey ones with black ring a / g patients .
vertical bars denote 0.95 confidence intervals moh can be considered as a clinical condition at the boundaries between drug addiction and chronic headache .
the hypothetical involvement of bdnf in the pathophysiology of moh was suggested by : ( 1 ) the association between the val66met polymorphism and drug abuse disorders , ( 2 ) the role of bdnf in modulating central and peripheral pain . in this study group ,
non - g / g patients showed a more severe clinical picture , because they had used a higher number of analgesic drugs in the previous 3 months , and had longer disease duration .
showing for the first time a role in moh , our findings confirm that somehow the examined bdnf polymorphism is involved , as an independent factor , in pathophysiology of this headache condition .
although it might sound too approximate , considering the association between met66 pro - bdnf and a reduced bdnf activity , it is tempting to speculate that in moh a lower bdnf activity is a worsening factor .
if moh is regarded as a drug abuse disorder , our data would agree with previous reports of an association between the val66met polymorphism and substance addition .
the proneness to abuse ( i.e. to have an impulse dyscontrol ) , previously reported in non - g / g subjects , would be expressed in moh patients by higher drug consumption . in light of this hypothesis ,
in fact , although the defining factor in moh is the number of days with drug intake , the variability of this value theatrically ranges from 10 to 30 ; on the contrary , a hypothetic upper limit for variability of monthly drug consumption does not exist and patients could be distributed on a wider range that better reflects their proneness to abuse ( and/or headache severity ) .
previous studies have attempted to evaluate bdnf role in headache disorders . in particular , it has been demonstrated an increased level of bdnf in cerebrospinal fluid ( csf ) of patients with a chronic daily headache ( with or without drug overuse ) , in comparison to healthy controls . furthermore , it has been shown a reduced level of platelet bdnf in the interictal phase of episodic migraineurs and cluster headache patients , in comparison to healthy controls .
more recently , a bdnf elevation in csf was demonstrated both in chronic migraine and in fibromyalgic patients .
these three studies were much different from our research for two reasons : the use of diverse classification criteria in the first study ; in general , the different study designs , with the lack of relations among bdnf levels , clinical markers of disease severity , and the patients bdnf genotype . however , these findings showed the involvement in chronic pain pathophysiology of bdnf up regulation , and might support indirectly our hypothesis that in moh patients a bdnf hypo - activity , related to val66met polymorphism , does not influence pain severity , but impulsive behaviour . to the best of our knowledge ,
pending specific studies on this topic , only speculations could be made about the chance that the higher drug intake is a consequence of putative bdnf - mediated higher pain sensitivity , or not .
however , the pain modulation is linked to bdnf release and up - regulation [ 23 , 24 ] , whereas val66met polymorphism is related with a reduction of bdnf activity .
these observations , though indirectly , might lend support the idea that bdnf influence on moh is played in terms of impulsivity modulation , as in other drug abuse disorder , and not pain hypersensitivity . moreover , a recent paper analysed influence of val66met polymorphism on episodic migraine .
examined polymorphism resulted neither associated to development of migraine ( comparing polymorphism frequency in a population of episodic migraineurs with a healthy control group ) , nor associated with differences in terms of clinical characteristics ( within the only migraine group ) .
consistent with these data , because val66met polymorphism seems to be unable to influence development and clinical features of migraine , we suppose that observed worsen effect of polymorphism on moh is likely driven by the a allele related proneness to abuse , and , in turn , that the monthly drug consumption in moh patients is not necessarily the expression of headache severity .
as reported above , we have also recorded the type of drug consumed by each patient and they were segregated in four groups : triptans , nsaids , association of different type of drugs and drugs of combination .
it is noteworthy that differences between g / g and non - g / g emerged only in the fourth group ( drugs of combination , all containing caffeine ) , and a trend was also found for the association of drugs . if one consider that non - g / g subjects are supposed to have a bdnf hypoactivity , and that bdnf is a kind of biological reward for presynaptic neurons , our observations could mean that the lack of reward induces non - g / g subjects in the continue search of different type of drugs , or in caffeine seeking ( for its psychotropic effect ) .
none of the patients reported to use drugs containing opioids ; therefore , we can not confirm our hypothesis with this class of drugs .
severity of depressive symptoms ( as measured by bdi score ) was found as another independent predictor of drug consumption in moh patients .
the role of depression in headaches has been previously reported and discussed elsewhere [ 26 , 27 ] .
although a relation between depression and val66met polymorphism was frequently found , in this study the g / g and non - g / g groups neither differ statistically for the severity of depressive symptoms , nor were they associated significantly with a psychiatric disorder .
maybe these associations are covered in our population by already described relationship between moh and psychiatric comorbidity that could not be further increasable by examined polymorphism . moreover , observed lack of association suggests that this bdnf polymorphism and depression might have an effect on moh monthly drug consumption through different and unrelated mechanisms . in summary ,
our results showed an influence of bdnf polymorphism val66met on drug consumption in moh patients .
the polymorphism is likely not related to severer forms of headache , but with a bdnf hypoactivity , in turn related to substance abuse behaviour , consistent with observed higher caffeine seeking .
therefore , aggravating effect of examined polymorphism in moh patients could be driven by its proneness to develop an impulsivity dyscontrol , the cornerstone of any substance abuse disorder .
in other words , we suppose that moh is not only a very severe headache , but also a complicated bio - behavioural disorder , not dissimilar to a kind of addictive disorder , preferentially developed by some migraineurs , aggravated by conditions unrelated to previous headache clinical features ; however , related to behavioural impulsivity dyscontrol , as other substance abuse disorders .
moreover , we would like to highlight that analysis of snps within functionally related genes appears as a good tool in headache research to identify mechanisms potentially involved in disease pathogenesis . | medication overuse headache ( moh ) can be considered a clinical condition at the boundaries between drug addiction and chronic pain disorder .
the common 196 g > a single - nucleotide polymorphism of bdnf gene , resulting in a valine 66 to methionine ( val66met ) , is related with behaviour disorders and substance abuse . with the aim of identifying a worsening factor in moh , rather than the detection of a specific risk factor for the development of the disease , we investigated whether the presence of a functional bdnf polymorphism might determine clinical differences within a group of 90 moh patients , particularly in monthly drug consumption , that is the hallmark of disease . directly comparing moh patients homozygous for g allele ( g / g ) with carriers of a allele ( non - g / g ) ,
we have observed 47 g / g genotypes and 60 non - g / g genotypes .
non - g / g had a higher consumption of monthly drug number ( cohen s d = 0.76 ) than g / g patients . at multiple regression analysis ,
the val66met bdnf polymorphism emerged as a significant independent predictor of analgesic drug consumption ( beta = 0.33 , cohen s f2 = 0.134 ) .
these findings showed an influence of examined bdnf polymorphism in the moh clinical features , supporting the idea that moh is a substance abuse disorder . |
accurate functional assessment of lesion severity is crucial for clinical decision making in patients with intermediate coronary stenosis .
fractional flow reserve ( ffr ) is determined by the ratio of pressure distal to the lesion ( pd)/pressure proximal to the lesion ( pa ) under maximal hyperemia .
the pressure gradient across the stenosis becomes greater as blood flow across the lesion increases during hyperemia induction .
ffr proves its value in diagnosing functionally significant stenosis , which is defined as an ffr threshold < 0.80 under maximal hyperemia . in europe ,
class ia recommendation is currently employed by ffr for the assessment of stable coronary stenosis ahead of revascularization when functional information is lacking .
the utility of the pressure wire remains under low level due to several limitations in measuring ffr , including the high cost , laboriousness of the manual procedure and possible adverse systemic effects of adenosine administration , such as bradycardia , chest pain and bronchospasm .
consequently , numerous research investigations have been carried out to evaluate the predictive value of the anatomic characteristics by intravascular ultrasound ( ivus ) and quantitative coronary angiography ( qca ) to the functional significance by ffr .
previous studies have showed moderate correlation between coronary stenosis severity assessed by qca / ivus and ffr .
however , functional significance of a coronary stenosis may not be determined by the severity of a stenosis alone , because in theory , maximal arteriolar vasodilatation is essential for minimal myocardial resistance in a given arterial territory , which ffr applies as a physiologic index of coronary artery stenosis .
ffr measurement is theoretically impossible when there are diabetes mellitus ( dm ) , hypertension , hyperlipidemia and other potential factors present , including smoking status , age and sex , etc .
, since associated functional and/or structural abnormalities in the coronary microvasculature could be caused by such factors .
when the functional significance of a lesion is assessed by qca or ivus , physicians should take demographic and anatomical variations of coronary artery into concern .
therefore , we performed this study to evaluate these factors that could influence ffr value .
the study enrolled 143 consecutive patients ( 203 lesions ) from december 2008 to april 2013 at peking university third hospital , which was approved by the peking university third hospital ethics committee in accordance with the declaration of helsinki , with informed consent waived due to the retrospective nature of the study .
all patients underwent anatomic and physiological assessment by off - line qca , ffr , respectively .
the 143 consecutive patients ( 203 lesions ) were referred to coronary angiography and found to have intermediate coronary lesions ( 40% to 70% stenosis by angiographic visual assessment ) .
exclusion criteria included : significant left main disease ( diameter stenosis > 50% ) , multiple lesions in the same vessel , myocardial infarction in the territory of the target coronary artery , angiographic evidence of collateral flow distally to the assessed lesion , significant valvular disease , bypass graft lesions , or left coronary dominant type .
reference segment and percent stenosis were measured in end - diastole in the projection where maximal narrowing was observed .
reference vessel diameter ( rvd ) was defined as the mean of diameters within the 5-mm proximal and distal non - affected segments in absence of collateral branches .
the following measurements were obtained by the qca software : lesion length ( ll ) , proximal and distal reference diameters ( acquired in the 5 mm non - affected segments next to the proximal and distal edge of the lesion ) , and minimum lumen diameter ( mld ) of the lesion .
average of proximal and distal rvd and percent diameter stenosis [ ds : ( rvd - mld)/rvd ] were obtained , and qangio xa software version 7.1.43.0 ( medis medical imaging systems , leiden , the netherlands ) was introduced for measurement . lesion location was determined according to the american heart association classification .
minor modification was made to reflect the amount of myocardium supplied by the target coronary segment .
proximal left anterior descending coronary artery ( lad ) was defined as the segment between the branching point of the left main stem and the first major branch ( septal or diagonal branch ) , and proximal left circumflex coronary artery ( lcx ) defined as the segment between the branching point of the left main stem and the first obtuse marginal branches .
proximal right coronary artery ( rca ) was defined as the segment between the origin of rca and the first major right ventricular branch .
ffr methodology was employed in a standard fashion , and adenosine or adenosine triphosphate ( atp : 140 g / kg per minute by intravenous infusion ) was for maximum hyperemia .
ffr was calculated as the ratio of the coronary pressure distal to the lesion measured by the pressure wire and the mean aortic pressure measured by the guiding catheter under maximum hyperemia .
student t - test was used for compare of continuous variables , and chi - square test used for analysis of discrete variables . in order to investigate independent predictors of ffr
< 0.8 , clinical variables were examined using multivariate analysis in a binary regression and linear regression analysis .
backward selection was introduced , and 0.05 was the significant level when entering an explanatory variable into the model .
the final model only included significant variables at p < 0.05 using the wald test .
receiver operating characteristic ( roc ) curve analysis was used to compare the diagnostic performance of mld and resting pd / pa with ffr , and the area under the curve ( auc ) was calculated ( spss , version 17.0 ) .
an area of 1 represents a perfect test , while an area of 0.5 represents a bad test .
accuracy of the mld and resting pd / pa to correctly predict the outcomes of ffr was calculated for predefined and clinically used cut - off values ( ffr = 0.80 ) .
the characteristics among different coronary artery trees were analyzed for statistically significant differences using the one - way anova , followed by tukey post - hoc test .
the study enrolled 143 consecutive patients ( 203 lesions ) from december 2008 to april 2013 at peking university third hospital , which was approved by the peking university third hospital ethics committee in accordance with the declaration of helsinki , with informed consent waived due to the retrospective nature of the study .
all patients underwent anatomic and physiological assessment by off - line qca , ffr , respectively .
the 143 consecutive patients ( 203 lesions ) were referred to coronary angiography and found to have intermediate coronary lesions ( 40% to 70% stenosis by angiographic visual assessment ) .
exclusion criteria included : significant left main disease ( diameter stenosis > 50% ) , multiple lesions in the same vessel , myocardial infarction in the territory of the target coronary artery , angiographic evidence of collateral flow distally to the assessed lesion , significant valvular disease , bypass graft lesions , or left coronary dominant type .
reference segment and percent stenosis were measured in end - diastole in the projection where maximal narrowing was observed .
reference vessel diameter ( rvd ) was defined as the mean of diameters within the 5-mm proximal and distal non - affected segments in absence of collateral branches .
the following measurements were obtained by the qca software : lesion length ( ll ) , proximal and distal reference diameters ( acquired in the 5 mm non - affected segments next to the proximal and distal edge of the lesion ) , and minimum lumen diameter ( mld ) of the lesion .
average of proximal and distal rvd and percent diameter stenosis [ ds : ( rvd - mld)/rvd ] were obtained , and qangio xa software version 7.1.43.0 ( medis medical imaging systems , leiden , the netherlands ) was introduced for measurement . lesion location was determined according to the american heart association classification .
minor modification was made to reflect the amount of myocardium supplied by the target coronary segment .
proximal left anterior descending coronary artery ( lad ) was defined as the segment between the branching point of the left main stem and the first major branch ( septal or diagonal branch ) , and proximal left circumflex coronary artery ( lcx ) defined as the segment between the branching point of the left main stem and the first obtuse marginal branches .
proximal right coronary artery ( rca ) was defined as the segment between the origin of rca and the first major right ventricular branch .
ffr methodology was employed in a standard fashion , and adenosine or adenosine triphosphate ( atp : 140 g / kg per minute by intravenous infusion ) was for maximum hyperemia .
ffr was calculated as the ratio of the coronary pressure distal to the lesion measured by the pressure wire and the mean aortic pressure measured by the guiding catheter under maximum hyperemia .
student t - test was used for compare of continuous variables , and chi - square test used for analysis of discrete variables . in order to investigate independent predictors of ffr
< 0.8 , clinical variables were examined using multivariate analysis in a binary regression and linear regression analysis .
backward selection was introduced , and 0.05 was the significant level when entering an explanatory variable into the model .
the final model only included significant variables at p < 0.05 using the wald test .
receiver operating characteristic ( roc ) curve analysis was used to compare the diagnostic performance of mld and resting pd / pa with ffr , and the area under the curve ( auc ) was calculated ( spss , version 17.0 ) .
an area of 1 represents a perfect test , while an area of 0.5 represents a bad test .
accuracy of the mld and resting pd / pa to correctly predict the outcomes of ffr was calculated for predefined and clinically used cut - off values ( ffr = 0.80 ) .
the characteristics among different coronary artery trees were analyzed for statistically significant differences using the one - way anova , followed by tukey post - hoc test .
there was no significant difference in baseline clinical characteristics between patients with ffr < 0.8 and those with ffr 0.8 , except that patients with ffr < 0.8 were younger ( p = 0.001 ) .
coronary lesions with ffr < 0.8 had greater ds and as , less rvd , mld and resting pd / pa in contrast to ffr 0.8 .
there was a trend towards higher frequency of lesion location in lad when ffr < 0.8 ( p = 0.09 ) .
ffr at maximum hyperemia was significantly correlated with qca - measured as ( r = 0.251 , p = 0.000 ) , mld ( r = 0.372 , p = 0.000 ) , and showed a weak correlation with ds% ( r = 0.210 , p = 0.000 ) and rvd ( r = 0.217 , p = 0.002 ) .
; ds : diameter stenosis ; ffr : fractional flow reserve ; lad : left anterior descending coronary artery ; lcx : left circumflex coronary artery ; ll : lesion length ; mld : minimum lumen diameter ; pd / pa : ratio of the pressures distal and proximal ; qca : quantitative coronary angiography ; rca : right coronary artery ; rvd : reference vascular diameter .
multiple linear and binary logistic regression analyses included age , male sex , hypertension , diabetes , hyperlipidemia , current smoker , family history of chd , different coronary lesion location , as , ds , rvd , ll , and mld . in the overall cohort of 203 lesions , mld ,
ds and different lesion location were predictors for ffr as continuous variables independent of possible confounding variables ( p < 0.05 ) , and the independent determinants of ffr < 0.80 were age , mld and different lesion location .
the cutoff value of mld to predict ffr < 0.80 was 1.565 ( 63.4% sensitivity , 62.1% specificity , auc = 0.703 ; 95% ci : 0.6300.776 ; p = 0.000 ) .
the resting pd / pa is the pressure gradient over coronary stenosis before the adenosine stress , which is surely correlated to ffr , thus was not listed as a variable in the predictive model of ffr .
we found that the resting pd / pa was positively correlated with ffr ( r = 0.723 , p = 0.000 ) , and the auc of resting pd / pa to predict ffr < 0.8 was 0.858 .
when we analyzed the independent factors that influenced resting pd / pa with a linear regression model , it was found that coronary artery and mld were still the predictors , and ds ( % ) and as ( % ) were the other two predictive factors ( data were not shown here ) .
the resting pd / pa value , similar to the ffr value , tended to be lower in lad compared to rca or lcx .
the functional significance of stenosis was different for lesions at different coronary artery locations , and there were 51 lesions ( 39% ) with ffr < 0.8 among the 130 intermediate lesions located in lad , 14 ( 32.6% ) in rca lesions and five ( 16.7% ) in lcx lesions .
although there was no statistical significance ( p = 0.09 ) , the ffr value in 130 lad lesions was significantly lower compared with lcx lesions ( 0.81 0.09 vs. 0.87 0.08 , p = 0.004 ) , and tended to be lower than that in rca lesions ( 0.81 0.09 vs. 0.84 0.10 , p = 0.172).there were no significant differences in the demographic characteristics among these three groups , and the differences in anatomic and functional characteristics are shown in table 3 .
ffr : fractional flow reserve ; lad : left anterior descending coronary artery ; mld : minimum lumen diameter .
as to the lesion location of different coronary arteries , there was no difference either in ffr value or percentage of ffr < 0.8 between proximal and distal lesion locations , as shown in table 4 .
. as : area stenosis ; ds : diameter stenosis ; ffr : fractional flow reserve ; lad : left anterior descending coronary artery ; lcx : left circumflex coronary artery ; ll : lesion length ; mld : minimum lumen diameter ; pd / pa : ratio of the pressures distal and proximal ; rca : right coronary artery ; rvd : reference vascular diameter .
there was no significant difference in baseline clinical characteristics between patients with ffr < 0.8 and those with ffr 0.8 , except that patients with ffr < 0.8 were younger ( p = 0.001 ) .
coronary lesions with ffr < 0.8 had greater ds and as , less rvd , mld and resting pd / pa in contrast to ffr 0.8 .
there was a trend towards higher frequency of lesion location in lad when ffr < 0.8 ( p = 0.09 ) .
ffr at maximum hyperemia was significantly correlated with qca - measured as ( r = 0.251 , p = 0.000 ) , mld ( r = 0.372 , p = 0.000 ) , and showed a weak correlation with ds% ( r = 0.210 , p = 0.000 ) and rvd ( r = 0.217 , p = 0.002 ) .
; ds : diameter stenosis ; ffr : fractional flow reserve ; lad : left anterior descending coronary artery ; lcx : left circumflex coronary artery ; ll : lesion length ; mld : minimum lumen diameter ; pd / pa : ratio of the pressures distal and proximal ; qca : quantitative coronary angiography ; rca : right coronary artery ; rvd : reference vascular diameter .
multiple linear and binary logistic regression analyses included age , male sex , hypertension , diabetes , hyperlipidemia , current smoker , family history of chd , different coronary lesion location , as , ds , rvd , ll , and mld . in the overall cohort of 203 lesions , mld ,
ds and different lesion location were predictors for ffr as continuous variables independent of possible confounding variables ( p < 0.05 ) , and the independent determinants of ffr < 0.80 were age , mld and different lesion location .
the cutoff value of mld to predict ffr < 0.80 was 1.565 ( 63.4% sensitivity , 62.1% specificity , auc = 0.703 ; 95% ci : 0.6300.776 ; p = 0.000 ) .
the resting pd / pa is the pressure gradient over coronary stenosis before the adenosine stress , which is surely correlated to ffr , thus was not listed as a variable in the predictive model of ffr .
we found that the resting pd / pa was positively correlated with ffr ( r = 0.723 , p = 0.000 ) , and the auc of resting pd / pa to predict ffr < 0.8 was 0.858 .
when we analyzed the independent factors that influenced resting pd / pa with a linear regression model , it was found that coronary artery and mld were still the predictors , and ds ( % ) and as ( % ) were the other two predictive factors ( data were not shown here ) .
the resting pd / pa value , similar to the ffr value , tended to be lower in lad compared to rca or lcx .
the functional significance of stenosis was different for lesions at different coronary artery locations , and there were 51 lesions ( 39% ) with ffr < 0.8 among the 130 intermediate lesions located in lad , 14 ( 32.6% ) in rca lesions and five ( 16.7% ) in lcx lesions .
although there was no statistical significance ( p = 0.09 ) , the ffr value in 130 lad lesions was significantly lower compared with lcx lesions ( 0.81 0.09 vs. 0.87 0.08 , p = 0.004 ) , and tended to be lower than that in rca lesions ( 0.81 0.09 vs. 0.84 0.10 , p = 0.172).there were no significant differences in the demographic characteristics among these three groups , and the differences in anatomic and functional characteristics are shown in table 3 .
ffr : fractional flow reserve ; lad : left anterior descending coronary artery ; mld : minimum lumen diameter . as to the lesion location of different coronary arteries
, there was no difference either in ffr value or percentage of ffr < 0.8 between proximal and distal lesion locations , as shown in table 4 .
. as : area stenosis ; ds : diameter stenosis ; ffr : fractional flow reserve ; lad : left anterior descending coronary artery ; lcx : left circumflex coronary artery ; ll : lesion length ; mld : minimum lumen diameter ; pd / pa : ratio of the pressures distal and proximal ; rca : right coronary artery ; rvd : reference vascular diameter .
the present study revealed that : ( 1 ) the functional significance of moderate coronary stenosis was influenced by many other factors except lesion severity ; ( 2 ) the different coronary artery tree of the lesion location did influence the ffr value , although the lesion location in the artery tree had no effect on the ffr value ; and ( 3 ) age of the patients should be taken into consideration when the functional significance of a lesion was evaluated by qca - measured anatomic parameters .
the relation between anatomic characteristics by qca and functional significance by ffr has been extensively studied .
it is suggested that lesion length , ds and minimum lumen diameter may be correlated with ffr. nevertheless , the correlation between stenosis severity and pressure - derived physiological parameters is either moderate or weak , but there is a large dispersion of ffr values for a similar angiographic degree of stenosis , consistent with our study .
we found that ffr at maximum hyperemia was significantly correlated with qca - measured as , mld , lesion length , ds , and the rvd , but the correlation was moderate or weak .
the hagen - poiseuille equation states that flow in a tube is determined by pr/8l , where l and r are the length and radius of the tube , respectively , p refers to the pressure gradient across the tube , and means the viscosity .
thus , there are also many other factors that may affect coronary blood flow except the intravascular - derived severity of a lesion . according to hagen - poiseulle equation ,
radius was much more powerful than length for the impact of flow in a tube .
this was consistent with our finding that mld was an independent factor in the multiple variable regression analysis , while length was not so powerful . in our study
, it was also found that a different coronary artery tree could influence the ffr value , which was an independent predictor of ffr < 0.8 .
the ffr value seemed higher in lcx lesions , lower in the rca lesions and lowest in lad lesions .
and the percentage of lad location in ffr < 0.8 appeared higher compared to non - lad location .
ffr : fractional flow reserve ; lad : left anterior descending coronary artery ; lcx : left circumflex coronary artery ; rca : right coronary artery .
the two independent factors of ffr value were also found by park , et al . in their study , it was revealed that the independent predictors for mismatch ( ds 50% and ffr > 0.80 ) were advanced age , non - left anterior descending artery location , and other anatomic characteristics , and conversely , reverse mismatch ( ds < 50% and ffr < 0.80 ) was independently associated with younger age , lad location , and other factors .
the importance of lad in the functional significance of a lesion has recently been reported by other investigators . , they found that an intermediate lesion was 3.44.4 times more likely to be functionally significant if it was located at the lad instead of any other arteries , attributed to the presence of myocardial ischemia which was determined by both the lesion severity and the amount of myocardium supplied , , as a much larger myocardial mass was supplied by lad compared with other coronary arteries .
but there was no difference in ffr value , or ffr < 0.8 between the proximal and distal stenosis along the same coronary artery tree in the present study , although koo , et al . found that the probability of a functionally significant intermediate lesion was further increased [ odds ratio ( or ) : 2.97 ] if the lesion was proximal .
furthermore , it was recently reported that the resting baseline pd / pa was linearly related to ffr in intermediate lesions , whereby a certain range of the resting baseline pd / pa values strongly predicted functional significance with excellent npv and high sensitivity , or excellent ppv and high specificity , similar to our study .
when we analyzed the independent factors that influenced resting pd / pa with a linear regression model , it was found that coronary artery and mld were still the predictors and the ds and as were the other two predictive factors ( data not shown here ) .
the resting pd / pa value , similar to the ffr value , tended to be lower in lad than in rca or lcx .
we saw the accordance of mld and the lesion location in different coronary arteries , which influenced the ratio of distal coronary pressure to aortic pressure under pre- or post - hyperemia .
it was implied that the mld and the lesion location in different coronary arteries were two of the most important factors related to the functional significance of an intermediate stenosis . while the discrepancy of age , ds and as maybe linked to the hyperemia .
the effect of ds and as on the ratio of distal to proximal coronary pressure can be deleted by that of vascular dilation by adenosine . with regard to age
adenosine is an important cardio - protective agent that works via several adenosine receptor subtypes to regulate cardiovascular activity .
have already found that , in rat heart , adenosine a1 and a2 receptor expression decreases with age .
therefore , we speculate that the decreased adenosine receptor may weaken the effect of hyperemia induced by adenosine at the same dosage , as may explain the p across the lesion decreased and ffr value tended to be higher .
first , this was a retrospective study with limitations inherent to this type of investigation .
second , it was a single - center study with a relatively smaller sample size and hence our findings might not be applicable to other study populations
. the lower prevalence of ffr < 0.80 ( n = 71 , 35% ) , rca ( n = 43 , 21% ) and lcx ( n = 30 , 17% ) in the study group may also affect statistical analysis .
third , the morphological assessment of lesions was exclusively qca . during the process of atherosclerosis
, fluid dynamic conditions may mold stenosis , making them progressively longer , or changed in shape in both inward and outward remodelling .
assessment of anatomic characteristics by qca may not be as accurate as that by ivus , and even ivus ca n't reliably evaluate the anatomic characteristics . in intermediate coronary artery stenosis , functional significance is related to many factors , including anatomic and demographic characteristics .
the correlation between anatomical and functional severity of coronary artery disease defined by qca and ffr is poor .
age and the lesion location in different coronary arteries can influence the functional significance , and therefore such factors should be taken into account when judging the benefit of revascularization , or performing functional assessment that overcomes limitations of simple stenosis quantification in making clinical decisions .
there are several limitations of the present study . first , this was a retrospective study with limitations inherent to this type of investigation .
second , it was a single - center study with a relatively smaller sample size and hence our findings might not be applicable to other study populations
. the lower prevalence of ffr < 0.80 ( n = 71 , 35% ) , rca ( n = 43 , 21% ) and lcx ( n = 30 , 17% ) in the study group may also affect statistical analysis .
third , the morphological assessment of lesions was exclusively qca . during the process of atherosclerosis , fluid dynamic conditions may mold stenosis , making them progressively longer , or changed in shape in both inward and outward remodelling .
assessment of anatomic characteristics by qca may not be as accurate as that by ivus , and even ivus ca n't reliably evaluate the anatomic characteristics .
in intermediate coronary artery stenosis , functional significance is related to many factors , including anatomic and demographic characteristics .
the correlation between anatomical and functional severity of coronary artery disease defined by qca and ffr is poor .
age and the lesion location in different coronary arteries can influence the functional significance , and therefore such factors should be taken into account when judging the benefit of revascularization , or performing functional assessment that overcomes limitations of simple stenosis quantification in making clinical decisions . | objectiveto analyze the influencing factors of the functional significance determined by fractional flow reserve ( ffr ) in intermediate coronary artery stenosis.methodsthe study enrolled 143 patients with 203 intermediate coronary lesions .
pressure - derived ffr of these lesions was gained at maximal hyperemia induced by intravenous adenosine infusion . an ffr < 0.80 was considered as abnormal functional significance .
anatomic parameters at the lesion sites were obtained by off - line quantitative coronary angiography analysis ( qca ) .
the predictive value of the demographic characteristics and anatomic parameters for ffr in these intermediate lesions was assessed using multiple linear and binary logistic regression analysis.resultsoverall , ffr < 0.8 was found in 70 ( 34% ) of the total 203 intermediate coronary lesions .
ffr values were positively correlated with qca - measured minimum lumen diameters ( mld , r = 0.372 , p = 0.000 ) and the reference vessel diameters ( rvd , r = 0.217 , p = 0.002 ) were negatively correlated with percent area stenosis ( as , r = 0.251 , p = 0.000 ) and percent diameter stenosis ( ds , r = 0.210 , p = 0.000 ) .
age , mld and the lesion location in different coronary arteries were the independent determinants of ffr < 0.8.conclusionsmld can predict the functional significance of intermediate coronary stenosis , while age and the lesion location in different coronary arteries should be taken into account as important influencing factors of ffr values . |
type 2 diabetes ( t2d ) is one of the leading causes of mortality worldwide , largely due to vascular complications including myocardial infarction and stroke [ 1 , 2 ] .
endothelial function , a key determinant of vascular health , is impaired in the diabetic vasculature primarily due to reduced nitric oxide ( no ) bioavailability that is brought about by a combination of uncoupling and inhibition of endothelial nitric oxide synthase ( enos ) and an excessive production of reactive oxygen species ( ros ) .
loss of no bioactivity , leading to endothelial dysfunction , is known to predispose patients to developing secondary complications such as stroke .
therefore understanding the mechanisms associated with vascular dysfunction is critical to the development of therapeutic targets in the hope of preventing t2d - related morbidity .
the nad - dependent deacetylase , sirtuin 1 ( sirt1 ) , is abundantly expressed in the vasculature and deacetylates a number of substrates that are key modulators of vascular function including enos and foxo1 .
sirt1 has been reported to be downregulated in peripheral blood mononuclear cells of obese humans and in the liver and pancreas of rodents on a high - fat diet .
high - fat feeding resulted in a significant decrease in mouse aorta sirt1 protein expression , while no changes were seen in the cerebral vasculature in obese zucker rats . sirt1
interacts with specific double - stranded repeat sequences of dna at the extremities of chromosomes [ 5-(ttaggg)n-3 ] , known as telomeres . during each round of cell division , telomeres shorten until they reach a critical length . at this point , the cell can no longer replicate and it becomes either senescent or apoptotic , thereby creating an accelerated aging phenotype and increasing risk of secondary disease .
telomere shortening can be prematurely accelerated in vitro in the face of oxidative stress , a hallmark of t2d . therefore restoring telomere
although epidemiological studies have suggested an association between t2d and shortened leukocyte telomeres [ 17 , 18 ] , a direct cause and effect relationship has never been established .
all animal procedures were carried out in accordance with the australian code for the responsible conduct of research and were approved by the flinders university animal ethics committee .
male hooded wistar rats ( n = 17 ) were bred and housed under controlled room temperature with a 12 : 12 hour light - dark cycle in the school of medicine animal care facility at flinders university . after weaning at three weeks of age
the diabetic group ( n = 9 ; t2d ) was placed on a high - fat diet which consisted of 45 kcal% fat , 35 kcal% carbohydrate , and 20 kcal% protein ( d12451 ; open source diet ) .
the chow was custom - made by gordon 's specialty stockfeed ( yanderra , australia ) .
the remaining group ( n = 8 ; control ) was maintained on a control diet which contained 10 kcal% fat , 70 kcal% carbohydrate , and 20 kcal% protein ( d12450h ; open source diet ) .
after 18 weeks on the diet regimen , the diabetic group was given an intraperitoneal injection of streptozotocin ( 30 mg / kg body weight ) dissolved in saline while the control group was injected with the same volume of vehicle .
previous studies have shown that a high - fat feeding followed by a low dose of streptozotocin induces a mild impairment of insulin secretion , similar to that seen in late stage t2d .
five days prior to sacrifice , rats were anesthetized with 3% isoflurane and their body temperature was maintained on a heating pad at 37c .
blood pressure was measured using mlt125/r pulse transducer / pressure cuff and in125 nibp controller ( ad instruments , australia ) and recorded using powerlab software ( ad instruments , australia ) .
systolic pressure was determined from the average of three consecutive readings obtained from each rat . on the day of sacrifice
blood glucose was measured from the tail vein using an accu - chek performa glucometer and test strips ( roche diagnostics , indianapolis , in , usa ) .
the animals were then decapitated using a rodent guillotine and trunk blood obtained for measurement of serum insulin and free fatty acids , plasma triglycerides , and c - reactive protein ( crp ) and for extraction of dna using a dneasy blood and tissue kit ( qiagen ) according to the manufacturer 's instructions .
the dna concentration of each extraction was measured in triplicate using a nanodrop 2000 ( thermo scientific ) .
the left and right femoral artery were excised from animals , cleaned of connective tissue , and washed to remove blood .
the size of each artery was 0.9 cm in length , 2 mm in en face width , and approximately 1 mg in weight .
dna was extracted using a qiaamp dna micro kit ( qiagen ) according to the manufacturer 's instructions and stored at 80c until analysis .
the brain was removed and the middle cerebral arteries were carefully harvested and pooled into 180 l modified radioimmunoprecipitation ( ripa ) buffer containing 50 mm tris , ph 7.8 with 150 mm nacl , 1% triton x-100 , 0.5% nonidet p-40 , 0.25% sodium deoxycholate , and 1 mm pmsf and protease inhibitor cocktail ( roche , indianapolis , in , usa ) . total protein was extracted as previously described and stored at 80c .
total protein was measured using ezq protein quantitation ( roche diagnostics , indianapolis , in , usa ) .
cerebral artery lysate was then combined with laemmli sample buffer and heated to 95c for 5 mins .
equal amounts of protein ( 18 micrograms ) were loaded onto each lane of a stain - free gel ( bio - rad laboratories , hercules , ca , usa ) alongside dual color precision plus protein standard ( bio - rad laboratories ) . total protein loaded on the gel and then transferred to the low fluorescence polyvinylidene difluoride ( pvdf )
membrane was imaged ( chemidoc mp imager , bio - rad laboratories ) and analysed using image lab 4.0.1 software ( bio - rad laboratories ) .
previous studies have shown that this method of protein normalization is superior to that of either total protein stains ( e.g. , sypro ruby , amido black , and coomassie blue ) or antibody loading controls .
membranes were probed with an antibody directed against either sirt1 ( abcam ) , enos ( bd transduction labs ) , nox2 ( bd transduction laboratories ) , manganese superoxide dismutase ( mnsod ; millipore ) , p66 ( millipore ) , or 3-nitrotyrosine ( abcam ) overnight at 4c .
the next day , membranes were washed and incubated with the appropriate secondary antibody ( jackson immunoresearch ) .
bands were visualized using enhanced chemiluminescence , captured with a digital acquisition system ( fujifilm , japan ) and quantified ( carestream molecular imaging software , rochester , nt , usa ) .
mean telomere length was measured using a singleplex assay similar to that described by cawthon .
the telomere and single - copy gene ( -actin ) were amplified in separate singleplex reactions because the efficiency of each amplicon was optimal in different mastermixes ( assessed in pilot studies ) .
the telomere primers were used as previously described by cawthon and used at a concentration of 900 nm . the single - copy gene ( -actin ) primer sequences ( written 5 3 ) were -actin - f , agg tca tca cta tcg gca atg a , and -actin - r , gag act aca act tac cca gga agg aa , and used at a final concentration of 2 m .
each qpcr reaction was conducted in a total volume of 20 l using 384-well plates and performed on a viia 7 real - time pcr instrument ( life technologies , foster city , ca ) .
the telomere assay was amplified in a custom - made mastermix as described by cawthon and the -actin assay was amplified in power sybr green pcr mastermix ( life technologies ) .
for the telomere assay , a standard curve was run alongside the experimental samples consisting of a calibrator dna sample which was diluted serially by 5-fold to produce five concentrations of dna ranging from 515 to 0.824 ng / ml . for the -actin assay
, a standard curve was run alongside the experimental samples consisting of the same calibrator dna sample which was diluted serially by 5-fold to produce five concentrations of dna ranging from 5150 to 8.24 ng / ml . the calibrator was rat dna extracted from either whole blood or femoral artery .
the amount of telomere was calculated as t = e , where e was the efficiency of the telomere primer calculated from the standard curve , cqcalibrator was the cq of the 1 : 5 dilution on the standard curve , and cqsample was the cq of the experimental sample .
the amount of -actin ( reference gene ) was calculated as s = e , where e was the efficiency of the -actin primer calculated from the standard curve , cqcalibrator was the cq of the 1 : 5 dilution on the standard curve , and cqsample was the cq of the experimental sample .
relative telomere length ( t / s ) was estimated as the ratio between the amount of telomere ( t ) and reference gene ( s ) .
the -actin and telomere primers were synthesized by geneworks ( thebarton , sa , australia ) and dissolved in water for injection ( glaxosmithkline ) .
statistical comparisons were performed using graphpad instat version 3 software ( graphpad software , la jolla , ca , usa ) using an unpaired t - test .
the initial power analysis was performed based on previous telomere length studies in rodents [ 24 , 25 ] .
the experiment was powered at 80% to detect a 20% change in telomere length , assuming a two - tailed alpha level of 5% .
rats fed a high - fat diet for 18 weeks weighed significantly more than age - matched control rats fed a control diet ( p = 0.029 ; see table 1 ) .
animals then received either a low dose of streptozotocin or vehicle and the diet regimen was continued for another eight weeks .
one rat died just prior to streptozotocin injection and two rats died the following week after streptozotocin injection .
systolic blood pressure was similar in both t2d rats and control rats ( p = 0.16 ; 113 8 mmhg versus 105 5 mmhg , resp . ) . on the day of sacrifice
, t2d rats weighed significantly less than the control group ( p = 0.0001 ; 358 12 g versus 432 7 g , resp . ) . fasting
plasma glucose levels were significantly higher in t2d rats compared to control rats ( p < 0.0001 ; 24.2 1.3 mm versus 8.6 0.3 mm , resp . ) . circulating free fatty acid concentration
was significantly elevated in t2d rats compared to control rats ( p = 0.012 ; 0.51 0.06 mm versus 0.34 0.02 mm , resp . ) .
plasma insulin concentration was significantly lower in t2d rats compared to the control rats ( p = 0.0028 ; 0.5 0.1 ng / ml versus 2.8 0.5 ng / ml , resp . ) , a reflection of streptozotocin - induced pancreatic -cell dysfunction . as a marker of systemic inflammation , plasma crp
was significantly elevated in t2d compared to control rats ( p < 0.0001 ; see table 1 ) . as shown in figure 2(a ) , arterial telomere length was comparable between t2d rats and control rats ( p = 0.28 ; 0.92 0.04 versus 0.81 0.08 , resp . ) . however leukocyte telomere
length was significantly shorter in t2d compared to the control group ( p = 0.034 ; 0.49 0.14 versus 1.09 0.19 , resp . ) .
the pcr amplification products were visualized by agarose gel electrophoresis , ethidium bromide staining , and uv transillumination ( see figure 2(b ) ) .
the -actin pcr product was observed at the expected size of 81 base pairs while the telomere pcr product showed a main product at 82 base pairs ( the sum of the length of the two telomere primers ) and a smear to 150 base pairs , reflecting longer pcr products due to staggered annealing of the primers .
vascular sirt1 and enos protein expression were significantly lower in t2d rats compared to their control counterparts ( p = 0.0082 and p = 0.011 , resp . ; see figure 3 ) .
mnsod and p66shc protein levels were significantly lower in t2d rats compared to control rats ( p = 0.039 and p = 0.028 , resp . ) ; however total 3-nitrotyrosine - containing proteins and nox2 were comparable between groups ( figure 4 ) .
firstly , we have demonstrated that t2d causes selective accelerated telomere shortening in circulating leukocytes but preserved telomere length in arteries . to our knowledge , this is the first direct evidence of leukocyte telomere shortening in t2d .
secondly , although hyperglycemia leads to blunted sirt1 and telomere shortening , we have shown in vivo that hyperglycemia and a deficit in vascular sirt1 per se are not sufficient to prematurely shorten vascular telomeres .
here we have shown that rats gained significantly more weight after an 18-week high - fat diet regimen ( see table 1 ) .
injection of a low dose of streptozotocin significantly increased blood glucose levels in t2d rats , which is consistent with other reports . circulating
insulin levels were significantly decreased , a feature of clinical late stage t2d when the pancreatic beta cells become impaired and insulin secretion is reduced .
the animals also showed evidence of dyslipidemia that is in line with elevated plasma free fatty acids observed in human t2d that has been linked to the onset of insulin resistance .
the t2d rats in our study had significantly elevated levels of crp , confirming that this animal model is a good representation of t2d in humans which is associated with prolonged low - grade inflammation .
we found that leukocyte telomere length is shorter in t2d rats compared to control rats .
our findings are in line with accumulating clinical studies showing a correlation between t2d and shorter leukocyte telomeres [ 17 , 18 , 34 , 35 ] .
one possible explanation for the shorter leukocyte telomeres in t2d is that it reflects increased leukocyte turnover due to the systemic low - grade inflammation .
however clinical studies have failed to show a significant inverse correlation between leukocyte telomere length and markers of inflammation [ 3537 ] .
tentolouris and colleagues have previously reported a correlation between telomere length and nitrosative stress .
taken together , we speculate that the observed leukocyte telomere attrition may be due , at least in part , to nitrosative stress which is absent within the vasculature in this animal model of t2d ( see below ) . shortened vascular telomere length has been previously documented in human coronary heart disease , atherosclerosis , and abdominal aortic aneurysms . because vascular biopsies can not be taken from obese or t2d patients without overt vascular pathology , leukocyte telomere measurements have been used as a surrogate for predicting vascular disease risk .
we have shown for the first time that arterial telomere length is preserved in t2d rats compared to control rats .
although sirt1 depletion and hyperglycemia have previously been shown to independently accelerate telomere shortening , our results have shown that hyperglycemia and blunted vascular sirt1 in vivo were not sufficient to prematurely shorten arterial telomeres .
our experiments were sufficiently powered at 80% to detect a 20% change in telomere length .
therefore a smaller change in telomere length would not have been detected and as a result we can not rule out the possibility that there are more subtle changes in telomere length within the t2d vasculature .
nevertheless our data suggests that leukocyte telomere length may not be an optimal surrogate for vascular telomere length in t2d .
sirt1 protein expression was significantly decreased in resistance - sized vessels harvested from t2d rats compared to control rats on a normal diet ( standard chow ) .
our findings are in line with studies showing a decline in sirt1 levels at the level of the macrovasculature ( aorta ) after six months of high - fat feeding .
however it is important to assess protein expression within the resistance - sized vessels , since these vessels are fundamental to the control of blood flow .
we have now confirmed for the first time that sirt1 protein expression is blunted within the microvasculature in t2d .
sirt1 activity has been shown to be controlled by both nutritional status and the cellular redox status ( nad / nadh ratio ) . in a nutrient rich environment ,
nad / nadh ratio is blunted due to an increased rate of reduction of nad to nadh , leading to a decrease in sirt1 activity .
endothelial progenitor cells , human umbilical vein endothelial cells , and mouse brain endothelial cells ( sokoya et al . , unpublished findings ) cultured in high glucose
recent studies have also shown that oxidative stress directly inhibits sirt1 activity via oxidative posttranslational modifications .
therefore , both a reduction in cellular redox status and an increase in oxidant stress may be working together to reduce vascular sirt1 expression .
the production of endothelial - derived no is key in mediating relaxation of the cerebral vasculature .
our study has found that t2d leads to significantly lower enos protein levels within cerebral arteries .
this may be mediated in part by crp , which has been shown to directly decrease enos protein expression and activity and/or hyperglycemia that promotes enos glycosylation thereby reducing the active form of no .
a reduction in total enos production suggests that no bioavailability may be reduced , potentially leading to blunted endothelial - mediated relaxations , which have previously been shown in this model .
our observation that vascular mnsod protein expression is blunted in t2d suggests that the antioxidant pathway is being overwhelmed , presumably due to an increase in superoxide production within the vasculature .
this is consistent with other data showing downregulation of mnsod in the vasculature after high - fat feeding .
vascular p66shc protein expression was also significantly lower in t2d rats compared to controls . as a redox mitochondrial enzyme
however more recent studies have localized p66shc to the cytoplasm where it is regulated by the redox sensitive transcription factor , nfe2-related factor 2 ( nrf2 ) .
nrf2 regulates the antioxidant response element ( are / epre ) mediated expression of antioxidant enzymes . in this way ,
our finding of blunted vascular p66shc protein levels in t2d is in accordance with a recent report of reduced nrf2 signaling in mouse brain after high - fat feeding .
interestingly , sirt1 itself has been shown to play an important role in regulating antioxidant gene expression in endothelial cells by enhancing the stability of the foxo3a / pgc-1 complex , which promotes antioxidant gene expression . in this way , blunted vascular sirt1 in t2d could mediate , at least in part , the observed decrease in antioxidant protein expression .
vascular nadph oxidases are one of the major sources of ros in the cardiovascular system . in our study
, we examined a subunit of nadph oxidase , nox2 , which has previously shown to be significantly increased in mouse aorta after high - fat feeding .
however , in our animal model of t2d , nox2 levels were uncompromised in cerebral artery homogenates .
nevertheless there are additional sources of superoxide including cyclooxygenase , xanthine oxidase , and other nadph oxidases such as nox1 and nox5 .
superoxide can react with no to form peroxynitrite which in turn promotes nitration of tyrosine residues in proteins leading to nitrotyrosine - containing proteins . in the present study
, we found that the abundance of nitrotyrosine - modified proteins was comparable in cerebral artery lysate from t2d rats compared to control rats .
this is in contrast to studies in mouse aorta , rat aorta , pig coronary artery , and rat epineurial arterioles that have shown increased nitrotyrosine expression following high - fat feeding . taken together , our data suggest that , although antioxidant defence capacity is reduced , there appears to be no vascular nitrosative stress in our experimental model of t2d .
a number of caveats need to be recognised here in terms of interpretation of our data .
firstly , markers of oxidative and nitrosative stress were measured within the cerebral vasculature while telomere length was measured in dna extracted from the peripheral vasculature . ideally ,
telomere length would have been measured within cerebral arteries ; however this was impossible given the small size of rodent cerebral arteries .
secondly , the femoral artery comprises a single layer of endothelial cells and a few layers of smooth muscle cells .
if there are changes in endothelial cell telomere length in the absence of smooth muscle cell changes , these would not be detected .
thirdly , the qpcr assay as used in the present study measures mean telomere length .
there is evidence that a cell may be triggered to enter senescence by a single critically short telomere .
therefore it remains a possibility that there is telomere dysfunction within the t2d vasculature , driven by a small number of critically short telomeres .
future studies could address this possibility using high - throughput quantitative fluorescence in situ hybridization . in summary ,
we have shown for the first time that leukocyte telomere length does not predict arterial telomere length in t2d rats .
our findings highlight the importance of measuring telomere dynamics in tissues other than circulating immune cells . | vascular dysfunction is an early feature of diabetic vascular disease , due to increased oxidative stress and reduced nitric oxide ( no ) bioavailability .
this can lead to endothelial cell senescence and clinical complications such as stroke .
cells can become senescent by shortened telomeres and oxidative stress is known to accelerate telomere attrition .
sirtuin 1 ( sirt1 ) has been linked to vascular health by upregulating endothelial nitric oxide synthase ( enos ) , suppressing oxidative stress , and attenuating telomere shortening .
accelerated leukocyte telomere attrition appears to be a feature of clinical type 2 diabetes ( t2d ) and therefore the telomere system may be a potential therapeutic target in preventing vascular complications of t2d .
however the effect of t2d on vascular telomere length is currently unknown .
we hypothesized that t2d gives rise to shortened leukocyte and vascular telomeres alongside reduced vascular sirt1 expression and increased oxidative stress .
accelerated telomere attrition was observed in circulating leukocytes , but not arteries , in t2d compared to control rats .
t2d rats had blunted arterial sirt1 and enos protein expression levels which were associated with reduced antioxidant defense capacity .
our findings suggest that hyperglycemia and a deficit in vascular sirt1 per se are not sufficient to prematurely shorten vascular telomeres . |
ribonucleotide reductases ( rnrs ) catalyze the conversion of nucleotides to deoxynucleotides in all organisms providing the monomeric precursors required for dna replication and repair.(1 ) the escherichia coli class ia rnr is composed of two subunits : r1 ( 2 ) and r2 ( 2 ) .
each subunit is homodimeric and a 1:1 complex of 2 and 2 is proposed to be the active form of the enzyme .
2 is structurally homologous to the class ii and iii rnrs and houses the catalytic machinery required for nucleotide reduction and the binding sites that regulate the specificity and efficiency of substrate turnover .
2 contains the di - iron - tyrosyl ( y122 ) radical cofactor that initiates catalysis on 2 by a process thought to involve long - range proton coupled electron transfer ( pcet ) .
the radical propagation process results in generation of a transient thiyl radical ( c439 ) in the active site of 2 .
in addition , there are two cysteines ( c225 and c462 ) , a glutamate ( e441 ) , and an asparagine ( n437 ) within the active site of 2 that are essential for catalysis ( scheme 1 ) .
n = a , g , c , or u. c462 and c225 provide the reducing equivalents required to make dndps . e441 and n437
the cysteines provide the reducing equivalents required to make deoxynucleotides , the glutamate functions as a general base and general acid catalyst(6 ) and the asparagine helps to position c225 and e441 in the appropriate conformation to effect the desired chemistry.(7 ) the mechanism of nucleotide reduction involves transformations employing both nucleotide and protein - derived free radicals.(8 ) the rate - limiting step in the class ia rnrs has been shown to be a conformational change that gates the pcet step , masking the chemistry.(9 ) studies to examine the chemical mechanism with nucleoside diphosphates ( ndps ) and the wild type ( wt ) rnr met with limited success , because of the rate limiting step.(10 ) to complement these studies we and others have successfully used mechanism based inhibitors and site - directed protein mutants to perturb the system so that the interesting chemical transformations can be studied .
this mutant is inactive in cdp reduction with either ttp or atp as allosteric effector .
their kinetic analysis led to the model where radical i gave rise to radical ii . to assign structures to each radical ,
studies using [ u--h ] cysteine 2 resulted in altered hyperfine interactions of the first radical monitored by epr spectroscopy and suggested that this radical was cysteine based . in a second set of experiments [ uc]-cdp replaced cdp .
the resulting broadening of the epr spectrum of the radical ii suggested that it was nucleotide based .
their hypothesis based on these studies was that the y122 in 2 gave rise to radical i , the c439 in 2 , which then gave rise to the 3-cdp radical ii ( scheme 1 , 1).(12 ) subsequently , the structures of these two radicals were reexamined using high field ( 140 ghz ) , pulsed epr methods.(13 ) this methodology facilitated deconvolution of the three radicals ( y and radicals i and ii ) with superimposed g values and revealed that the cysteine based radical ( i ) is in fact , a disulfide radical and not the c439 initially proposed .
mechanistically this was an important result as our proposed mechanism for nucleotide reduction ( scheme 1 ) , requires that mutation of e441 to a q , would prevent reduction of intermediate 3 by the disulfide radical anion and hence promote build up of this intermediate .
model studies had previously demonstrated that this reduction step requires protonation of the 3-ketone by e441 , concomitant with electron transfer .
identification of a disulfide radical anion thus provided the first insight into the mechanism of reduction of the 3-ketodeoxynucleotide . in our original report we suggested that radical ii was a 4-ketyl radical of the 3-deoxynucleotide ( 5 , scheme 2 ) . the assignment was based on the g values obtained from high field epr ( g1 = 2.0072 , g2 = 2.0061 , and g3 = 2.0021 ) , and the observation of hyperfine interactions with two nuclei ( i = 1/2 ) originally reported by perrson et al . in the present paper
we describe our efforts using isotopically labeled cdp analogs , e441q 2 , 2 , high field epr , endor analysis and dft calculations ( b3lyp / iglo - iii / b3lyp / tzvp ) to establish the structure of radical ii . the structure is not 5 ( scheme 2 ) but is proposed to be an off pathway radical with an additional oxygen in the nucleotide ( structure q , figure 6 ) .
2 and 2 were isolated as previously described and had specific activities of 2000 mol / min / mg and 6000 mol / min / mg respectively.(16 ) e441q-2 was isolated as previously described . [ -h ] tyrosine ( 99% enriched ) was obtained from cambridge isotopes laboratory .
[ 2-h ] cytidine was synthesized by the procedure of cook and greenberg(17 ) nmr analysis revealed 78% deuterium incorporation .
dideuterocytidine was synthesized by a modification of the procedure of rabi and fox.(18 ) nmr analysis revealed 83% and 96% incorporation at c-5 and c-6 , respectively . for nucleoside chromatography ,
merck kieselgel 60-f254 sheets were used for tlc and products were detected by uv or by development of color with ce(so4)2/(nh4)6mo7o24h2o / h2so4/h2o .
h nmr ( 400 mhz ) was carried out in the department of chemistry instrumentation facility .
1,2;5,6-di - o - isopropylidene--d - ribo - hexafuranose-3-ulose ( 7a ; 2 g , 7.7 mmol ) in d2o ( 8 ml ) and pyridine ( 50 ml ) was heated for 5 min at 95 c and then kept at ambient temperature for 20 h. the solvents were removed in vacuo and the deuterium exchange procedure repeated five times to achieve deuterium incorporation at > 97% ( h nmr ) .
this step was followed by reduction of the oily residue with nabh4 ( 0.58 g , 15.38 mmol ) in etoh ( 20 ml ) at 0 c .
after 2 h , excess nabh4 was decomposed by acoh ( 0.5 ml ) and the volatiles were evaporated under vacuum .
the residue was partitioned between h2o and ch2cl2 and the water layer was extracted with ch2cl2 ( 3 ) .
the combined organic layer was washed ( brine ) , dried ( na2so4 ) and evaporated .
the residue was purified by column chromatography ( 25 35% etoac / hexanes ) to give 8a ( 1.44 g , 72% ) : h nmr 1.38 ( s , 3 , ch3 ) , 1.39 ( s , 3 , ch3 ) , 1.48 ( s , 3 , ch3 ) , 1.59 ( s , 3 , ch3 ) 4.014.12 ( m , 3 , h5 , 6,6 ) , 4.32 ( t , j = 6.5 hz , 1 , h3 ) , 4.64 ( dd , j = 3.9 , 5.2 hz , 1 , h2 ) , 5.83 ( d , j = 3.8 hz , 1 , h1 ) .
benzoyl chloride ( 0.67 ml , 0.81 g , 57.5 mmol ) was added to a stirred solution of 8a ( 1.0 g , 3.83 mmol ) in pyridine ( 8 ml ) and stirring was continued for 3 h at ambient temperature .
the volatiles were evaporated and the residue was partitioned between etoac//nahco3/h2o . the organic layer was separated and was washed ( brine ) , dried ( na2so4 ) , evaporated and purified by column chromatography ( 8 18% etoac / hexanes ) to give 8b ( 1.3 g , 95% ) : h nmr 1.35 ( s , 3 , ch3 ) , 1.36 ( s , 3 , ch3 ) , 1.42 ( s , 3 , ch3 ) , 1.57 ( s , 3 , ch3 ) , 4.01 ( dd , j = 5.8 , 8.6 .
hz , 1 , h6 ) , 4.13 ( dd , j = 6.8 , 8.6 .
hz , 1 , h6 ) , 4.38 ( t , j = 6.5 hz , 1 , h5 ) , 4.99 ( dd , j = 3.9 , 5.1 hz , 1 , h2 ) , 5.10 ( d , j = 5.1 hz , 1 , h3 ) , 5.91 ( d , j = 3.8 hz , 1 , h1 ) , 7.467.50 ( m , 2 , ar ) , 7.597.63 ( m , 1 , ar ) , 8.078.09 ( m , 2 , ar ) .
h5io6 ( 225 mg , 0.99 mmol ) was added to a stirred solution of 8b ( 300 mg , 0.82 mmol ) in dried etoac ( 9 ml ) at ambient temperature .
a precipitate appeared within the first five min and the resulting solution was stirred for 2 h. the precipitate was removed by filtration and was washed with etoac ( 2 5 ml ) .
the filtrate was evaporated and the crude 5-aldehyde was dissolved in absolute etoh ( 10 ml ) and nabh4 ( 47 mg , 1.23 mmol ) was added .
stirring was continued for 2 h , acoh ( 0.5 ml ) was added and volatiles were evaporated to give 9a ( 210 mg , 87% ) : h nmr 1.37 ( s , 3 , ch3 ) , 1.59 ( s , 3 , ch3 ) , 3.74 ( d , j = 12.8 , hz , 1 , h5 ) , 4.02 ( d , j = 12.8 hz , 1 , h5 ) , 4.97 ( t , j = 4.3 hz , 1 , h2 ) , 5.05 ( d , j = 4.8 hz , 1 , h3 ) , 5.92 ( d , j = 3.7 hz , 1 , h1 ) , 7.297.50 ( m , 2 , ar ) , 7.597.63 ( m , 1 , ar ) , 8.088.10 ( m , 2 , ar ) .
standard acetylation of 9a ( 160 mg , 0.54 mmol ) in pyridine ( 5 ml ) with ac2o ( 0.13 ml , 138 mg , 1.36 mmol ) gave 9b ( 180 mg , 82% ; tlc one spot ) of sufficient purity to use in next step : h nmr 1.36 ( s , 3 , ch3 ) , 1.58 ( s , 3 , ch3 ) , 2.11 ( s , 3 , ch3 ) , 4.23 ( d , j = 12.3 , hz , 1 , h5 ) , 4.45 ( d , j = 12.3 hz , 1 , h5 ) , 4.93 ( d , j = 4.6 hz , 1 , h3 ) , 4.97 ( t , j = 3.9 hz , 1 , h2 ) , 5.92 ( d , j = 3.7 hz , 1 , h1 ) , 7.487.50 ( m , 2 , ar ) , 7.607.63 ( m , 1 , ar ) , 8.068.09 ( m , 2 , ar ) .
a solution of 9b ( 180 mg , 0.44 mmol ) in cf3co2h / h2o ( 9:1 , 2.8 ml ) was stirred at 0 c ( ice - bath ) for 45 min and then the volatiles were evaporated and coevaporated with toluene .
the oily residue was dissolved in pyridine ( 5 ml ) and dmap ( 3 mg ) and ac2o ( 0.21 ml , 224 mg , 2.20 mmol ) were added , and the mixture was stirred at 5 c overnight .
volatiles were evaporated and residue was dissolved ( etoac ) and washed ( dilute hcl / h2o , saturated nahco3/h2o , brine ) , dried ( na2so4 ) and evaporated .
column chromatography ( 30% 35% etoac / hexanes ) gave 10 ( 150 mg , 89% ; / , 1:4 ) .
the anomer had : h nmr 2.10 ( s , 3 , ch3 ) , 2.11 ( s , 3 , ch3 ) , 2.15 ( s , 3 , ch3 ) , 4.25 ( d , j = 12.0 , hz , 1 , h5 ) , 4.42 ( d , j = 12.0 hz , 1 , h5 ) , 5.50 ( dd , j = 1.3 , 5.0 hz , 1 , h2 ) 5.61 ( d , j = 4.9 hz , 1 , h3 ) , 6.25 ( d , j = 1.3 hz , 1 , h1 ) , 7.477.50 ( m , 3 , ar ) , 8.018.11 ( m , 2 , ar ) .
the anomer had : h nmr 2.03 ( s , 3 , ch3 ) , 2.12 ( s , 3 , ch3 ) , 2.17 ( s , 3 , ch3 ) , 4.32 ( d , j = 12.1 , hz , 1 , h5 ) , 4.39 ( d , j = 12.1 hz , 1 , h5 ) , 5.36 ( dd , j = 4.5 , 6.4 hz , 1 , h2 ) 5.55 ( d , j = 6.4 hz , 1 , h3 ) , 6.53 ( d , j = 4.5 hz , 1 , h1 ) , 7.617.65 ( m , 3 , ar ) , 8.098.11 ( m , 2 , ar ) .
4-n - acetylcytosine ( 84 mg , 0.55 mmol ) was suspended in hexamethyldisilazane ( 2 ml ) and me3sicl ( 0.1 ml ) was added and the resulting mixture was stirred at 120 c under argon atmosphere for 4 h. volatiles were evaporated and the residue was kept under vacuum for 30 min .
compound 10 ( 140 mg , 0.37 mmol ) was dissolved in dry clch2ch2cl ( 4 ml ) and this solution and tmsotf ( 0.07 ml ) were added to the presililated cytosine and the reaction mixture was heated at 70 c for 7 h under n2 .
volatiles were evaporated and residue was dissolved ( etoac ) and washed ( nahco3/h2o , brine ) , dried ( na2so4 ) and evaporated .
column chromatography ( chcl3 1% meoh / chcl3 ) gave 11a ( 130 mg , 75% ) : h nmr 2.06 ( s , 3 , ch3 ) , 2.17 ( s , 3 , ch3 ) , 2.30 ( s , 3 , ch3 ) , 4.40 ( d , j = 12.5 , hz , 1 , h5 ) , 4.47 ( d , j = 12.5 hz , 1 , h5 ) , 5.57 ( t , j = 4.5 hz , 1 , h2 ) , 5.60 ( d , j = 5.8 hz , 1 , h3 ) , 6.23 ( d , j = 4.5 hz , 1 , h1 ) , 7.447.52 ( m , 3 , h5 and ar ) , 7.62 ( t , j = 7.5 hz , 1 , ar ) , 7.95 ( d , j = 7.5 hz , 1 , h6 ) 8.03 ( d , j = 7.9 hz , 2 , ar ) , 9.55 ( brs , 1 , nh ) . a solution of 11a ( 130 mg , 0.27 mmol ) in nh3/meoh ( 10 ml ) was stirred at 0 c for 6
h. volatiles were evaporated and the residue was dissolved in h2o and extracted with ch2cl2 ( 3 ) and then with et2o .
the aqueous layer was concentrated and applied to a column of dowex 1 2200 ( oh form ) .
elution ( h2o 40% meoh / h2o ) and concentration of the appropriate fractions gave 11b ( 55 mg , 82% ) as a white powder ( with deuterium incorporation of > 98% ) : h nmr ( dmso - d6 ) 3.52 ( dd , j = 5.2 , 12.1 , hz , 1 , h5 ) , 3.64 ( dd , j = 5.2 , 12.1 hz , 1 , h5 ) , 3.923.94 ( m , 2 , h2 , 3 ) , 4.96 ( d , j = 4.5 hz , 1 , 3 oh ) , 5.03 ( t , j = 5.1 hz , 1 , 5 oh ) , 5.27 ( d , j = 4.7 hz , 1 , 2 oh ) , 5.70 ( d , j = 7.4 hz , 1 , h5 ) , 5.76 ( d , j = 3.7 hz , 1 , h1 ) , 7.12 ( br d , j = 6.2 hz , 2 , nh2 ) , 7.84 ( d , j = 7.4 hz , 1 , h6 ) .
deuterium incorporation was 93% based on h nmr and esi - ms.(23 ) synthesis of [ 5 , 5-h ] cdp was carried out as described in figure 2 sm .
nine ghz epr spectra of radical ii generated from e441q-2/2 /ttp with cdp and [ 1-h ] , 2-h ] and [ 4-h]- cdp taken at 77 k and hand quenched at 3 min reaction time .
all isotopically labeled cytidine analogs , except [ 5 , 5-h ] cytidine were converted enzymatically to cmp using hexahistidine tagged - human deoxycytidine kinase ( hdck ) by a modification of the procedure of usova and eriksson.(25 ) a typical reaction contained in a final volume of 5 ml : 1 mm cytidine , 1.33 mg / ml 6xhis - hdck ( specific activity 170 nmol / mg / min ) , 2 mm atp , 2 mm dtt and 0.5 mg / ml bsa in 50 mm tris , 100 mm kcl , 5 mm mgcl2 at ph 7.6 .
the reaction was initiated by the addition of enzyme and incubated at 37 c for 10 min .
the reaction mixture was then loaded onto a deae sephadex a25 column ( 1 14 cm ) and eluted with a 100 ml 100 ml linear gradient from 0.005 to 0.4 m triethylammonium bicarbonate ( teab ) ph 7.8 .
the cmp eluted at 0.2 m teab , the appropriate fractions were pooled and the teab removed in vacuo .
all isotopically labeled cytidine analogs were converted to cdp using a glutathione s - transferase tagged ump / cmp kinase by a modification of the procedure of van rompay et al.(26 ) a typical reaction contained in a final volume of 5 ml : 1 mm cmp , 0.065 mg / ml gst - ump / cmp kinase ( specific activity 4.8 mol / mg / min ) , 4 mm atp , 2 mm dtt in 50 mm tris , 5 mm mgcl2 at ph 8.0 .
the reaction was initiated by the addition of enzyme and incubated at 37 c for 30 min .
the reaction mixture was then loaded onto a deae sephadex a25 column ( 1 14 cm ) and eluted with a 120 ml 120 ml linear gradient from 0.005 to 0.6 m teab .
the reaction mixtures contained in a final volume of 0.23 ml : 45 m e441q-2 , 45 m 2 , 1 mm cdp , 0.8 mm ttp , 5 mm dtt , 50 mm hepes ph 7.6 , 15 mm mgso4 , and 1 mm edta .
all components except for substrate were combined and equilibrated at 25 c for 5 min .
the reaction was started by the addition of cdp and then transferred to an epr tube and frozen in liquid nitrogen after 1 to 4 min incubation at 25 c .
continuous wave epr spectra at 9 ghz were acquired on a bruker esp-300 spectrometer equipped with a bruker high sensitivity 4119hs cylindrical cavity and an er-041 g microwave bridge containing an internal frequency counter .
spectra were acquired at either 77 k using a liquid - helium cooled esr-900 cryostat and an oxford llt 650/1.0 transfer line .
typical parameters were as follows : microwave power 20 w ; modulation frequency 100 khz ; modulation amplitude 1 g ; conversion time 20.48 ms ; time constant 5.12 ms .
the reaction mixture contained in a final volume of 15 l : 300 m e441q-2 , 300 m 2 , 1 mm cdp ( or 0.3 mm gdp ) , 0.8 mm ttp , 5 mm dtt , 50 mm hepes ph 7.6 , 15 mm mgso4 , and 1 mm edta .
after equilibration of the mixture in the absence of substrate at 25 c , the reaction was started by the addition of cdp and the mixture was drawn into a suprasil capillary by capillary action and frozen in liquid nitrogen 3 min after initiation .
spectra were acquired at 70 k on a custom - designed pulsed spectrometer.(27 ) the stimulated echo sequence was used with typical pulse length of t/2 = 60 ns and pulse spacing of = 200 ns .
the pulsed method allowed us to filter out the radical ii from the y , as previously demonstrated.(13 ) the external magnetic field was swept with the assistance of a field lock system .
the number of shots per point , scans and recycle delays were adjusted for different signal intensities and temperatures .
reactions were prepared as described for the 140 ghz experiments except that the final volume was 60 l .
pulsed epr and endor data were obtained at 35 ghz with an instrument(28 ) described earlier . during the course of experiments several components of the spectrometer were modified .
a solid - state 4w microwave amplifier ( microwave power inc . ) was used in place of the twta 40 w amplifier , and data acquisition was handled using the software package specman(29 ) ( http://www.speman.4epr.com ) in conjunction with a spincore pulseblaster esr_pro 400 mhz word generator and an agilent technologies acquiris dp235 500 ms / sec digitizer .
first order endor spectra from i = 1/2 nuclei ( h , c , n ) exhibit pairs of features at = |n a/2| .
geometry optimizations and calculation of hyperfine couplings ( hfc ) have been performed with gaussian 03 ( http://gaussian.com/citation_g03.htm ) using the becke3lyp functional(30 ) in combination with the tzvp basis set.(31 ) thermochemical parameters to predict enthalpies at 298k have been obtained using the unscaled force constants computed at this latter level .
single point calculations have then been performed using the iglo - iii basis sets(32 ) in combination with the becke3lyp functional in order to predict isotropic and anisotropic hyperfine coupling ( hfc ) parameters .
these latter calculations use a pruned grid of 99 radial shells and 590 angular points for numerical integration , while all geometry optimizations have been performed with the default pruned 75/302 grid . spin and charge densities have been calculated using the natural population analysis ( npa)(33 ) of the b3lyp / iglo - iii//b3lyp / tzvp kohnsham orbitals .
hyperfine tensors for small reference compounds were calculated with the adf - dft package to test those obtained from gaussian 03 .
the g - tensor calculations were carried out on the b3lyp / tzvp optimized structures and employed a second - order perturbation approach,(34 ) which has been demonstrated to provide excellent accuracy in calculations of g - tensors for organic radicals . the kohnsham molecular orbitals ( mo )
were obtained with the turbomole ( v. 5.6 ) program at the ri - bp86/dzvp level .
the unrestricted kohnsham molecular - orbital information from turbomole was transferred by appropriate interface routines to the mag ( magnetic resonance ) property module of the in - house program respect.(42 ) low symmetry of the structures and a variety of spin distribution patterns together with small g - anisotropy imposed use of an explicit description of all one- and two - electron breit - pauli spinorbit integrals ( full ) and distributed gauges with gauge - including atomic orbitals ( giaos ) . in the calculations ,
the g - tensor is defined as g = ge(1 ) + g , where ge = 2.002319 .
g - shift components ( gi ) defined as corrections to the free electron value in ppm ( that is , in units of 10 ) are presented and discussed .
the approach presented includes not only the dominant second - order spinorbit / orbital - zeeman cross terms but also the relativistic mass correction ( rmc ) and the one - electron part of the spinorbit gauge correction ( gc ) terms .
to test our initial proposal that the 3 min radical is a 4-ketyl radical(13 ) and to obtain more experimental information , we generated a series of specifically , isotopically labeled cdps and investigated the effect of the isotopic substitution on the hyperfine couplings with 9 and 140 ghz epr spectroscopy .
given our proposed structure for radical ii ( 5 , scheme 2 ) and that the triplet epr line shape reported by persson et al . at 9 ghz ( see figure 3 ) had been attributed to the hyperfine couplings with two almost equivalent protons,(11 ) we initially examined the effect of selective cdp deuterated at c5,5. the synthetic method , reduction of the 5-aldehyde with nabh4 , resulted in incorporation of a single h ( > 98% ) , randomly distributed between h5 and h5. the results from an experiment with this compound taken at 140 ghz are shown in figure 1c .
no alterations in the observed hyperfine are apparent relative to the control with cdp ( figure 1a ) .
this result demonstrates that radical ii is not 5 ( scheme 2 ) as we had proposed .
such a radical would exhibit substantial hyperfine coupling to at least one of the adjacent protons at c5 and this coupling would be decreased by deuteration .
140 ghz pulsed epr spectra of radical ii generated from e441q-2/2/ttp with cdp and [ 1-h ] , [ 2-h ] and [ 4-h]- cdp taken at 60 k and hand quenched 3 min after mixing .
the features in the region between 49700 and 49740 ( bottom two spectra ) are associated with remaining y due to difficulty in temperature control during the pulsed experiment .
140 ghz pulsed epr spectra of radical ii generated from e441q-2/2/ttp : ( a ) with cdp ; ( b ) with cdp and exchange into d2o ; ( c ) with [ 5,5-h]-cdp ; ( d ) with [ 5,6 h]-cdp ; ( e ) with [ un]-cdp .
( f ) with [ h y ] 2 ; and ( g ) with [ uc , n]-cdp .
the spectra were recorded at 70 k. a point by point derivative of the ese - spectra was built to better visualize the hyperfine structure .
our previous analysis eliminated the possibility that 2 ( scheme 1 ) was radical ii as the observed hyperfine interactions from the protons attached to c1 and c2 of 2 must be substantially different , in contrast to the experimental results . to eliminate the possibility that one or both of the hyperfine interactions is associated with an exchangeable proton , an experiment in d2o with cdp was carried out.(8 )
no changes in the hyperfine structure were observed ( figure 1b ) , except for the expected sharpening due to the exchange of the protein matrix protons , eliminating this possibility .
the unusual g values of radical ii and our previous identification of a 3-ketone during catalysis,(45 ) seemed to leave few options for assignment of nuclei giving rise to the observed hyperfine interactions .
one option was that the nucleic acid base could interact with the sugar radical resulting in the delocalization of the radical onto the base.(46 ) this proposal seemed unlikely as perrson had reported ( no data was shown ) that interaction of e441q-2 with gdp / ttp gave rise to a similar radical and a purine base is distinctly different from the pyrimidine base .
however , at this stage , the identity of ii was a real mystery , thus the importance of the base was further investigated using [ 5 , 6-h ] , [ un ] , [ uc , n ] cdp with e441q-2 . once again as revealed in figure 1d and e no altered hyperfine interactions were observed .
the spectrum with [ uc , n]-cdp ( figure 1 g ) was slightly broadened reproducing the previous results described by perrson and requiring that ii is nucleotide based.(11 ) finally , previous studies on galactose oxidase , an enzyme with a o - thioether substituted tyrosyl radical essential for catalysis , revealed a spectrum very similar to that of radical ii.(47 ) one could image a scenario where a similar radical could be generated given the importance of the c439 radical in nucleotide reduction and its propinquity to y730 ( scheme 1 ) . thus , based on previous studies with galactose oxidase , we incorporated [ -h ] tyrosine into all of the tyrosines of e441q-2 .
the results of incubation of this protein with cdp , figure 1f , revealed no altered hyperfine structure . at this point we believed we had a nucleotide based radical , but that it was probably generated off pathway or in a parallel pathway relative to the disulfide radical anion formation and not directly from the disulfide radical anion as perrson et al .
proposed.(12 ) we thus synthesized 1 , 2 , 4-[h ] cdps and examined the spectra resulting from these compounds at both 9 and 140 ghz by epr spectroscopy .
deuteration at either c1 or c4 collapses the triplet to a doublet ( compare figure 2a with b and d and figure 3a with b and d ) , while no such collapse occurs by deuteration at c2 ( compare figure 2a and c and figure 3a and c ) .
these surprising findings suggest that the radical structure is unusual and led us to search for other possible couplings to protons , carbons , and nitrogens in more detail in the effort to determine the structure .
the epr measurements assigned the two protons giving rise to the triplet epr pattern as those associated with c1 , c4 of cdp . to obtain precise measurements of their hyperfine coupling tensors , 35 ghz h davies and h mims and
remims endor studies were carried out on ii prepared with unlabeled cdp and with c1-h labeled cdp ( figure 4 ) .
the h spectra collected at 2 k ( not shown ) are dominated by h peaks from y , which overlap all the signals associated with radical ii except that from c4-h .
however , at 80 k the phase memory of y is shorter than that of ii and y does not contribute to the davies h pulsed endor spectra collected with a pulse spacing = 800 ns .
the 80 k h spectrum of unlabeled ii shows intensity from + of the two strongly coupled protons with 64 + 71 mhz , and with corresponding intensity ( figure 4 , top ) .
h spectra collected at 2k from the radical with [ 1h ] cdp ( figure 4 , bottom ) isolate the h signal from that site .
simulations of the h pattern yielded the hyperfine tensor , a(c1h ) = [ 21.5 , 21.5 , 28 ] mhz .
35 ghz pulsed davies h ( top ) and h ( bottom ) endor spectra of radical ii generated from e441q-2/2/ttp with cdp and [ 1-h]-cdp hand quenched 3 min after mixing .
simulation of c1-,h peaks . experimental conditions : davies endor pulse lengths 80 ns , = 800 ns , rf pulse length 20 s , repetition rate 5 ms , 15,000 transients / point .
remims endor /2 pulse lengths 32 ns , = 144 ns , rf pulse length 60 ms , repetition rate 20 s , 192 transients / point .
simulation parameters : h(top ) a = [ 21.5 , 21.5 , 28 ] mhz , = 45 , line width = 0.5 mhz .
h(bottom ) a = [ 3.3 , 3.3 , 4.3 ] mhz , = 45 ; p = 0.08 mhz and line width = 0.2 mhz .
the remaining h endor intensity from the strongly coupled protons must come from c4-h ; it has larger couplings , with maximum and minimum hyperfine tensor values of a = 38 , 30 mhz .
the observation of large and roughly isotropic hyperfine couplings to c1h and c4h suggest that these protons are adjacent to sites of substantial spin density . to search for possible smaller couplings to the c2 or c5 protons , h
mims spectra were collected on samples prepared with [ c2h ] cdp and [ c5h ] cdp ( not shown ) .
the search was hampered by the presence of ( mi = 2 ) nitrogen peaks in the region of the deuteron larmor frequency , but nonetheless , no h peaks were seen for [ c2h ] .
only very weak possible peaks were seen for [ c5h ] at a = 800 ns ; if real , these would correspond to a h coupling of 0.2 mhz ( corresponding to a h coupling of 1.3 mhz ) .
overall , the h experiments definitively establish that no protons , other than those associated with c1h or c4h , have a coupling larger than 2 mhz .
n / c : the 35 ghz davies pulsed endor spectrum of a sample with [ uc , un ] cdp at 80 k is shown in figure 5 .
peaks are observed at 5 ( weak ) , 67 , 10 , 20 , and 22 mhz , and an edge at 24 mhz .
none of these features are seen in an unlabeled sample , demonstrating that they are all due to coupling to c /
first , the 10 and 20 mhz peaks are separated exactly 2(n ) ; suggesting an assignment to a n doublet centered at a(n)/2 15 mhz .
however , such a doublet would correspond to a n coupling in the unlabeled sample of a(n ) 21 mhz .
such an n would have given rise to observable features in endor spectra of the unlabeled cdp and would have prevented observation of the well - resolved triplet in the epr spectrum of the unlabeled radical , figure 2a .
thus , we reject the assignment of the 20 mhz peak to n. similar arguments lead us to reject the assignment of features between 20 and 24 mhz to n. thus , we assign the endor intensity between 20 and 24 mhz to + features from c with 14 < a < 22 mhz .
attempts to simulate this signal in terms of a single c failed , so we conclude that it arises from at least two strongly coupled c. given these conclusions , it is plausible to assign the 7 and 5 mhz peaks as their - partners.(50 ) additional mims endor measurements do not support an alternate assignment of these peaks as a +/ - doublet from a weakly coupled n. with these assignments , then the 10 mhz peak can only be a + peak from n with a 9 mhz ; its - partner is at 1 mhz , and not detected .
as shown in figure 5 , this peak can be simulated as arising from a nearly isotropic n coupling , n hyperfine tensor a = [ 8.0 , 8.0 , 9.5 ] mhz.(51 ) in addition mims endor spectra ( supporting information , figure 5 ) show weak signals near the c and n larmor frequencies that correspond to c and n with very small coupling(s ) , a 0.5 mhz .
35 ghz endor spectrum at 77 k of e441q-2/2/ttp and [ uc / n ] cdp ; ( --- ) simulation of the n peak .
experimental conditions : pulse lengths 80 ns , = 800 ns , rf pulse length 20 s , repetition rate 5 ms , 10000 transients / point .
simulation parameters : a = [ 8.0 , 8.0 , 9.5 ] mhz , line width = 0.5 mhz . to summarize , the h endor and epr measurements suggest that c1 and c4 are adjacent to sites with substantial spin density , while the c / n measurements show that the spin density of ii is distributed so as to give relatively strong hyperfine couplings to at least two c and one n nucleus .
however , with multiple labels in a single cdp , the measurements do not define the sites involved .
further , the spin density required to generate the observed signals would be a small fraction of an electron ; to locate this spin density and to constrain a structure assignment , more nuclear couplings are required . in principle , the problem could be solved by the synthesis of cdp with additional , specific labels ( c , n , o , h ) , but this is chemically impractical . as a result , quantum chemical calculations were undertaken .
a new model structure is required to explain the equivalency of the h hyperfine couplings associated with c1 and c4 and the observed g values .
initially we focused on model systems descriptive of plausible intermediates in the reaction of cdp with e441q - rnr ( figure 6 , am ) . the hyperfine couplings ( hfc ) and g - tensor data
these models differ from the actual substrates as the phosphate groups have been removed from c5. to avoid artifacts caused by the truncation , conformational searches excluded all structures in which the c5 hydroxyl group acted as a hydrogen bond donor .
the first three models ac correspond to transient intermediates proposed for the normal mechanism of nucleotide reduction .
their calculated hfc data ( table 1 ) differ substantially from those measured for ii .
the c3-keto radical d ( figure 6 ) was included here as a model for the off - pathway radical 5 ( scheme 2 ) and was previously examined computationally by siegbahn et al.(33 ) while their results clearly favored formation of radical d on thermodynamic grounds , their analysis failed to recapitulate the observed experimental g - factor and hfc pattern .
our calculations also suggested that the hydrated versions of the two keto - substituted radicals b and d , e and f , are not good candidates for ii .
as shown in scheme 1 , a primary function of the e441 carboxylate involves deprotonation of the c3 hydroxyl group .
should this deprotonation step be impaired in the e441q mutant one might expect formation of radical cation g ( instead of radical b ) in the course of the reaction .
this species shows hyperfine couplings to the hydrogens at c1 and c4 similar to those measured experimentally , but lacks the proper g - factor characteristics .
the lifetime of radical cations such as g may also be limited , as conformational searches induced a number of side reactions with minimal to no enthalpic barrier .
the products formed on this occasion include the base radical cation h , the cyclized radical cation i , and the allylic , distonic radical cation j. isomer j is the energetically most favorable of these radical cations , the isomers g , i and h being less stable by 15.2 , 16.9 , and 65.4 kj / mol , respectively , at the ub3lyp / tzvp level of theory
. the calculated spectroscopic data of j are , however , far from experimental values . the most relevant interaction between a radical cation such as g with the mutant enzyme
binding pocket will most likely involve the q441 amide side chain and calculations on radical cations gj have therefore been repeated using acetamide as a mimic for this side chain .
the energetically most favorable structure obtained in this system was n , in which proton transfer has occurred from the radical cation to acetamide . only 3.3 kj / mol less favorable than n is the formal base - adduct m , while the localized radical cations l and m are 42.5 and 49.8 kj / mol less stable .
none of these four systems fits very well with the experimentally measured spectroscopic data , the smallest deviation being present in radical cation m. a survey of g - factor data for systems an ( see table 1 ) shows that gav values are usually smaller than measured experimentally .
sufficiently high values of gav only result for those systems in which the unpaired spin localizes on highly oxidized carbon centers .
this observation inspired further calculations on the more highly oxidized substrate radicals o and p that derive from a through formal elimination of h2 .
the g - factor data for o are indeed significantly larger than those of a , and show a large hyperfine coupling to h(c1 ) and a small coupling to h(c4 ) .
the situation is similar in p , but reverses the size of the hfc values for h(c1 ) and h(c4 ) .
rapid exchange between o and p through hydrogen transfer between the oxygen atoms at c2 and c3 may thus generate an average spin distribution at c2 and c3 , which effectively generates the experimentally observed hfc data .
this type of intermediate has been observed in dioldehydratases and recent calculations by radom et al.(52 ) indicate that the barrier for interconversion between the localized tautomeric forms drops substantially in the presence of neutral and basic residues and practically vanishes in the presence of anionic residues .
deprotonation of o / p may thus generate a delocalized spin system across c2 and c3 , thereby avoiding the need for any assumptions with respect to the rate of interchange of two tautomeric forms of the substrate radical .
calculations on the corresponding semidione radical anion were performed in the presence of a water molecule in order to provide a hydrogen bonding environment for the charge - carrying oxygen atoms .
the spectroscopic values predicted for radical anion q are highly similar to those observed experimentally , giving particularly good agreement with the g - factor anisotropies .
further agreement between experiment and theory can be derived from analysis of spin dipolar couplings for the most relevant hydrogen atoms .
the smaller of the experimentally measured isotropic couplings of 8.5 g shows a notable anisotropy with ax = 10.0 g , and ay = az = 7.7 g. combination of the calculated isotropic coupling for c1h of + 8.3 g with the anisotropic spin dipolar couplings calculated for this nucleus ( table 2 ) yields ax = 9.90 g , and ay = 7.45 and az = 7.55 g , in practically quantitative agreement with the experiment .
similar isotropic and dipolar couplings have been calculated for the hydrogen at c4 position , also in reasonable agreement with the experiment , but in this case the calculation is 3 g too low .
the values of ay ( c4h ) and az ( c2/3 ) are not determined by experiment .
uniform c - substitution in q is predicted to lead to small isotropic hyperfine coupling interactions with the carbons in the ribose moiety ( c1 : 3.7 g , c2 : 0.7 g , c3 : 2.0 g , c4 : 4.0 g , c5 : + 4.8 g ) , while there are practically no interactions with the carbon atoms in the cytosine base .
the n nuclei in the base lead to substantial isotropic hfc interactions only from the n1 position ( 2.16 g ) , but not from the other two positions ( 0.08 g from n3 and 0.007 g from nh2(c4 ) ) . assuming a conversion factor for
n / n coupling constants of 1.4028 , this equates to an isotropic n hfc interaction of 3.04 g at the n1 position .
the spin dipolar couplings for carbon and the nitrogen centers in q are summarized in table 2 .
the two carbon atoms carrying most of the unpaired spin density , c2 and c3 , have not only small isotropic hyperfine couplings , but show large anisotropies in their couplings .
c1 and c4 , adjacent to c2 and c3 , as well as c5 show somewhat larger isotropic couplings and have , in contrast , much smaller anisotropic dipolar couplings .
the most remarkable structural characteristic of radical q ( figure 7 ) is the almost perfectly planar arrangement of the ribose ring carbon atoms with d(c1c2c3c4 ) = 0.6. the oxygen atoms attached to carbon atoms c2 and c3 deviate from this plane by no more than 1. the ribose ring oxygen extends from this plane slightly upward with d(oc1c2c3 ) = + 5.8. front and side view of radical an ion q ( optimized at ub3lyp / tzvp level ) .
the electronic structure of q is fully compatible with the delocalized lewis structure shown in figure 6 with spin density coefficients of 0.21 and 0.18 at the c2 and c3 carbon atoms .
the remaining part of the unpaired spin density is distributed over the two oxygens attached to the c2 and c3 atoms with spin density coefficients of 0.30 and 0.27 , respectively .
a test of structure q is provided by comparison of the hyperfine parameters measured by endor spectroscopy and those calculated theoretically .
the calculated n1 coupling for q is very similar to the largest n coupling observed experimentally , and the calculated coupling for the n3 and nh2 ( largest principal values 0.7 and 0.1 mhz , respectively ) could account for the very small couplings observed in mims endor .
the calculation suggests that all five c atoms of the ribose should have easily observable couplings , which can be divided into two types .
c1 , c4 and c5 have similar , largely isotropic dominated couplings of 1014 mhz .
a simulation carried out using the calculated tensors is remarkably close to the experimental data , figure 3sm . with slight adjustments to the values , one tensor of each type
the adjusted simulation shown in figure 3sm is a summation of two c tensors of ( )[14.5 , 14.5 , 12 ] mhz and [ 21.5 , 17.5 , ( 26 ) ] mhz , with relative intensities chosen to best match the experimental data .
the largest principal value of the dipolar tensor is not determined by the experiment , and was chosen to match the calculated value for c3. all data obtained from the analysis of the endor spectra are summarized in table 2 together with the calculated data .
the c hyperfine tensors thus arrived at are quite different from earlier experimental studies of reference systems such as malonic acid ( a = [ 30 , 30 , 210 ] mhz,(53 ) or ch2oh radical ( aiso 130 mhz,(54 ) figure 8) . our observation is consistent with a recent report of a semidione radical,(55 ) in which the c hyperfine couplings also appeared to be different from those expected for carbon - centered radicals .
this led us to test the validity of the computed tensors for malonic acid and ch2oh radicals , and of the unsubstituted semidione radical by use of the adf dft package as well as gaussian 03 to calculate the c couplings ( figure 8 , table 3 ) .
the hyperfine couplings obtained from both packages for the malonic acid and ch2oh radicals agreed satisfactorily with experiment and with each other , while the adf value for the semidione radical are quite similar to those calculated for ii with gaussian 03 .
calculated at ub3lyp / igloiii// ub3lyp / b3lyp/631g(d ) level using the energetically most favorable conformer .
calculated at blyp / tz2p level with adf2007.1 ( scm , theoretical chemistry , vrije universiteit , amsterdam , the netherlands ) .
the calculated and observed hyperfine tensor of the semidione radical q can be rationalized by using the theory of karplus and frenkel(56 ) for the interpretation of the isotropic hyperfine interaction of c nuclei in -organic radicals .
the theory considers - bond interactions which polarize both the 1s and 2s electrons of the observed carbon atom .
these polarization effects are caused not only by the spin density on the observed c nucleus , but also by neighboring atoms on which unpaired spin density is present , leading to polarization contributions of different signs that partially cancel each other . for a sp hybridized carbon atom that is bonded to three atoms xi ( i = 1 , 2 , 3 ) , the isotropic c hyperfine constant a has the form :
where and are the -electron spin densities on the atom c and xi respectively .
the contribution of the 1s electrons is determined by s and that of the 2s electrons by the qabs , where the index ab denotes the contribution of the polarized bond between the atoms ab due to the spin density on the atom a. using the above formula with the constants s = 12.7 g , qcc = 14.4 g and qcc = 13.9 g from karplus(56 ) and our calculated spin density distribution on the semidione radical q of = 0.21 , = 0.18 and at c2 , c3 = 0.3 and 0.27 respectively , we arrive at a = qc3c4 * 2.5 and similarly a = 2.9 since only one term of eq
for an estimate of a at c2 and c3 , the coefficients qco and qoc are required which are unknown . however ,
if all other values are inserted , we arrive at the expression : a = 0.3 + qc3o 0.18 + qoc3 0.27 . as qco and qoc
might have similar values but opposite signs , just as qcc and qcc , here too the contributions likely would cancel , leading to an almost vanishing c isotropic hyperfine coupling at o - bonded carbon atoms carrying the spin density .
this suggestion is in agreement with our observation and demonstrates the difficulties of predicting c hyperfine couplings a priori , for a radical whose structure is not known .
finally , it remains difficult to rationalize the predicted positive isotropic hyperfine coupling on c5. since c5 is not part of the -radical semidione fragment , the positive value of a may arise from a more complex polarization mechanism .
the c endor measurements exhibit two different c signals , but do not indicate how many cs correspond to each individual signal . given the narrow and well - defined features for these signals , if one were to arise from two c nuclei , their couplings would have to be essentially identical ( < 1 mhz difference in principle values ) .
examination of the computation results suggests that c1,4 , 5 and c2 , 3 of the proposed model have sufficiently similar tensors that they might ( accidentally ) be even more similar in reality , and show unresolved endor signals . to help count the number of cs that contribute to the c endor signals , and thereby test the validity of the proposed model , x - band epr spectra from the natural - abundance ( figure 2a ) and [ uc / n ] labeled samples ( figure 4sm ) were carefully simulated .
the experiment was carried out at x band so that broadening of the spectrum due to g dispersion is minimized ; by carrying out the experiments at 80 k , interference from the rapidly relaxing y is eliminated .
simulation of the x - band cw spectrum obtained with unlabeled cdp was first carried out to establish an inherent line width . a good fit ( not shown )
was obtained using the h and n hyperfine couplings established by endor , plus the small c5h calculated couplings , which had little effect on the simulation .
the simulation established a maximum line width of 5.0 g. simulations of the spectrum of [ uc / n ] cdp ( figure 4sm ) were then performed by including the n replacement and two or more c couplings , as required by endor ; simulations were done with up to two of the dipolar type , and up to three of the isotropic type .
simulations with only two c were poor , showing more structure to the spectrum than observed and/or insufficient peak to peak width of the spectrum , depending on the choice of intrinsic line width . adding a second dipolar coupling into the simulation improves the fit substantially .
the peak to peak width is a much better match , as is the line shape , provided that the line width used is near the upper bound suggested by the unlabeled simulations , and noncoaxial tensors are used . finally , including one or both of the two remaining isotropic couplings arguably improves the fit further , and allows a smaller line width to be used .
the epr simulations thus support the quantum chemical computation in showing that ( at least ) three c nuclei have large couplings , and confirm that there is an accidental equivalence of the c that is permitted , but not actually indicated , by the computations .
the experiments with e441q - r1 provided the first mechanistic insight into the intermediates involved in reductive half - reaction of the nucleotide and formation of the disulfide ( scheme 1 ) in the rnr catalyzed reaction.(13 ) our mechanistic model predicted that this mutation would likely slow down the conversion of ndp to 2 as e441 could no longer function as a general base catalyst and would prevent the reduction of 3 to 4 , as the electron transfer from the disulfide radical anion requires concomitant protonation of the 3-ketone oxygen .
e441q mutation removes the proton source and hence the disulfide radical anion should and does accumulate . while the spectroscopy is sufficient to firmly establish formation of a disulfide radical anion , the kinetic connection to the normal mechanism of reduction is absent .
we hoped that the kinetic relationship between this radical ( i ) and radical ii would provide further support for the functional role of the disulfide radical anion .
the initial studies of perrson reported that the first radical gave rise to the second radical.(11 ) thus determination of the structure of the second radical , established to be nucleotide based by perrson , was important.(12 ) our current studies confirm that the new radical is in fact nucleotide based . the data
, however rule out many mechanistically interesting radicals and are most consistent with structure q ( figure 6 ) , an unexpected result .
while we have not carried out an extensive kinetic analysis of this reaction , it now seems unlikely that there is a direct kinetic connection between the two radicals as originally proposed.(11 ) the question is then raised as to whether the radicals occur within the same or in parallel pathways .
if the radicals occur within the same pathway , the disulfide radical anion , in equilibrium with the thiyl radical / thiolate , has limited reactivity options . with the e441q mutant
, electron transfer can not occur to ketone 3 ( scheme 1 ) , as this step requires concomitant protonation to make the reaction thermodynamically feasible .
thiyl radicals can add to carbonyls and can also mediate hydrogen atom abstraction such as the c4-radical formation ( 5 , scheme 2 ) we originally proposed .
neither of these options , however , gives rise to a nucleotide radical structure consistent with our epr and endor spectroscopy . in a preliminary kinetic experiment , there appears to be no lag phase in the production of radical ii .
a lag phase would be expected if radical ii is on the same pathway as i , given the complex chemistry required to generate q ( figure 6 ) from 1 ( scheme 1 ) .
instead radical ii is present in the earliest time points ( 200 ms ) , suggesting that it is most likely generated in a parallel reaction sequence and is off pathway from the normal reaction .
the difficulties in identifying radicals generated on the second time scale in rnr has previously been observed with the nitrogen centered radical formed on inactivation of e. coli rnr with 2-azido-2-deoxynucleotide.(57 ) the chemistry occurs within a closed active site cavity and is unlikely to be recapitulated in homogeneous solution . in the case of the nitrogen centered radical ,
this species is at least three steps from the initial 3 hydrogen atom abstraction and the observed radical is very stable . in the case of radical ii
, one possibility is that the observed nucleotide radical is also many steps removed from the initial 3 radical ( 1 , scheme 1 ) , with the resulting radical also being very stable .
several experiments , in addition to our spectroscopic analysis , support the assignment of structure q ( figure 6 ) to the radical ii .
the first is the recent report from the frey and reed laboratories that hydroxyacetaldehyde inactivates the adenosylcobalamin requiring dioldehydratase , giving rise to a stable radical whose structure is proposed to be analogous to ii.(55 ) unfortunately this radical interacts with cob(ii)alamin and thus the detailed hyperfine interactions have not been reported .
second , 2,2 difluoro-2-deoxycytidine 5-triphosphate is a potent mechanism based inhibitor of all ribonucleotide reductases we have examined.(23 ) inactivation of the lactobacillus leichmannii ribonucleoside triphosphate reductase with this analog resulted in trapping with nabh4 of a 2 , 3 diketone nucleotide.(23 ) during the course of the enzymatic inactivation , two fluorines were lost , an oxygen was added and an oxidation of the nucleotide occurred !
thus , as noted above , the chemistry in the active site cavity is very complex .
one possible mechanism is that oxygen addition must also be occurring to a cytidine intermediate with the e441q mutant to generate radical ii .
initially we thought that a radical such as 2 ( scheme 1 ) or 5 ( scheme 2 ) might be trapped by o2 , however , persson in their original paper reported that both radicals i and ii were produced under anaerobic conditions.(12 ) thus the mechanism by which an additional oxygen , presumably derived from water , is incorporated into the nucleotide is not understood and is likely many steps removed from the initially generated radicals .
this state along with altered general base / acid chemistry associated with the e441q substitution , might allow 1 ( scheme 1 ) to be oxidized directly by the active site disulfide .
our previous studies with a mechanism based inhibitor , 2-chloro-2-deoxynucleotide showed that 3 hydrogen atom abstraction could occur with oxidized rnr.(45 ) however , the mechanism by which hydrogen atom abstraction by the putative thiyl radical ( c225 ) on the face of the ribose to give q , remains a challenge due to stereochemistry .
this proposed pathway requires that a substantial amount of rnr is in the oxidized state , despite the fact that enzyme is prereduced directly before each experiment and that 10 mm dtt is present during the inactivation . in conclusion ,
the present paper describes another example(57 ) of the difficulty in assigning a structure to a radical detected in an enzymatic reaction that occurs within an enzyme active site , when rerouted from normal catalytic pathway either by a mutation of the protein ( e to q ) or the substrate .
it also demonstrates the power of combining high field spectroscopic epr and endor spectroscopic methods in conjunction with computational methods , to elucidate such a structure . | the escherichia coli ribonucleotide reductase ( rnr ) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric - tyrosyl radical cofactor for catalysis .
rnr is composed of a 1:1 complex of two homodimeric subunits : and . incubation of the e441q- mutant rnr with substrate cdp and allosteric effector ttp results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale . the first radical was previously established by stopped flow uv / vis spectroscopy and pulsed high field epr spectroscopy to be a disulfide radical anion .
the second radical was proposed to be a 4-radical of a 3-keto-2-deoxycytidine 5-diphosphate . to identify the structure of the nucleotide radical [ 1-2h ] , [ 2-2h ] , [ 4-2h ] , [ 5-2h ] , [ u13c , 15n ] , [ u15n ] , and [ 5,6 -2h ] cdp and [ -2h ]
cysteine- were synthesized and incubated with e441q-22 and ttp .
the nucleotide radical was examined by 9 ghz and 140 ghz pulsed epr spectroscopy and 35 ghz endor spectroscopy .
substitution of 2h at c4 and c1 altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted .
dft calculations ( b3lyp / iglo - iii / b3lyp / tzvp ) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data .
the results indicate , unexpectedly , that the radical is a semidione nucleotide radical of cytidine 5-diphosphate .
the relationship of this radical to the disulfide radical anion is discussed . |
systemic lupus erythematosus ( sle ) is a chronic autoimmune disease of unknown cause which can affect several parts of the body even central nervous system ( cns ) .. among cns manifestations , cerebrovascular accident ( cva ) is one of the severe complications .
previous cohort studies revealed that 3 - 20% of sle patients may experience an episode of cva during their course of the disease .
data about differential features and various subtypes of cva in iranian sle patients are lacking . in this study
, we aimed to determine the prevalence and prognosis of cva and its subtypes among iranian sle patients .
this study was a cross - sectional hospital clinic - based study that performed in isfahan , iran . during the period of 1 year , since february 2010 - 2011 , sle patients attending the rheumatology clinic of alzahra hospital ( a tertiary referral center in isfahan , iran ) enrolled in the study .
their medical documents have been reviewed and patients who had developed acute cva include of ischemic attack , hemorrhagic attack , cerebral vasculitis and cerebral vein thrombosis in their course of disease were extracted .
drug - induced vasculitis , generalized vasculopathy , transverse myelitis and loss of consciousness due to metabolic disorders were causes of exclusion . in patients who enrolled the study demographic data ,
sle related clinical and laboratory findings and neurological symptoms in the disease duration were recorded .
sle was diagnosed according to the revised criteria which suggested by american college of rheumatology at 1997 .
diagnosis was based on the clinical features and on data collected by brain imaging . in patients with cva , demographic features , history of hypertension , dyslipidemia and diabetes
mellitus , anti - phospholipid antibodies ( apla ) , duration of the disease until cva , cva manifestation(s ) and follow - up duration were extracted . in the next step , patients with any kinds of cva were summoned to neurology clinic to identify the grade of disability .
the grade of disability of cva determined for each patient through a precise neurological exam by a single expert neurologist using modified rankin scale ( mrs ) .
imaging study of each patient ( which was done at the time of hospitalization ) was studied again and the subtype of cva was confirmed by a single neurologist .
we used spss 20.0 software ( ibm spss statistics ) for analysis of the data .
this study was a cross - sectional hospital clinic - based study that performed in isfahan , iran . during the period of 1 year , since february 2010 - 2011 , sle patients attending the rheumatology clinic of alzahra hospital ( a tertiary referral center in isfahan , iran ) enrolled in the study .
their medical documents have been reviewed and patients who had developed acute cva include of ischemic attack , hemorrhagic attack , cerebral vasculitis and cerebral vein thrombosis in their course of disease were extracted .
drug - induced vasculitis , generalized vasculopathy , transverse myelitis and loss of consciousness due to metabolic disorders were causes of exclusion . in patients who enrolled the study demographic data ,
sle related clinical and laboratory findings and neurological symptoms in the disease duration were recorded .
sle was diagnosed according to the revised criteria which suggested by american college of rheumatology at 1997 .
diagnosis was based on the clinical features and on data collected by brain imaging . in patients with cva , demographic features , history of hypertension , dyslipidemia and diabetes
mellitus , anti - phospholipid antibodies ( apla ) , duration of the disease until cva , cva manifestation(s ) and follow - up duration were extracted . in the next step ,
patients with any kinds of cva were summoned to neurology clinic to identify the grade of disability .
the grade of disability of cva determined for each patient through a precise neurological exam by a single expert neurologist using modified rankin scale ( mrs ) .
imaging study of each patient ( which was done at the time of hospitalization ) was studied again and the subtype of cva was confirmed by a single neurologist .
we used spss 20.0 software ( ibm spss statistics ) for analysis of the data .
of the total number 575 patients ( 64 men and 511 women ) enrolled in the study , 38 were identified to have cva . 6 ( 15.8% ) were men and 32 ( 84.2% ) were women .
their ages at the time of cva ranged from 19 to 69 years , with the mean of 35.1 1.2 years .
small vessels thrombosis was the most common subtype , which includes eight patients ( 21.05% ) .
cva subtypes among iranian sle patients who enrolled in the study 12 ( 31.6% ) patients had a history of hypertension ; positive history of diabetes mellitus was present in two ( 5.7% ) patients and seven ( 18.4% ) had hyperlipidemia .
duration of sle before cva in the study patients ranged from 0 to 16 years , with the mean of 4.25 4.59 years . in 11 ( 28.9% )
patients sle was initiated with cva and in three ( 7.9% ) patients cva was happened in the 1 year of sle .
in fact , in 88.2% of all ischemic attacks ( include of large vessels thrombosis , small vessels thrombosis and cardio emboli ) aplas were positive . with the mean follow - up of 4.4 3.4 years ,
the cva had recurred and in two patients transient ischemic attacks was recorded prior to cva .
the comparison of mrs and sle duration before cva among cva patients with positive and negative apla the most common cva manifestation in our study was hemi paresis , which occurred in 15 ( 39.47% ) patients .
previously , it has been demonstrated that age of sle onset and sex ratio in iranian patients are similar to those from other racial groups .
this prevalence is also within the range that reported from other parts of the world . of the 38 cva patients , in 11 cases
futrell et al . implied that most sle patients who develop cva have their first cva during the first 5 years of their disease and the highest rate is found during the 1 year .
in fact , it seems that in some patients sle has special tendency to involve cerebral vessels , which is not related to the duration of sle , but the nature of sle .
the current study implies that a large proportion of all acute cvas in iranian sle patients are constituted by ischemic attacks .
this fact may reflect the influence of apla and other autoantibodies , premature atherosclerosis , small vessel vasculopathy , arterial and venous thrombosis , embolism and vessel spasm in sle .
although cvt , posterior reversible encephalopathy syndrome ( pres ) and cva secondary to hypercoagulable state are not in toast classification , but in that classification there is a category for stroke of other determined etiology .
we expanded the classification for more exact recognition of stroke subtypes in sle patients . among our patients , we identified three cases of cvt .
our data showed that cvt is constituted 7.9% of cvas in iranian sle patients , which is higher than the prevalence of cvt in the normal population .
there is no study about the rate of pres in sle patients . although pres is not one of the classic cva subtypes , but as the mechanism of pres is cerebral vasospasm and because of endothelium dysfunction in sle patients , we report pres as the cause of 7.9% of cvas in iranian sle patients . as following the average follow - up of 4.4 years ,
mean mrs of patients were less than 1 , we may construe that if a patient with sle survives cva and his or her 5 year rehabilitation would not be unfavorable .
significant relationship between apla and mrs ( p = 0.006 ) shows that in patients with negative serology for apla , morbidity rate of cva is even less .
our study also has some limitations ; this study was not hospital - based so we were unwitting about the mortality rate of cva in sle patients .
thus , the prevalence of cva in iranian sle patients that we have identified is likely to be an underestimation of the true frequency .
second , due to positive history of some factors such as hypertension , hyperlipidemia and diabetes mellitus in some stroke patients , it was favorable to operate proper statistical tests for eliminating their effects on the occurrence of stroke .
however , as our recorded data about the presence of these factors in other non - stroke patients was not sufficient this step was not possible .
disease activity of sle and the type of apla ( anti - cardiolipin antibody or lupus anticoagulant ) at the time of stroke were absent in our medical records which might have a correlation with the stroke subtypes .
however , the study showed positive apla in sle patients with stroke had more prevalence and was associated with a worse prognosis .
according to the significant prevalence of cva among sle patients , cva prevention should start at the early phase of disease and be considered as an essential component of therapeutic strategies .
moreover , we showed that the prognosis of cva in iranian sle patients is not unfavorable . | background : the aim of the present study was to determine the prevalence and prognosis of cerebrovascular accident ( cva ) and its subtypes among iranian patients with systemic lupus erythematosus ( sle).methods : in a cross - sectional study , medical records of 575 sle patients were reviewed .
the patients developing cva in their course of disease were extracted . in these patients , data about demographic features , lupus anti bodies , duration of the disease until cva , cva manifestation(s ) and follow - up duration were gathered . in the next step ,
patients with any kinds of cva were summoned to neurology clinic to identify the grade of disability in each patient with cva.results:we identified 38 patients with cva of which 6 ( 15.8% ) were men and 32 ( 84.2% ) were women .
the most common subtype of cva was small vessels thrombosis ( 21.05% ) among the study patients and hemi paresis was the most prevalent initial presentation ( 39.47% ) . in 11 ( 28.9% ) patients ,
sle was initiated with cva and in 3 ( 7.9% ) patients cva had happened in the 1st year of sle .
anti - phospholipid antibodies ( apla ) were positive in 29 ( 76.3% ) patients .
mean modified rankin scale in patient with positive and negative serology for apla was 0.93 1.11 and 0.22 0.66 respectively ( p = 0.006).conclusions : our study shows that 6.6% of iranian sle patients have cva during their course of the disease .
small vessels thrombosis is the most common cva subtype and hemi paresis is the most prevalent initial presentation .
moreover , we showed that the prognosis of cva in iranian sle patients is not unfavorable . |
illumina hi - seq 2500 data is deposited at the ncbi gene expression omnibus ( geo ) database under geo series and is available at the following link : http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse64507 .
s2 cells were maintained at 28 c in schneider 's drosophila medium ( invitrogen ) , supplemented with 10% heat - inactivated fetal bovine serum ( life technologies ) . for bacterial stimulation ,
heat - killed s. typhimurium was added to s2 cells at a final concentration of 10 cells / ml . at 0 , 30 min , 1 h , and 4 h after stimulation
, 3 10 s2 cells were harvested each time point , and cross - linked by incubating with 1% formaldehyde at room temperature for 10 min . to stop the protein - dna cross - links , 125 mm glycine was added .
nuclei were isolated by re - suspending cells in nuclei isolation buffer ( 0.3 m sucrose , 2 mm mgoac2 , 1 mm cacl2 , 1% nonidet p-40 , 10 mm hepes , ph 7.6 ) and spinning at 800 g for 10 min .
s2 nuclei were digested with 40 u / ml micrococcal nuclease ( mnase ) ( worthington biochemical corp ) in mnase cleavage buffer ( 4 mm cacl2 , 25 mm kcl , 4 mm mgcl2 , 12.5% glycerol , 50 mm hepes , ph 7.4 ) at 37 c for 5 min .
rna was removed by treating samples with 100 g / ml rnase a ( thermo scientific ) at 55 c for 10 min .
samples were incubated at 65 c over night with 0.2 g/l proteinase k ( worthington biochemical corp ) and 1% sodium dodecyl sulfate to remove the cross - links .
dna was purified by phenol chloroform extraction and ethanol precipitation . to isolate mononucleosomal dna ,
the area of the gel containing mononucleosomal dna ( 200 bps and below ) was excised and purified by the freeze - squeeze method , , .
libraries of mononucleosomal dna were prepared using nebnext ultra dna library prep kit for illumina ( neb #
e7370s / l ) , starting with 30 ng of mononucleosomal dna for each reaction .
the samples were end - prepared , adaptor - ligated and indexed ( neb # e7335s / l , neb #
e7500s / l ) according to the manufacturer 's instructions , then purified with ampure xp beads ( beckman coulter , inc . # a63881 ) without size selection .
the quantity and quality of the library were checked with qubit 2.0 fluorometer ( life sciences ) and agilent 2100 bioanalyzer ( agilent technologies )
resulting libraries were plated using the illumina cbot and run on the illumina hiseq 2500 platform configured for 50 bp paired - end reads .
nucleosome occupancy was calculated by reads per million that mapped to the fly genome with bedtools 2.17 .
analyses of nucleosome occupancy were performed in r environment using our lab - developed software package : rage.r .
we created heatmaps for each time point by plotting the nucleosome occupancy of all fly genes and centering the genes at the tss .
we found a significant loss of nucleosome occupancy at the 4 h time point post s. typhimurium stimulation ( k - s test 2.2 10 ) ( fig . 1 ) .
these results suggest that widespread nucleosome loss may be an instrumental part of genome regulation during the immune response to s. typhimurium .
s2 cells were maintained at 28 c in schneider 's drosophila medium ( invitrogen ) , supplemented with 10% heat - inactivated fetal bovine serum ( life technologies ) . for bacterial stimulation ,
heat - killed s. typhimurium was added to s2 cells at a final concentration of 10 cells / ml . at 0 , 30 min , 1 h , and 4 h after stimulation
, 3 10 s2 cells were harvested each time point , and cross - linked by incubating with 1% formaldehyde at room temperature for 10 min . to stop the protein - dna cross - links , 125 mm glycine was added .
nuclei were isolated by re - suspending cells in nuclei isolation buffer ( 0.3 m sucrose , 2 mm mgoac2 , 1 mm cacl2 , 1% nonidet p-40 , 10 mm hepes , ph 7.6 ) and spinning at 800 g for 10 min .
s2 nuclei were digested with 40 u / ml micrococcal nuclease ( mnase ) ( worthington biochemical corp ) in mnase cleavage buffer ( 4 mm cacl2 , 25 mm kcl , 4 mm mgcl2 , 12.5% glycerol , 50 mm hepes , ph 7.4 ) at 37 c for 5 min .
rna was removed by treating samples with 100 g / ml rnase a ( thermo scientific ) at 55 c for 10 min .
samples were incubated at 65 c over night with 0.2 g/l proteinase k ( worthington biochemical corp ) and 1% sodium dodecyl sulfate to remove the cross - links .
dna was purified by phenol chloroform extraction and ethanol precipitation . to isolate mononucleosomal dna ,
the area of the gel containing mononucleosomal dna ( 200 bps and below ) was excised and purified by the freeze - squeeze method , , .
libraries of mononucleosomal dna were prepared using nebnext ultra dna library prep kit for illumina ( neb #
e7370s / l ) , starting with 30 ng of mononucleosomal dna for each reaction .
e7500s / l ) according to the manufacturer 's instructions , then purified with ampure xp beads ( beckman coulter , inc . # a63881 ) without size selection .
the quantity and quality of the library were checked with qubit 2.0 fluorometer ( life sciences ) and agilent 2100 bioanalyzer ( agilent technologies )
resulting libraries were plated using the illumina cbot and run on the illumina hiseq 2500 platform configured for 50 bp paired - end reads .
nucleosome occupancy was calculated by reads per million that mapped to the fly genome with bedtools 2.17 .
analyses of nucleosome occupancy were performed in r environment using our lab - developed software package : rage.r .
we created heatmaps for each time point by plotting the nucleosome occupancy of all fly genes and centering the genes at the tss .
we found a significant loss of nucleosome occupancy at the 4 h time point post s. typhimurium stimulation ( k - s test 2.2 10 ) ( fig . 1 ) .
these results suggest that widespread nucleosome loss may be an instrumental part of genome regulation during the immune response to s. typhimurium . | in eukaryotes , nucleosomes participate in all dna - templated events by regulating access to the underlying dna sequence .
however , nucleosome dynamics during a genome response have not been well characterized [ 1 ] , [ 2 ] .
we stimulated drosophila s2 cells with heat - killed gram - negative bacteria salmonella typhimurium , and mapped genome - wide nucleosome occupancy at high temporal resolution by mnase - seq using illumina hiseq 2500 .
we show widespread nucleosome occupancy change in s2 cells during the immune response , with the significant nucleosomal loss occurring at 4 h after stimulation .
data have been deposited to the gene expression omnibus ( geo ) database repository with the dataset identifier gse64507 . |
collapse of the hard and soft - tissues occur following extraction and the severity of the horizontal and vertical loss of tissue may reach up to 60% after 2 years .
considerable volume of post extraction ridge loss is encountered in the 1 year of tooth loss .
these localized ridge defects are characterized by regional deficit of bone and / gingival tissue .
these defects may result from tooth extraction , local trauma , periodontal lesions , congenital developmental disturbances .
such defects present a challenge for implant placement , soft - tissue adaptation and finally prosthetic rehabilitation .
implant supported rehabilitation has the final goal of achieving a soft and hard tissue integrity with optimal esthetics .
an accurate soft - tissue treatment may facilitate peri - implant soft - tissue stability over time .
localized ridge defect can be due to either bone and or / soft - tissue deficit .
type 1 and 2 ridge defects ( soft - tissue ) have been shown to have a predictable success rate post augmentation .
the importance of a thick keratinized peri - implant mucosa has been indicated for prevention of mucosal recession and maintenance of peri - implant health .
various techniques to augment keratinized tissue on implant sites have been described in the literature : roll flap , connective graft , epithelial and connective graft , coronally advanced flap .
this case report presents a surgical modification of the roll technique in the management of deficient soft - tissue around the implants . at 2 years
the present case report is about two patients age group 23 - 32 years presented with an edentulous space in relation to 22 , 23 . after successful placement of the implant , clinical presentation as after 4 months was : ( 1 ) deficient buccolingual width ( 2 ) thin keratinized mucosa .
bone gauge was used to estimate the crestal bone thickness around the implant to rule out the hard tissue defect .
transgingival probing was done to measure the soft - tissue thickness before surgery with acrylic stent and graded periodontal probe in millimeters ( mm ) with a rubber stop .
soft - tissue augmentation was planned to improve the buccal tissue thickness around the future implant supported prosthesis . after procuring adequate anesthesia , pedicle flap
was outlined starting as partial thickness horizontal incision 3 - 4 mm away from diameter of the underlying cover screw from the palatal side .
two vertical partial thickness incisions , 1.5 mm away from the adjacent interdental papilla , were marked out from the horizontal incision up to the crest and descended buccally .
partial thickness pedicle flap was reflected palatally . on approaching the crest over the cover screw , it was further reflected as a full thickness flap extending buccally to create a pouch dissection with the buccal periosteum intact .
once the buccal dissection was complete , the palatal tissue of the pedicle graft was rolled and tucked into the buccal pouch .
( 4 - 0 ) ( cytogenix ) and silk sutures ( 5 - 0 , ethicon ) 3 mm high healing abutments were screwed - in to provide support for the rolled pedicle graft [ figures 113 ] schematic representation of the surgical steps .
oral antiseptic rinse ( 0.12% chlorhexidine ) twice a day for 4 weeks and analgesic ( ibuprofen ) 8 hourly for 5 days was prescribed . at 5 weeks , sutures were removed .
after intermediate healing period of 10 months , final prosthesis was placed [ figure 7 ] .
pre - operative thin keratinized mucosa de - epithelised palatal donor site papilla saving incision and reflection of partial thickness palatal flap suturing of the rolled palatal flap into the buccal pouch 5 week follow up with temporary abutments in place temporary crowns cemented final prosthesis in place de - epithelization of the donor site papilla saving incisions marked partial thickness palatal incisions partial thickness flap raised creation of the buccal pouch rolled flap into the buccal pouch and sutured mean mucosal thickness as achieved after 2 years was 2.6 mm and mean increase in thickness was 1.75 mm [ table 1 ] .
after procuring adequate anesthesia , pedicle flap was outlined starting as partial thickness horizontal incision 3 - 4 mm away from diameter of the underlying cover screw from the palatal side .
two vertical partial thickness incisions , 1.5 mm away from the adjacent interdental papilla , were marked out from the horizontal incision up to the crest and descended buccally .
partial thickness pedicle flap was reflected palatally . on approaching the crest over the cover screw , it was further reflected as a full thickness flap extending buccally to create a pouch dissection with the buccal periosteum intact .
once the buccal dissection was complete , the palatal tissue of the pedicle graft was rolled and tucked into the buccal pouch .
( 4 - 0 ) ( cytogenix ) and silk sutures ( 5 - 0 , ethicon ) 3 mm high healing abutments were screwed - in to provide support for the rolled pedicle graft [ figures 113 ] schematic representation of the surgical steps .
oral antiseptic rinse ( 0.12% chlorhexidine ) twice a day for 4 weeks and analgesic ( ibuprofen ) 8 hourly for 5 days was prescribed . at 5 weeks , sutures were removed .
after intermediate healing period of 10 months , final prosthesis was placed [ figure 7 ] .
pre - operative thin keratinized mucosa de - epithelised palatal donor site papilla saving incision and reflection of partial thickness palatal flap suturing of the rolled palatal flap into the buccal pouch 5 week follow up with temporary abutments in place temporary crowns cemented final prosthesis in place de - epithelization of the donor site papilla saving incisions marked partial thickness palatal incisions partial thickness flap raised creation of the buccal pouch rolled flap into the buccal pouch and sutured mean mucosal thickness as achieved after 2 years was 2.6 mm and mean increase in thickness was 1.75 mm [ table 1 ] .
approximately two - thirds of bone width resorption occurs within the first 3 months after tooth extraction . in the maxillary frontal region ,
the resorption is characterized by a marked horizontal component and variable amount of vertical deficit .
any alteration in the gingival morphology post extraction plays a vital role in determining the final esthetic outcome .
these localized ridge deformities used to be corrected with prosthetic materials in order to reestablish the natural arch contour .
these prosthesis were acceptable from a functional viewpoint but resulted in poor esthetics . with the advent of periodontal plastic surgical procedures and their successful performance in implantology
a thin biotype has less interdental papilla fill with increased risk of peri - implant recession . in a study by chung et al .
observed that mucosal inflammation and plaque accumulation were significantly higher around implants with km < 2 mm .
thick mucosa ( 1 mm ) is associated with less mucosal recession as compared with a thin mucosa ( < 1 mm ) .
cardaripolli in a prospective study measured the dimensional alterations of the soft and hard tissues around 11 single - implant restorations over a 1-year postloading period .
the degree of mucosal collapse depend on the biotypes of the peri - implant mucosa .
hence the need to transform a thin mucosal biotype to a thick keratinized tissue is vital in order to have stable periimplant dimensions .
free mucosal graft and free connective tissue grafts have been used to augment the soft tissues around the implant .
these grafts are harvested from the palate of the same patient and transferred to the recipient site . in the aesthetic zone
it is usually necessary to provide a band of keratinized gingiva in order to harmonize with the surrounding adjacent natural teeth . connective tissue graft with a collar of epithelium attached
the results of the pediculated connective tissue graft with its rich vascular supply helps in augmenting and thickening of the marginal tissue better than the free connective tissue graft . achieving and maintaining an adequate marginal gingiva thickness and sufficient width of keratinized tissue at an early phase of the implant uncovery surgery is important for the maintenance of peri - implant health and esthetics .
speroni in a 3 year retrospective study on the augmented soft - tissue observed a mean mucosal thickness of 2.89 mm and mean increase in mucosal thickness of 1.75 mm at 12 months when compared from baseline .
this case report has presented a modified roll technique for soft - tissue augmentation around a dental implant during the second stage of implant surgery .
the mean mucosal thickness as achieved after 2 years was 2.6 mm and mean increase in thickness as 1.75 mm from baseline .
the advantage of this technique resulted in preserving the interproximal tissue , less discomfort and faster healing of the palatal donor site . and enhance the marginal gingiva associated with the dental implant . in a systemic review on surgical procedures for soft tissue augmentation
, all autogenous tissue graft procedures were found to be effective in increasing tissue volume .
it would therefore appear that treatment success is not a function of the choice of a surgical technique and the choice of a specific surgical technique is not as important as the adherence to sound biologic principles . if the aim is to achieve a good clinical outcome .
the procedure as describe in this case report is indicated for correction of mild to moderate buccal ridge deficiency or to increase the bulk of the marginal gingiva at the stage two implant surgery .
average increase in the mucosal thickness was 2.5 mm and increase in keratinized tissue was up to 1.8 mm in both the cases after 2 years post loading .
this procedure provided a good healing and stability of the peri - implant tissues after the maturation time of the soft - tissue augmentation . | the aim of the article is to describe a modification of the roll flap , performed at the implant second surgical stage , allowing the correction of small horizontal defects by enhancing the soft - tissue thickness and improving the buccal soft - tissue profile .
the advantages for this modification lies in the fact to preserve the papilla morphology and enhancement of the soft - tissue esthetic results . |
the majority of the developed countries try to associate economic growth with the provision of certain social services and , in this manner , aim at progressively improving the well - being of citizens .
accordingly , a social welfare model was developed to guarantee that everyone had , among other things , access to health care and education .
however , with regards to health care a disproportionate growth of the public expenditure in this sector was verified , for various motives , especially population ageing . in 2010 , 85.5 % of the health care expenditure in norway was supported by the tax payers , in denmark 85.1 % and in the united kingdom 83.2 % .
this fact inevitably implies that limits have to be established for the public provision of health care .
also in most developed countries , namely in oecd , the proportion of total government expenditure that goes to health care is growing steadily . indeed , in 2010 health expenditure was in norway 9.4 % of the gdp , in denmark 11.1 % and in the united kingdom 9.6 % ( medium of 9.5 % in oecd countries ) . with the establishment of priorities in health care
explicit forms of rationing have already been implemented in some countries , as is the case of the scandinavian countries , the netherlands , england , canada , among others , to improve the management of the considerably high public health expenditure .
many of these prioritization systems resort to a proposal by norman daniels known as accountability for reasonableness , an indispensable tool to approach this matter .
nevertheless , if this method provides the required legitimacy to make decisions regarding options in health care , the degree of fairness involved in these choices has been questioned .
firstly , that the health concept of the world health organization is too vast in order to achieve the appropriate choices in health care .
the second presupposition , which follows from the latter , is that fairness in health care basically implies attaining a
normal level of human performance ; the citizens preferences that do not correspond to the real health requirements do not need to be met .
accountability for reasonableness to the world health organization s holistic view of health and propose an evolutionary method in relation to the normal functioning standard proposed by norman daniels .
thus , the study will contribute to an even more just and equitable prioritization system in health care .
there are several solutions to overcome the problem of lack of sustainability in the public health care systems , though the rationalization and efficiency measures should not be neglected and should continue to be implemented .
but , considering the presupposition that the possibility of increasing the contributions in the form of co - payments is very limited , the combination between efficiency and prioritization in health care has enabled the principle of equality , regarding the access to health care by everyone , to be maintained in the developed countries .
therefore , in the latter years , the establishment of priorities in health care has been considered in many western countries regarding the reform of the public health care systems .
the citizens increasing needs , associated to ageing and the consequent demographic inversion have led to a discrepancy between demand and supply in health care .
accountability for reasonableness is probably the most widespread model of priority setting in health care in the western world . in the united kingdom , for instance , accountability for reasonableness helped shape thinking about how the national institute for health and clinical excellence ( nice ) should incorporate social value judgments into its evidence based clinical proposals . indeed ,
public health systems with public accountability , such as those of canada , the united kingdom , new zealand , and sweden , are now explicitly applying this framework of accountability for reasonableness [ 10 , 13 , 17 ] . in the universal coverage systems of most developed countries such decisions
are made by public agencies . in mixed systems , such as in the united states , decisions whether to fund new technologies drugs , devices , procedures are made both by public agencies and by private insurers and managed care organisations .
although the problem of formal ( democratic and public ) legitimacy is usually guaranteed by accountability for reasonableness there still remains the problem of substantive legitimacy that is if justice as fairness is really considered .
for instance with regards the national institute for health and clinical excellence and other regulators that have direct impact on limit setting in health care it has been overtly suggested that a more inclusive process might lead us to a solution to the problem of substantive legitimacy . a decision - making process based on inclusive deliberation as an accepted standard of decision - making by the overall democratic society might add fairness to the system .
it follows that rational decision - making can not be achieved on the near - exclusive basis of quantitative evidence and regulators should abide to communitarian traditions including most ethical , moral and religious traditions . assuming that states in a pluralistic society are at least partially neutral in the sense that every perspective of human happiness has the same relative weight most regulators adopt a procedural approach to ethics not a substantive one .
and justice is more related to fair procedures and public accountability than to any specific view of distribution of benefits and burdens .
indeed , a better framework for prioritizing health care can be achieved through the inclusion of ethical traditions that give more weight to features specific to particular persons , communities , families and political units . in a secular pluralistic society
it is desirable to channel those different views of a good life resorting to democratic deliberation through democratic institutions and to the direct empowerment of the people .
qualitative evidence will only partially allow for procedural justice in health care regulators deliberative process .
this process in a democratic society should be objective , comparable , accountable and externally evaluated .
evidence based medicine ( ebm ) is in accordance with these principles , at a global level .
this is the reason why ebm is so appealing and a determinant factor in accountability for reasonableness .
as stated by daniels and sabin , public accountability means a robust disclosure of relevant information about benefits and performance as well as a demand for a due process . a specific array of conditions should be met to comply with the principle of accountability [ 57 ] , namely : publicity condition : decisions regarding both direct and indirect limits to care and their rationales must be publicly accessible;relevance condition : the rationales for limit - setting decisions should aim to provide a reasonable explanation of how the organization seeks to provide
value for money in meeting the varied health needs for a defined population under reasonable resource constraints .
specifically a rationale will be reasonable if it appeals to evidence , reasons , and principles that are accepted as relevant by fair - minded people who are disposed to finding mutually justifiable terms of cooperation;revision and appeals condition : there must be mechanisms for challenge and dispute resolution regarding limit setting decisions , and , more broadly , opportunities for revision and improvement of policies in the light of new evidence or arguments;regulative condition : there is either voluntary or public regulation of the process to ensure that previous conditions are met .
publicity condition : decisions regarding both direct and indirect limits to care and their rationales must be publicly accessible ; relevance condition : the rationales for limit - setting decisions should aim to provide a reasonable explanation of how the organization seeks to provide value for money in meeting the varied health needs for a defined population under reasonable resource constraints .
specifically a rationale will be reasonable if it appeals to evidence , reasons , and principles that are accepted as relevant by fair - minded people who are disposed to finding mutually justifiable terms of cooperation ; revision and appeals condition : there must be mechanisms for challenge and dispute resolution regarding limit setting decisions , and , more broadly , opportunities for revision and improvement of policies in the light of new evidence or arguments ; regulative condition : there is either voluntary or public regulation of the process to ensure that previous conditions are met .
this framework has as a genetic fingerprint both the deliberative process necessary to establish the legitimacy of the decision - making process as well as the fairness of such decisions .
daniels claims that accountability for reasonableness makes limit - setting decisions in health care not only legitimate , but also fair .
daniels argues that claims to equality of opportunity may be limited by scarcity of resources , but nevertheless choices and priorities in health care must be accountable by democratic procedures .
this perspective of distributive justice as well as its democratic accountability is responsible for the scope and limits of health care services .
it follows that particular entitlements to health care namely expensive innovative treatments and medicines may be fairly restricted as long as this decision is socially accountable and imposed by financial restrictions of the system .
this framework has been used , for instance , in rationing pharmaceuticals in a accountable way as the process facilitates a broader public discussion about fair limit setting .
the starting point of daniel s account of fairness is that disease and disability restricting the range of opportunities that would otherwise be open to individuals are properly seen as unjust and not only as unfortunate circumstances . as this argument goes a right to health care access and delivery does exist as a determinant to the exercise of the equal opportunity rule .
this claim for an equal opportunity of all citizens intends to ensure normal and not a truly equal functioning .
this distinction seems to be paramount because each one of us is not equal to a fellow citizen in the strict sense ; in fact , we are entitled to some primary goods that would allow us to function physically , psychologically and socially at a basic level .
then , our talents and capacities , following our will , may be expressed or not depending of particular circumstances . as a positive welfare right , the right to health care access and delivery imposes on society the duty to allocate resources to health related needs with maximal efficiency .
this perspective is based on a health concept distinct from the world health organization proposal since 1946 . concerning
this aspect norman daniels refers that since we believe that health is a distinct concept from happiness , we also reject the overly broad view of health as a state of complete physical , mental , and social well - being , and not merely the absence of disease or infirmity , that is embodied in the world health organization definition . by
normal performance we mean the performance of the average citizen ( criterion of the reasonable person ) .
the excellent or truly equal performance is not the case since such a radical view of the citizens equal rights contradicts the proper essence of the human diversity .
that is , and according to daniels , we correct for some natural effects on that distribution disease and disability but we do not attempt to eliminate all differences in the name of a radically expansive view of equal opportunity .
the question then is how distributive decision - making in health care might gain in fairness by giving wider representation to the diversity of perspectives in contemporary western society .
this is the reason why the world health organization definition of health is such a broad concept . on the other hand
there is no hierarchy in the provision of health when the standard is normal versus non - normal functioning .
it is these authors conviction that even in the normal functioning criteria some treatments and medical interventions have priority upon others .
also , justice might be incremented with a progressive approach because in principle every health service ( treatment , diagnosis , and so on ) might be negotiated in the deliberative process .
but fairness is also optimised because the confluence between vertical and horizontal equity implies that , even within the basic package , a progressive approach originates a true hierarchy of priorities meaning that a particular treatment has precedence over another one , although the latter may still be an integral part of the basic package .
to enable a better compatibility between accountability for reasonableness and the integrated view of health of the world health organization ,
the authors suggest a change of paradigm regarding the referential all or nothing such as proposed by norman daniels ( normal vs. non - normal functioning ) . although the logical consequence of justice as fairness being the implementation of an effective opportunity for everyone to have access to health care and , thus , the issue being the existence of a universal and general health system , its revaluation in the light of a distributive justice may dictate a different future ( fig . 1 ) .
equal opportunities may be represented graphically in the form of a function : ( eo)f equal opportunity function .
these concepts in their philosophical and economic scope do not specify the relevant properties to characterize an agent as
this is to say , if equals should be treated in the same manner , with regards to distribution of material resources , and others in an unequal manner , it is essential to define a method which enables this distinction to be established . the graphic representation of ( eo)f , in the theoretical plan , resorts to two variables : the x - axis represents the variable hierarchy of needs in accordance with the maslow pyramid ( physiological , safety , belongingness and love , esteem , self - actualization and self - transcendence needs);the y - axis indicates the health care performance level .
this variable reaches a maximum value through the universality and generality of the performance ( public services ) and a minimum value in which the access to health care services should be imputed to the individual responsibility of each citizen.fig .
d health care delivery , t tiering , n needs ( maslow ) , b&l belongingness and love , s.a self - actualization , s.t self - transcendence the x - axis represents the variable hierarchy of needs in accordance with the maslow pyramid ( physiological , safety , belongingness and love , esteem , self - actualization and self - transcendence needs ) ; the y - axis indicates the health care performance level .
this variable reaches a maximum value through the universality and generality of the performance ( public services ) and a minimum value in which the access to health care services should be imputed to the individual responsibility of each citizen .
d health care delivery , t tiering , n needs ( maslow ) , b&l belongingness and love , s.a self - actualization , s.t self - transcendence when the ( eo)f intersects the x axis ( point eo * ) , in accordance with the combination of : a ) the needs criteria , and b ) the financial restrictions imposed by the system , appears the point from which the provision of health care through the public services is not mandatory . from this point ,
any citizen may have access to health care ( at the cost of his own individual responsibility or the professional group which he belongs to ) without damaging the principle of justice as fairness , if he resorts to alternative coverage schemes , namely private health insurance or out of pocket payments .
but , only if the fulfilment of the second order needs may be at issue which may be situated , schematically , and in a variable form , as from the point that maslow defined as esteem needs ( in comparison with the normal functioning criteria fig . 2).fig
. 2equal opportunity function ( eo)fnormal function version . 1 normal function curve .
2 who curve equal opportunity function ( eo)fnormal function version . 1 normal function curve .
2 who curve norman daniels establishes a distinction between health need and health preference , this means between the health care needs in order to reach a level of functioning physical and psychological typical of our species , and mere preferences , conveniences , which are beyond a reasonable and consensual level of normality . though it is not possible to satisfy all the esteem and self - actualization needs , nor all the individual preferences , it is up to each person to resort to his financial resources ( individual responsibility ) to achieve this objective . as from this point eo * , and even though in an ideal society , with absolute availability of resources in which these needs could be met , in the present state of economic and financial restriction it is not only an option but , probably , a duty to proceed with this priority . in practice , and presupposing a maximum efficiency of the public health systems ,
the ( eo)f allows the establishment of priorities in health care as far as the health care services at issue aim at meeting the needs which do not interfere with the normal performance of a citizen .
the universality of access is not subject to any objection but the claim that there are no limits to public provision ( having as major criteria the individual needs , by definition , almost unlimited ) is today contested since the allocation of society s resources to health has to compete with the supply of other social goods . indeed , one of the major challenges of modern societies is to converge economic development with human development and therefore it has been overtly suggested that a true developed society promotes every aspect of human fulfilment , such as health , education , gender equality , and so on .
inevitably a delicate balance emerges between the social goods which the citizens may , or may not , be entitled to , in conformity with the social and economic situation of a particular society . and as referred by penelope mullen and peter spurgeon
, so prioritizing has become the conceptual paradigm of the health policies of the developed countries . .
in fact , inequality ( treating two people in a different way ) becomes an inequity ( treating two people in an unfair way ) if a solid motive does not exist to justify the negative discrimination of a citizen in detriment of another .
however , the opposite is equally valid , that is , an apparent inequity becomes a mere inequality ( ethically acceptable and , therefore , fair ) if it is in conformity with the restrictions imposed on the system with the application of the economic constraints in accordance with the ( eo)f .
but if , and only if , that situation of inequality does not harm the citizens basic rights and , furthermore , if the process that led to the decision is democratically determined in conformity with the public accountability criteria . reformulating this appreciation of the problem , the restrictions of an economic and financial nature transform a decision for allocation of resources from iniquitous ( unfair ) to acceptable ( fair ) as far as it does not negatively discriminate any class of citizens ; even though a particular class of services may be legitimately restricted .
this seems to be a huge dilemma of the health systems : to articulate in a fair and impartial manner the right of access to the necessary health care using the resources available in society [ 1 , 29 ] . to illustrate this proposal
we are going to refer to the application of equal opportunity function on elective caesarean delivery .
evidence - based medicine ( ebm ) has focused its attention on particular aspects of medicine , and specific areas such as elective on - demand caesarean delivery were left behind because there is some difficulty to perform this kind of ebm research .
although there is still no absolute evidence with regards the risk / benefit analysis of on - demand caesarean delivery ebm must be carefully evaluated in this setting if adequate health policies are to be promoted .
evidence should be obtained by international co - operation , by independent agencies , so that its goals are truly achieved .
this evidence is necessary so that a fair resource allocation policy is accomplished . indeed , even if it is accepted that there is a right to reproductive autonomy allowing a woman to make informed choices about the pregnancy and birth another issue is related to the social costs of this kind of decision . and ,
therefore , it should be clearly determined if it should be included in the basic health care package or if it should be left to individual responsibility . from an ethical perspective on - demand caesarean delivery
faces the problem of justice in allocation of health care resources . in a changing economic environment
it is not surprising that new approaches to health care may limit the provision of care as it is not possible to deliver everything to everyone .
as there is no absolute evidence about the comparative risks and benefits of vaginal versus abdominal deliveries , the authors will focus specifically in the relevance condition as far as the rational and the reasons and principles for limit - setting decisions are concerned .
indeed , choices and priorities in health care are fair if they are accountable by democratic procedures .
norman daniels states quite clearly that in any health care system , then , some choices will have to be made by a fair , publicly accountable , decision - making process .
just what constitutes a fair decision - making procedure for resolving moral disputes about health care entitlements is itself a matter of controversy .
our rights are not violated , however , if the choices that are made through fair decision - making procedures turn out to be ones that do not happen to meet our personal needs , but instead meet needs of others that are judged more important .
this perspective of distributive justice as well as its democratic accountability is responsible for the scope and limits of health care services .
it follows that particular entitlements to health care namely access to a particular type of delivery in a public service may be fairly restricted as long as this decision is socially accountable and imposed by financial restrictions of the system . in many countries , for instance , upper and middle - class women choose regularly abdominal deliveries when having babies since they can afford to pay for it in private clinics . in the private health care system ,
where women come from higher income and education levels , caesarean delivery prevails . on the other hand , in the public sector , where women belong to less privileged social classes , vaginal deliveries are the usual practice due to medical care routines that are imposed on them .
as far as the rational and the reasons for limit setting is concerned , and although in principle , the pregnant woman s reproductive autonomy should be respected ( see for instance the 2011 nice s guideline that states that for women requesting a cs , if after discussion and offer of support a vaginal birth is still not an acceptable option , offer a planned cs ) , daniels relevance condition implies that a thorough economic analysis is performed .
it follows that restrictions to the basic package in health care imply necessarily that the competitive medical treatment ( on - demand caesarean delivery ) is more expensive than the usual one ( vaginal delivery ) .
if the price is the same or lower then there is no reason based on fairness to restrict its use in public services ( although it might be restricted by a risk / benefit analysis ) .
this economic analysis should take into consideration not only direct costs but also indirect ones . in caesarean versus vaginal delivery
it is usually considered only the unitary price of each procedure as determined by each country health authority ( or insurance company ) . in general
the unitary price reflects mainly the aggregated costs of the professionals , the operating room and the materials used .
this analysis may be biased because the cost of a caesarean delivery is usually calculated for caesareans performed with medical indications ( crash caesareans for instance ) which tend to be much more expensive than caesarean on - demand , that are planned and elective .
indeed most reports show that the cost of caesarean delivery is higher than vaginal birth .
therefore , it can be claimed that the practical application of the equal opportunity function can place caesarean delivery in the t segment ( tiering ) of the y - axis that is outside the basic health care package .
it follows that public health care systems have the responsibility of promoting good quality planned parenthood services but some interventions such as on - demand caesarean delivery may be legitimately positioned outside the basic package .
functioning and therefore it is a health need not a mere preference , the proposed methodology equal opportunity function allows that in this circumstance a fairer decision - making process is obtained . on the other hand
if , in the future , it is technically demonstrated that the cost of vaginal birth is higher than that of a caesarean delivery a straightforward application of the equal opportunity function might led to the conclusion that cs might be included in the basic package .
the ( eo)f curve then has a great elasticity with regard the amount of services available to all citizens allowing to fairly adjust the basic package to the amount of resources available .
there is no doubt that health systems must be explicit about prioritizing and norman daniels theory of justice and health is the most widespread tool to accomplish this goal .
however , it faces a practical problem that is how to allocate resources fairly when they are especially scarce .
daniels claims that accountability for reasonableness makes limit - setting decisions in health care not only legitimate , but also fair .
the paper discusses different options for resolving this lack of clarity and examines how they apply to daniels accountability for reasonableness framework . in general
this theory holds that treatments that accomplish a species typical behaviour ( normal ) should be in the basic package and that this is accepted by reasonable people . and
therefore a treatment that does not relate to a health need , but only to an individual preference , should be left outside .
for instance pre implantation genetic diagnosis is not strictly speaking essential for the normal function of a person .
nevertheless , if there are enough resources available this method of diagnosis could still be offered to the general public .
it would depend contingently on the balance reached by the social contract between the global amount of resources and the public services desired .
it follows that the ( eo)f curve is not static but slides to the left or to the right depending of particular social arrangements .
it means that in principle with growing resources more health services could be in the basic package ( ( eo)f curve to the right ) and in a period of scarcity the amount of health services is shorter ( ( eo)f curve to the left ) . and what about prioritizing treatments that originate a species typical ( normal ) behaviour but that are more expensive than less expensive alternatives ( such as on - demand caesarean section , some medical assisted procreation technologies , some blood alternatives or snoring surgery in non - pathological cases ) ?
moreover , does the traditional framework of accountability for reasonableness help to prioritize pharmaceuticals of proven medical benefit but extremely expensive in a just and fair way [ 12 , 16 ] ?
moreover , how far should society be involved in these decisions so that they are legitimate and fair ? it can be claimed that applying the who definition of health in priority setting would allow access to a wider range of health care , without violating the four conditions underlying
accountability for reasonableness . indeed , publicity , revision and appeals , and regulative conditions are mainly procedural ones but are extremely useful in legitimating health care choices applying the
might even be optimized with this rationale because it can be claimed that not only a reasonable explanation but a fair explanation is given of how value for money is optimized .
namely in the european tradition equal opportunity function appeals to evidence , reasons , and principles that might be accepted as fair by most citizens .
the manchester triage system ( mts ) is a practical example of the equal opportunity function at a micro level . from a justice perspective
the mts algorithm implies that more fundamental needs ( such as protecting life ) have priority over less fundamental ones , although these might also be considered . at a macro level the equal opportunity function
could be not only fairer but also more practical because it would also allow a more equitable managing of waiting lists , if the reasons and the rationale for choices are made public and are truly accountable .
accountability for reasonableness is an extremely valuable tool to address the issue of setting limits in health care ; second , that what is called equal opportunity function can reflect how
accountability for reasonableness results in fair limit - setting decisions ; and third , that this methodology must be further specified to best achieve fair limit - setting decisions .
indeed , when resources are especially scarce the methodology suggested in this paper will allow not only prioritizing in an all or nothing basis but can contribute to a more systematic and fair approach .
it might even be considered as an evolutionary perspective in relation to daniels account of fairness . in developed societies
most citizens are aware of the need to establish priorities in health care but there is a strong disagreement about the ethical legitimacy of many choices .
functioning standard suggested by norman daniels is very appealing because the reasonable and prudent person can easily determine what services should be included in the basic package . but this standard may leave important modalities of diagnosis and treatment outside public services .
the equal opportunity function evolves from this perspective in the sense that keeping in mind that a distinction should be made with regard the treatments that may or not be included in the basic package it allows a gradual approach and a true hierarchic system of priorities in health care . | explicit forms of rationing have already been implemented in some countries , and many of these prioritization systems resort to norman daniels
accountability for reasonableness methodology .
however , a question still remains : is accountability for reasonableness not only legitimate but also fair ?
the objective of this paper is to try to adjust
accountability for reasonableness to the world health organization s holistic view of health and propose an evolutionary perspective in relation to the normal functioning standard proposed by norman daniels . to accomplish this purpose
the authors depart from the normal functioning standard to a model that promotes effective opportunity for everyone in health care access , because even within the normal functioning criteria some treatments and medical interventions should have priority upon others .
equal opportunity function is a mathematical function that helps to hierarchize moral relevant necessities in health care according to this point of view .
it is concluded , first , that accountability for reasonableness is an extremely valuable tool to address the issue of setting limits in health care ; second , that what is called in this paper equal opportunity function might reflect how accountability for reasonableness results in fair limit - setting decisions ; and third , that this methodology must be further specified to best achieve fair limit - setting decisions .
indeed , when resources are especially scarce the methodology suggested in this paper might allow not only prioritizing in an all or nothing basis but can contribute to a hierarchy system of priorities in health care . |
platform switching , also known as diameter shifting , is a technique combining an implant with a reduced diameter abutment.12 the concept was introduced in the early 1990s after development of a wide diameter implant that was connected to the standard abutment.2345 since it was introduced , the technique has been evaluated by many researchers ; it was proposed that connecting the smaller diameter abutment to a larger implant could help prevent crestal bone loss.26789101112 in addition , it is reported that platform switching is beneficial in establishing biological width and produces excellent esthetic results.131415 other than the suggestion that platform switched implants produce satisfactory esthetic results , their advantages can be summarized in terms of biological and biomechanical aspects.910161718 biologically , marginal bone preservation is explained by the change in the micro - gap location , which might be related to inflammatory cell infiltration and reformation of biological width.1016 the relocation of the micro - gap might serve as a defense mechanism against bacterial penetration and limit inflammation in marginal bone.917 in addition , platform switching generates a larger surface area for soft tissue attachment.18 mechanically , platform switched implants are known to redistribute stress and ultimately affect peri - implant marginal bone loss.19 stress redistribution is reported to be achieved by centralizing stress.20 it was also stated that prosthetic loading forces that transmitted to the bone - implant interface was reduced.20 however , the biomechanical properties of the platform switching concept have not been explained clearly and there seems to be no consensus on its mechanical advantages .
platform switched implants reportedly decrease the stress concentration on the implant - bone interface , while increasing stress on the abutment and screw.19 in another study , platform switching was found to reduce bone strain.2122 the value and range of stress concentration decreased as the implant diameter increased in a further study.323 previous studies suggested that platform switching reduced stress on the marginal bone crest.2425 however , some argued that these favorable results were related to the increased diameter of the implant , not the platform switching concept.1 when the platform switching concept was introduced , it was compared with regular platform implants because there was no matching abutment for wide diameter implants.2 currently , the wide platform implant system is used clinically and a comparison of platform switched and wide platform implants is necessary . according to a previous study , although the relocation of the micro - gap via platform switching affected the mechanical properties of the implant - abutment connection , platform switching did not alter the stress concentration in the bone , suggesting that marginal bone preservation is related to biological advantages , and not platform switching.126 this was supported by a study using finite element analysis , in which a circumferential horizontal mismatch of 0.5 mm did not make an important contribution to the biomechanical environment of implants.27 finite element analysis is a computational method that can calculate the stress and displacement of the structure using discretization.28 one of the advantages of finite element analysis is that the analysis of complex structures is possible .
there have been many studies that used finite element analysis to analyze the stress distribution of implants and the surrounding structures.293031 finite element analysis has been used to verify the platform switching concept and to show stresses on internal structures.11932 the purpose of this study was to analyze the influence of the platform switching concept , which was thought to have biomechanical advantages in peri - implant bone , using three - dimensional finite element analysis .
two three - dimensional finite element models , wide platform and platform switching models , were designed .
they included an external hex implant system and peri - implant bone tissue . in the wide platform model
the platform switching model was created by connecting a regular platform abutment to a wide platform implant . in each model , the implant was placed in a segment of the mandible and a gold crown was placed on the abutment . a vertical load of 300 n was applied on the occlusal surfaces of the crowns .
the maximum von mises stress values and displacements of the components in the two models were compared to analyze the biomechanical behavior of the models .
the models for three - dimensional finite element analysis were assumed to have the implant - prosthesis complex for the mandibular first molar .
finite element models of cortical bone , cancellous bone , implant , abutment , abutment - screw , and crown were separately generated and then combined .
the bone block that was similar to the shape of the partial mandible was created using mesh generation software ( visual - mesh , esi group , paris , france ) .
the cancellous bone was surrounded by the cortical bone of approximately 2 mm thickness . to minimize the influence of the model size on the results of the finite element analysis ,
the mandible model was designed to be 35 mm in length , 15 mm in width , and 30 mm in height.33 the implant used for finite element model was based on an external hex system , with 5 mm in diameter and 10 mm in height ( usii , osstem , pusan , korea ) .
the geometries of the implant system in the initial graphics exchange specification ( iges ) file format were provided by the manufacturer .
they were changed to meshes for finite element analysis using meshing software visual - mesh ( esi group , paris , france ) . during the conversion process from raw geometry data to finite element
meshes , the implant and abutment screws were simplified into a cylinder shape for the reduction of calculation time .
tetrahedral meshes for the simulation were generated using meshing software ( visual - mesh , esi group , paris , france ) .
the wide platform model was composed of cortical bone , cancellous bone , a wide diameter implant , a wide diameter abutment , an abutment screw , and a crown .
the wide diameter abutment had the same external hex structure as the regular platform abutment .
the platform switching model had a regular diameter abutment instead of the wide diameter abutment .
except for the abutment , the wide platform model was identical to the platform switching model .
for the wide platform model , 21,915 nodes and 86,183 tetrahedral elements were used , and , for the platform switching model , 21,057 nodes and 86,119 elements were used ( fig .
the material properties of each component from previous studies were used.3435 the materials were assumed to be homogeneous , isotropic , and linearly elastic .
the gold crown and the abutment screw were assumed to be made of gold alloy .
the medial and distal ends of the mandible segment were constrained in six degrees of freedom .
the surfaces between the top of the implant and the bottom of the abutment was set as frictional contact surface and the coefficient of friction between two components was 0.16.36 the other contact surfaces was located between the lower part of the abutment screw head and the abutment .
the coefficient of friction was set to be 0.2.36 other components were tied to each other at the contact surfaces . as a loading condition , a total vertical load of 300
n was gradually distributed to the occlusal surfaces of the crowns over 2 milliseconds ( table 2).37 the distributed load was applied at the nodes on the buccal and occlusal surfaces of the crown model .
a simulation was performed using finite element structural analysis software ( virtual performance , esi group , paris , france ) .
the maximum von mises values in the components and stress distribution patterns of the wide platform and platform switching model were compared .
during the gradual loading period , stress appeared in the components . at the end of loading ,
stress concentrations were mainly observed at the bottom of the abutment and on the top surface of the implant in both models .
the platform switching model showed higher von mises stress values in the prosthodontic components than the wide platform model ( fig .
the maximum von mises stress value of the platform switching model was 830.00 mpa and that of the wide platform model was 107.41 mpa .
the highest von mises stress value of the platform switching model was approximately eight times greater than that of the wide platform model .
in other prosthodontic components , platform switching model also showed higher von mises stress values ( table 3 ) .
the maximum von mises stress value at the crown of platform switching model was 1.99 times higher than that of the wide platform model , 12.06 times higher at the screw , and 12.89 times higher at the implant .
the stress distribution patterns in the prosthesis and implant components were not symmetrical . on the loading side ,
the contact area between the abutment and implant showed the highest stress concentration among all components in the models .
the von mises stress values in the abutment screws were lower than those in the abutment or implant .
other than the abutment and the implant , stress concentrations were observed at the crown and in cortical bone . in the bone ,
relatively lower von mises stress values were observed than those in other components and the highest von mises stress values and stress distribution patterns of the two models were similar ( fig .
the von mises stress values were higher than those in wide platform model , although the difference was not significant .
the highest value of the cortical bone in the platform switching model was 45.25 mpa , and 45.75 mpa in the wide platform model . in both models ,
the maximum von mises stress value of cortical bone was higher than that of cancellous bone .
in cortical bone , the stress was concentrated around the implant and spread along the upper cortical bone portion . in cancellous bone ,
the maximum von mises stress values were 1 mpa in both models and stress concentrations occurred in areas close to the cortical bone and implant .
components of the platform switching model showed greater displacement than those of the wide platform model ( table 4 ) .
the maximum displacement of the abutment in the platform switching model was 11.17 times greater than that of the wide platform model . at the abutment screw , the maximum displacement of the platform switching model was 10.73 times greater than that of the wide platform model and 1.21 times greater at the implant .
in this study , there were similar stress distribution patterns in both wide platform and platform switching models .
the stress concentration occurred at the abutment - implant interface , and these results were in accordance with the previous studies.12426 however , the platform switching model had different stress distribution features compared to the wide platform model .
the highest von mises stress values of the abutment , the abutment screw , and the implant in the platform switching model were much higher than those in the wide platform model . in the cortical bone , the von mises stress values in the platform switching model were similar to those in the wide platform model . in the cancellous bone , there was no difference between the models .
this means that platform switching might not affect stress in the marginal bone , which is in agreement with the results of the previous studies.2627 the result that displacement of the bone did not vary greatly between the models was also in accordance with other studies.2027 in several previous studies using finite element analysis , it was reported that platform switching reduced bone strain and affected the stress distribution in the marginal bone , as well as increasing the stress on the abutment and abutment screw.19202124 considering the displacements and stresses in the cortical and cancellous bone and the implant components in this study , there seemed to be no noticeable influence of platform switching on peri - implant bone structure .
the cause of decreased bone resorption of marginal bone in platform switching has been thought to be related to the changes of a biological width,38 inflammatory cells,2 the distance between the implant - abutment gap and the bone crest,39 and stress concentration.4041 platform switching has been suggested to be advantageous in preventing bone resorption ; one report found that bone resorption in marginal bone was related to overload.41 however , the results of the present study suggested that the change in stress distribution in the platform switched implant might not explain differences in marginal bone resorption , although platform switching could impact the mechanical components of the implant system.26 this is supported by the research suggesting that the reduction of bone stress in the platform switched implant is related to the increased implant diameter , not the platform switching technique.20 previously reported differences in marginal bone loss in the platform switched implant might be biological in nature rather than biomechanical .
the reason why the results in this study varied from other studies reporting decreased stress in bone might be related to differences in methods of finite element analysis . in one study , the contact condition was not set and all of the components were presumed to be bonded completely.24 in another study , separate prosthodontic components did not exist , unrealistic bonding between the components was set , and the occlusal force applied was very small.19 another explanation for the varied results of finite element studies is that , in most of the studies , the concept of preload was not introduced .
with preload , stress distribution patterns would be different from that without preload.42 in this study , the results showed that displacements and stress distribution patterns of prosthodontic components in the platform switching model were different from those in the wide platform model . considering these results ,
the platform switched implant might have mechanical disadvantages compared to the wide platform implant , which had larger components . in the wide platform implant , more favorable mechanical features including a wide external hex structure can be expected .
although there have not been any reports of remarkable drawbacks in using the platform switched implant,27 the stress concentration on the prosthodontic components can be a serious disadvantage .
centralized forces to the axis of the implant might be transmitted through the abutment screw and thus might concentrate stress on implant components , increase the possibility of component fracture , and lead to implant failure.24 considering the stress concentrations and large displacements of the prosthodontic components in this study , platform switching seemed undesirable in terms of mechanical properties and might lead to mechanical complications including screw loosening and component fracture in implant system.24 the related stress concentration might be greater due to the increased difference in diameter between the implant and abutment interface.1926 besides the stress concentration on the prosthodontic components , there might be other disadvantages to the platform switching concept . in platform switching
, there is the limitation of having the same screw access hole size and requiring sufficient space for the development of the emergence profile.19 although finite element analysis has been developed since it was first introduced to the dental field , it still has limitations related to reproduction of complex structure , mesh size , boundary conditions , introduction of preload , and non - linear material properties . in this study ,
the geometry of the implant was simplified for the reduction of calculation time and the convenience of modeling . with the threaded implant design
, the stress distribution pattern and the displacements may be different because threads can contribute in dissipating stress , increase implant - bone contact area , and resist external force.4344 further finite element studies with more sophisticated conditions could reveal the quantitative influence of the stress concentration on marginal bone loss in the platform switched implant , accurate structural analysis of platform switching concept , and the mechanism of peri - implantitis .
finite element analysis with these advanced features would contribute to solving problems including reproduction of dynamic masticatory simulation and the influence of different restorative materials on the implant system .
although von mises stress values in the implant components were much higher in the platform switching model than those in the wide platform model , the stress values in the bone of the platform switching model were similar to those of the wide platform model .
biomechanical advantages of the platform switched implant in marginal bone loss was thought to be doubtful . due to the stress concentration generated in the implant and the prosthodontic components of implant system | purposethe purpose of this study was to analyze the influence of the platform switching concept on an implant system and peri - implant bone using three - dimensional finite element analysis.materials and methodstwo three - dimensional finite element models for wide platform and platform switching were created . in the wide platform model , a wide platform abutment was connected to a wide platform implant . in the platform switching model ,
the wide platform abutment of the wide platform model was replaced by a regular platform abutment .
a contact condition was set between the implant components . a vertical load of 300 n was applied to the crown .
the maximum von mises stress values and displacements of the two models were compared to analyze the biomechanical behavior of the models.resultsin the two models , the stress was mainly concentrated at the bottom of the abutment and the top surface of the implant in both models .
however , the von mises stress values were much higher in the platform switching model in most of the components , except for the bone .
the highest von mises values and stress distribution pattern of the bone were similar in the two models .
the components of the platform switching model showed greater displacement than those of the wide platform model.conclusiondue to the stress concentration generated in the implant and the prosthodontic components of the platform switched implant , the mechanical complications might occur when platform switching concept is used . |
mitochondria are essential organelles whose replication , development , and physiology depend upon coordinated interactions between gene combinations from both the mitochondrial and the nuclear genomes . when genes from both genomes interact , their differing modes of inheritance , bi - parental versus maternal , can result in genomic conflict ( werren 2011 ) .
there is direct evidence of the genomic conflict generated by epistatic interactions between the mitochondrial genes and nuclear genes from studies of mitochondria involvement in sperm development ( wang 2004 ; rajender et al .
furthermore , introgression of foreign mitochondria into drosophila populations has revealed sex - biased epistatic effects with x - linked nuclear genes ( rand et al .
2011 ) . when organismal fitness depends on the epistatic interactions between the nuclear and mitochondrial genomes ( rand et al .
2007 ) , the cotransmission of nuclear mitochondrial gene combinations facilitates epistatic selection ( wade and goodnight 2006 ; brandvain and wade 2009 ) .
cotransmission is important to epistatic selection because it maintains gene combinations across generations . by increasing the heritability of gene combinations
the question remains , as to how these genome interactions have shaped the geography of the genome .
theory predicts that the nuclear genome should evolve in a way to maintain nuclear mitochondrial gene combinations ( wade and goodnight 2006 ; brandvain and wade 2009 ) .
one means of increasing the heritability of nuclear mitochondrial gene combinations involves changing the physical location of nuclear genes within the nuclear genome as opposed to changing location between the nuclear and mitochondrial genomes .
the probability of cotransmission of x - linked genes and mitochondria is significantly higher than that of mitochondria and autosomal genes ( 0.67 versus 0.50 in a randomly mating population ) ( rand et al . 2001 ) .
therefore , the evolution of coadapted ( ca ) nuclear mitochondrial gene combinations would be facilitated if nuclear genes interacting with the mitochondria were located on the x - chromosome instead on the autosomes because of the increased probability of cotransmission ( rand et al .
mitochondrial gene combinations and against poor ones is facilitated by the location of nuclear genes located on the x chromosome relative to the autosomes .
a clear prediction of the ca hypothesis is that nuclear genes which interact with the mitochondrion ( n - mt genes ) , should be overrepresented on the x relative to autosomes .
here , we tested the prediction of the ca by investigating the chromosomal distribution of nuclear genes that interact with the mitochondria .
using the online genome database biomart ( http://www.biomart.org ) via their martview interface ( smedley et al .
2009 ) , we collected genomic information on the following organisms using the available ensembl genes 63 datasets : homo sapiens ( grch37.p3 ) ( international human genome sequencing consortium 2004 ) , pan troglodytes ( chimp2.1 ) ( chimpanzee sequencing and analysis consortium 2005 ) , gorilla gorilla ( gorgor3 ) ( scally et al .
2012 ) , pongo pygmaeus ( ppyg2 ) ( locke et al . 2011 ) , macaca mulatta ( mmul_1.0 ) ( rhesus macaque genome sequencing analysis consortium et al . 2007 ) , callithrix jacchus ( caljac3 ) ( genbank assembly i d gca_000004665.1 ) , mus musculus ( ncbim37 ) ( gregory et al . 2002 ) , rattus norvegicus ( rgsc3.4 ) ( gibbs et al . 2004 ) , oryctolagus cuniculus ( orycun2.0 ) ( lindblad - toh et al .
2005 ) , bos taurus ( btau_4.0 ) ( bovine genome sequencing analysis consortium et al . 2009 ) , sus scrofa ( sscrofa9 ) ( genbank assembly i d gca_000003025.4 ) , equus caballus ( equcab2 ) ( lindblad - toh et al .
2011 ) , monodelphis domestica ( mondom5 ) ( mikkelsen et al . 2007 ) , gallus gallus ( washuc2 ) ( hillier et al . 2004 ) , and taeniopygia guttata ( taegut3.2.4 ) ( warren et al . 2010 ) .
we focused on mammalian datasets because they contain the greatest level of annotation , including chromosomal location . also , we restricted our analysis to this single database so that we could make comparisons between genomes while minimizing data collection artifacts .
for each of the 16 genomes , we collected two gene sets : 1 ) complete list of all genes with annotated function ; 2 ) including only those nuclear genes with mitochondrion annotation ( n - mt genes ) as specified by the gene ontology i d 0005739 . from the database , we obtained for each gene : chromosomal location , ensemble gene name , and gene i d .
we processed this output with scripts written in matlab ( the mathworks , natick , ma ) to calculate the counts of genes per chromosome in each genome for the two different datasets .
we compared the density of n - mt genes on the x or z chromosomes with that of the autosomes relative to the expectation based on overall gene density for 14 vertebrate genomes with male heterogamety and two vertebrate genomes with female heterogamety ( the birds , g. gallus and t. guttata ) .
we performed all of our statistical analysis in matlab . to determine if the set of n - mt genes was under- or overrepresented on each chromosome , we compared it with an expected count .
for a particular genome , we used the following method to determine the expected number of n - mt genes per chromosome .
we calculated the product of the total number of n - mt genes and the fraction of all annotated genes found on each chromosome .
this expected gene count per chromosome assumes that the n - mt genes are distributed across the chromosomes in the same pattern as the total set of genes in the genome .
the ratio of observed count to expected count will equal one when there is no under- or overrepresentation of n - mt genes on a particular chromosome .
when the value is less ( greater ) than one , then there is an under representation ( over representation ) of n - mt genes on a chromosome .
we computed the comparisons as described above to limit any potential biases in how the data were collected . in order to create a bias in the density of this particular group of genes
, some chromosomes would need to be over- or underannotated for n - mt genes in particular .
we are unaware of any feature of the n - mt genes which make them more or less likely to be sequenced and subsequently recognized as genes relative to any other subset of genes .
our method is also robust to differences in annotation level between chromosomes within a genome .
if for some reason there was a bias in the annotation level of autosomes compared with the sex chromosome , we would expect more genes to be annotated with the n - mt term as well .
our density of n - mt genes ( rather than an absolute count ) would not be affected .
we tested the significance of the underrepresentation on the sex chromosome by calculating a confidence interval of the mean over-/underrepresentation for the autosomes based on 10,000 replicate bootstrap samples and then determining if the sex chromosome representation fell below this confidence interval at a idk corrected alpha of 0.032 , based on an individual alpha of 0.05 and 16 different comparisons ( idk 1967 ) .
using the online genome database biomart ( http://www.biomart.org ) via their martview interface ( smedley et al .
2009 ) , we collected genomic information on the following organisms using the available ensembl genes 63 datasets : homo sapiens ( grch37.p3 ) ( international human genome sequencing consortium 2004 ) , pan troglodytes ( chimp2.1 ) ( chimpanzee sequencing and analysis consortium 2005 ) , gorilla gorilla ( gorgor3 ) ( scally et al .
2012 ) , pongo pygmaeus ( ppyg2 ) ( locke et al . 2011 ) , macaca mulatta ( mmul_1.0 ) ( rhesus macaque genome sequencing analysis consortium et al . 2007 ) , callithrix jacchus ( caljac3 ) ( genbank assembly i d gca_000004665.1 ) , mus musculus ( ncbim37 ) ( gregory et al . 2002 ) , rattus norvegicus ( rgsc3.4 ) ( gibbs et al . 2004 ) , oryctolagus cuniculus ( orycun2.0 ) ( lindblad - toh et al .
2005 ) , bos taurus ( btau_4.0 ) ( bovine genome sequencing analysis consortium et al . 2009 ) , sus scrofa ( sscrofa9 ) ( genbank assembly i d gca_000003025.4 ) , equus caballus ( equcab2 ) ( lindblad - toh et al .
2011 ) , monodelphis domestica ( mondom5 ) ( mikkelsen et al . 2007 ) , gallus gallus ( washuc2 ) ( hillier et al . 2004 ) , and taeniopygia guttata ( taegut3.2.4 ) ( warren et al . 2010 ) .
we focused on mammalian datasets because they contain the greatest level of annotation , including chromosomal location . also , we restricted our analysis to this single database so that we could make comparisons between genomes while minimizing data collection artifacts .
for each of the 16 genomes , we collected two gene sets : 1 ) complete list of all genes with annotated function ; 2 ) including only those nuclear genes with mitochondrion annotation ( n - mt genes ) as specified by the gene ontology i d 0005739 . from the database , we obtained for each gene : chromosomal location , ensemble gene name , and gene i d .
we processed this output with scripts written in matlab ( the mathworks , natick , ma ) to calculate the counts of genes per chromosome in each genome for the two different datasets .
we compared the density of n - mt genes on the x or z chromosomes with that of the autosomes relative to the expectation based on overall gene density for 14 vertebrate genomes with male heterogamety and two vertebrate genomes with female heterogamety ( the birds , g. gallus and t. guttata ) .
if the set of n - mt genes was under- or overrepresented on each chromosome , we compared it with an expected count . for a particular genome , we used the following method to determine the expected number of n - mt genes per chromosome .
we calculated the product of the total number of n - mt genes and the fraction of all annotated genes found on each chromosome .
this expected gene count per chromosome assumes that the n - mt genes are distributed across the chromosomes in the same pattern as the total set of genes in the genome .
the ratio of observed count to expected count will equal one when there is no under- or overrepresentation of n - mt genes on a particular chromosome .
when the value is less ( greater ) than one , then there is an under representation ( over representation ) of n - mt genes on a chromosome .
we computed the comparisons as described above to limit any potential biases in how the data were collected . in order to create a bias in the density of this particular group of genes
, some chromosomes would need to be over- or underannotated for n - mt genes in particular .
we are unaware of any feature of the n - mt genes which make them more or less likely to be sequenced and subsequently recognized as genes relative to any other subset of genes .
our method is also robust to differences in annotation level between chromosomes within a genome .
if for some reason there was a bias in the annotation level of autosomes compared with the sex chromosome , we would expect more genes to be annotated with the n - mt term as well .
our density of n - mt genes ( rather than an absolute count ) would not be affected .
we tested the significance of the underrepresentation on the sex chromosome by calculating a confidence interval of the mean over-/underrepresentation for the autosomes based on 10,000 replicate bootstrap samples and then determining if the sex chromosome representation fell below this confidence interval at a idk corrected alpha of 0.032 , based on an individual alpha of 0.05 and 16 different comparisons ( idk 1967 ) .
using the online genome database biomart ( http://www.biomart.org ) via their martview interface ( smedley et al .
2009 ) , we examined the distribution of genes that have a mitochondrion cellular component annotation ( gene ontology i d 0005739 ) across chromosomes in 16 vertebrates .
we found a strong and significant underrepresentation of nuclear genes interacting with the mitochondrion on the sex chromosomes in all of the mammals ( fig .
, although the sex chromosome did not always have the lowest density of n - mt genes relative to expectation , it was always in the lower end of the distribution of chromosomes .
in both bird species , the sex chromosomes ( z ) had the expected density of n - mt genes based on overall gene density .
expected counts of genes are calculated based on the distribution of all annotated genes in the genome . the fraction of all genes present on a particular chromosome is then multiplied by the total number of n - mt genes in a particular genome to give the expected count .
ratios close to one show no over- or underrepresentation on a chromosome . each chromosomal value for each genome
is represented by a blue ( autosomes ) or red and pink ( sex chromosome [ x / z ] ) circle .
values in parenthesis after the genome name indicate the number of identified n - mt genes in the genome .
sex chromosomes that are significantly different from the autosomes are highlighted in red and nonsignificant values are pink ( h. sapiens , p < 0.0001 ; p. troglodytes , p < 0.0001 ; g. gorilla , p < 0.0001 ; p. pygmaeus , p < 0.0001 ; m. mulatta , p < 0.0001 ; c. jacchus , p < 0.0001 ; m. musculus , p < 0.0001 ; r. norvegicus , p < 0.0001 ; o. cuniculus , p < 0.0001 ; c. familiaris ,
p < 0.0001 ; b. taurus , p < 0.0001 ; s. scrofa , p = 0.0001 ; e. caballus , p < 0.0001 ; m. domestica , p < 0.0001 ; g. gallus , p = 0.7621 ; and t. guttata , p = 0.5281 ) .
expected counts of genes are calculated based on the distribution of all annotated genes in the genome . the fraction of all genes present on a particular chromosome is then multiplied by the total number of n - mt genes in a particular genome to give the expected count .
ratios close to one show no over- or underrepresentation on a chromosome . each chromosomal value for each genome
is represented by a blue ( autosomes ) or red and pink ( sex chromosome [ x / z ] ) circle .
values in parenthesis after the genome name indicate the number of identified n - mt genes in the genome .
sex chromosomes that are significantly different from the autosomes are highlighted in red and nonsignificant values are pink ( h. sapiens , p < 0.0001 ; p. troglodytes , p < 0.0001 ; g. gorilla , p < 0.0001 ; p. pygmaeus , p < 0.0001 ; m. mulatta , p < 0.0001 ; c. jacchus , p < 0.0001 ; m. musculus , p < 0.0001 ; r. norvegicus , p < 0.0001 ; o. cuniculus , p <
0.0001 ; c. familiaris , p < 0.0001 ; b. taurus , p < 0.0001 ; s. scrofa , p = 0.0001 ; e. caballus , p < 0.0001 ; m. domestica , p < 0.0001 ; g. gallus , p = 0.7621 ; and t. guttata , p = 0.5281 ) .
we have discovered a striking pattern in the distribution of genes among chromosomes : nuclear genes which interact with the mitochondrion are significantly underrepresented on the x - chromosome in mammals , but not in birds ( fig .
1 ) . these data are inconsistent with the predictions of the ca nuclear mitochondrial gene hypothesis which proposes that due to the increased heritability of gene combinations , the n - mt genes should be overrepresented on the x - chromosome .
we discuss other hypotheses that may help to explain the observed pattern of gene distributions .
we propose that the observed pattern may be explained by sexual conflict ( sc ) .
sc occurs when genes beneficial to the fitness of one sex are deleterious to the fitness of the other .
the evolution of maternally transmitted organelles can lead to sc when mitochondrial mutations that interact epistatically with nuclear genes have favorable effects in females but are deleterious in males ( rice 1984 ; rice et al . 2006 ; vicoso and charlesworth 2006 ) . with epistatic selection , the effects of nuclear genes interacting with mitochondria manifest only on certain mitochondrial backgrounds and not on others ( fig .
importantly , changing the mitochondrial background alters the effects of the interacting nuclear genes without changing the nuclear gene sequence .
epistatic fitness interactions between nuclear genes and the mitochondrion can result in sexually antagonistic effects for nuclear genes .
a schematic of the hypothesized process generating selection for the movement of n - mt genes off the x chromosome .
a mitochondrial mutation occurs ( purple circle ) with epistatic sexually antagonistic fitness effects ( red arrow ) on the current nuclear genetic background .
( b ) adaptive spread of the mitochondrial mutation leads to the eventual fixation of the female beneficial mutant mitochondria despite male deleterious effects .
a schematic of the hypothesized process generating selection for the movement of n - mt genes off the x chromosome .
a mitochondrial mutation occurs ( purple circle ) with epistatic sexually antagonistic fitness effects ( red arrow ) on the current nuclear genetic background .
( b ) adaptive spread of the mitochondrial mutation leads to the eventual fixation of the female beneficial mutant mitochondria despite male deleterious effects . with nuclear
mitochondrial epistasis , the force of genomic conflict ( between the nucleus and the mitochondrion ) is aligned with that of intralocus sc ( between males and females ) . because mitochondria have nearly exclusive maternal inheritance , selection can favor those mitochondrial genotypes that enhance female fitness , even if at the expense of male fitness ( partridge and hurst 1998 ; rand et al .
that is , mitochondrial mutations with epistatic effects favorable to females will spread owing to maternal inheritance ( fig .
because the mitochondrial mutations are not selected on the basis of their effect in males , even mutations which carry unfavorable effects in males will spread as long as those mutations are favorable in females . as a result
, a nuclear gene interacting with the mitochondria will become more deleterious to males over time .
there is direct evidence of sexually antagonistic epistatic interactions between the mitochondrial and nuclear genes from studies of the involvement of mitochondria in sperm development ( wang 2004 ; rajender et al .
furthermore , introgression of foreign mitochondria into drosophila populations has revealed sex - biased epistatic effects for x - linked nuclear genes ( rand et al .
although sc based on segregating nuclear genes depends upon the nature of allelic dominance ( rice 1984 ) , selection on the mitochondrial mutations is independent of the dominance relationships among alleles of its nuclear partners , requiring only positive fitness effects on females in some nuclear backgrounds .
furthermore , the sc process we are proposing does not depend on the simultaneous polymorphism of both nuclear and mitochondrial genes or the cotransmission of nuclear mitochondrial gene combinations , which was a key to the alternative ca hypothesis .
sc due to the spread of mitochondrial mutations with epistatic effects with nuclear loci can occur whether the nuclear partner of the mitochondrion is autosomal or x - linked . as a mitochondrial mutation causing the sc spreads within the population , there is increasing selective pressure to resolve the sc .
several mechanisms for the nuclear resolution of the sc have the potential to alter the genomic distribution of n - mt genes .
an evolutionary means for resolving such sc ( among many ) is via gene movement off of the x and onto the autosomes .
this is a process involving several steps , including gene duplication , fixation and subsequent gene loss , and requiring significant chromosomal bias at one or all of these steps to account for the observed pattern ( wu and yujun xu 2003 ) .
an alternative process is the creation of duplicate copies on the autosomes with sex - specific or sex - biased expression ( connallon and clark 2011 ; gallach and betrn 2011 ) .
both proposed mechanisms involve the fixation of nuclear gene duplications which ameliorate the male detrimental effects caused by the mitochondrial mutation .
theory and empirical evidence suggest that these duplicate copies accumulate on the autosomes ( connallon and clark 2011 ; gallach and betrn 2011 ) .
the bias in the location of the duplicate copies creates the overrepresentation on the autosomes relative to the x chromosome .
female beneficial ( male deleterious ) epistatic effects with mitochondrial mutations are not favored equally at the x and autosomal locations because x - linked loci spend two - thirds of their time in females and only one - third in males ( rice 1984 ) .
autosomal loci , on the other hand , spend half their time in males and such genes with male beneficial ( female deleterious ) epistatic effects would be expected to fix more easily on autosomes compared with the x chromosome .
this autosomal - bias in selection efficiency may be particularly important for male traits involved in reproductive competition , where selection on male fertility can be several times stronger than opposing selection on females ( wade 1979 ; shuster and wade , 2003 ; miller et al .
, the difference in selection efficiency would lead to a greater cumulative deterioration of x - linked relative to autosomal nuclear genes as a result of mitochondrial mutation pressure ( fig .
2 ) . empirical support for this mechanism comes from drosophila where gallach et al .
( 2010 ) find that n - mt gene duplications accumulate additional copies on autosomes more readily than the x chromosome .
interestingly , these gene duplicates also show testis - specific expression that the authors suggest is evidence of sc resolution ( gallach et al . 2010 ) .
other hypotheses regarding the resolution of sc suggest that changes in the sex - specific expression of sexually antagonistic genes , possibly via cis - acting expression modifiers , resolve intralocus sc ( rice 1984 ; rice and chippindale 2001 ; gibson et al .
our data are based on the genomic distribution of n - mt genes and not on sex differences in gene expression .
although our data in mammals are consistent with resolution of sc via gene movement or duplication , they do not exclude the possibility that sc antagonism may be resolved by alternative means .
our data suggest that mammalian n - mt genes may be useful for elucidating the mechanistic basis by which sc is resolved ( vibranovski et al . 2009 ; gallach et al .
we hypothesize that the pattern we observe in mammals differs from that observed in birds , because of one or a combination of differences affecting the proposed process based on mitochondrial mutation pressure and gene movement .
second , there is a near absence of nuclear gene retro - transposition in birds relative to mammals ( haas et al .
third , if the fitness effect is caused by epistatic interactions with the mitochondrion , then strict maternal inheritance of the mitochondria results in reduced cotransmission of the mitochondria and z chromosomes compared autosomes .
additionally , all of the cotransmission occurs from mother to sons at which point that mitochondrial - z chromosome combination has a zero probability of being passed along to the next generation .
the combination these factors suggests that our proposed process will be significantly weaker in birds , relative to mammals and that intralocus sc in birds may be resolved differently than it is in mammals .
we appreciate that our sample of genomes does not meet the criterion of phylogenetic independence .
one could argue that we have a single contrast between two types of male heterogametic vertebrates ( mammals and marsupials ) and one type of female heterogametic vertebrate ( birds ) .
although other vertebrate genomes are available , none meet the criteria of having sex chromosomes ( e.g. , danio rerio and fugu rubripes ) and the insect gene annotations in this database are not coincident with their annotations in other databases ( e.g. , drosophilids go annotation in biomart relative to flybase ) .
it is well recognized that genomes are highly fluid , with frequent translocations among chromosomes , even though a recognizable amount of synteny is present ( hillier et al .
thus , despite its taxonomic limitations , our data suggest that the signal of a lack of n - mt genes on the x chromosomes of mammals is present and has been maintained across a diversity of species for tens of millions of years .
our results significantly extend the single - gene theory for the evolution of sexually antagonistic genes to involve epistatic interactions between nuclear and mitochondrial genes .
it also synergistically combines sc theory with results from genomic conflict theory , wherein , mitochondrial genes with deleterious effects on males spread if they are favorable to females owing to uniparental , maternal transmission .
the high rate of such mitochondrial mutations and their subsequent adaptive spread leads to the continual accumulation of male deleterious fitness effects on x - linked genes ( rand et al .
our theory does not preclude patterns of sex - limited gene expression for sex - linked genes with direct ( instead of epistatic ) fitness effects .
our empirical finding adds to our knowledge of the forces affecting gene clustering and gene expression on sex chromosomes .
our results are consistent with a synergistic interaction between the forces of sc and genomic conflict affecting the distribution of n - mt genes on the sex chromosomes relative to the autosomes in vertebrates . | mitochondria are essential organelles whose replication , development , and physiology are dependent upon coordinated gene interactions with both the mitochondrial and the nuclear genomes .
the evolution of coadapted ( ca ) nuclear mitochondrial gene combinations would be facilitated if such nuclear genes were located on the x - chromosome instead of on the autosomes because of the increased probability of cotransmission . here , we test the prediction of the ca hypothesis by investigating the chromosomal distribution of nuclear genes that interact with mitochondria . using the online genome database biomart , we compared the density of genes that have a mitochondrion cellular component annotation across chromosomes in 16 vertebrates .
we find a strong and highly significant genomic pattern against the ca hypothesis : nuclear genes interacting with the mitochondrion are significantly underrepresented on the x - chromosome in mammals but not in birds .
we interpret our findings in terms of sexual conflict as a mechanism that may generate the observed pattern .
our finding extends single - gene theory for the evolution of sexually antagonistic genes to nuclear mitochondrial gene combinations . |
the only naturally
fluorescent amino acids are the aromatics , i.e. ,
tryptophan , tyrosine , and phenylalanine , but in general tryptophan
fluorescence dominates tyrosine fluorescence , and both dominate phenylalanine
fluorescence .
the endogenous fluorescence from
tryptophan has been used to study properties of proteins since the
original observation by weber .
tryptophan usually dominates
protein emission , not only because it has a higher extinction coefficient
than tyrosine ( 5579 m cm at
279 nm for tryptophan versus 1405 m cm at 275 nm for tyrosine ) but also because tyrosine can be so readily
quenched in the protein matrix .
fluorescence methodologies have been
used for several decades to study protein oligomerization and aggregations . a comparatively modern method to determine protein oligomerization
and association dissociation equilibrium at low concentration
levels originates from the analysis of fluctuations in fluorescence
intensity .
techniques based on fluctuations in fluorescence intensity ,
namely single point fluorescence correlation spectroscopy ( fcs ) , raster
image correlation spectroscopy ( rics ) , photon counting histogram ( pch ) ,
and number and brightness ( n&b ) , have been used to study protein
interactions both in vitro and in vivo .
one of the main drawbacks to these methods , however , is that they
almost always require addition of an external fluorophore , either
an organic or inorganic molecule or a fluorescent protein .
tryptophans
are endogenously present in almost all proteins , and hence it would
be useful if the fluorescence from tryptophan residues could be used
for the fluorescence fluctuation based techniques
. the main
difficulties of using tryptophan as a fluorescent probe
in the fluctuation based techniques are severalfold .
these difficulties
include the need for ultraviolet excitation , the optical transmission
properties of the components of the commercial laser scanning microscope ,
and the relative low brightness of the tryptophan fluorescence .
the
laser sources directly capable of excitation at 280 nm are very limited ,
and fcs requires a highly focused excitation source , like that provided
by a laser .
a second option involves using an optical parametric oscillator
( opo ) to achieve two - photon excitation by doubling an original wavelength
of 560 nm .
instead ,
we chose to use three - photon excitation by utilizing the 840 nm emission
of a commercially available titanium
the three - photon excitation has a very low probability
of absorption and requires higher power than the two - photon excitation .
however , this power is still easily achievable with commercial ti sapphire
lasers .
as mentioned , the transmission
properties of the optics of commercial
laser scanning microscopes present a problem for work with ultraviolet
light wavelengths .
the scanning mirrors and the lenses have good transmissions
above 400 nm , but the transmitted signal decreases dramatically in
the uv .
the objectives usually used for the excitation and collection
of fluorescence in the epi configuration of a microscope do not transmit
below 370 nm , except for quartz objectives . combinations of these
optical elements result in low efficiency of photon detection in the
uv range of tryptophan emission . thus , for excitation using uv lasers ,
both excitation and emission efficiencies are adversely affected .
however , for multiphoton excitation , the excitation is not in the
uv and only the emission suffers . to circumvent this difficulty
, we
used the home - built diver ( deep imaging via enhanced - photon recovery )
microscope , originally designed for deep tissue imaging , to measure intensity fluctuations resulting from tryptophan fluorescence .
the excitation part of the diver microscope has the regular upright
configuration of a commercial microscope .
the three - photon excitation
of tryptophan requires an excitation wavelength near 840 nm , and this
wavelength transmission is not affected by the objective .
the high
uv sensitivity of diver comes from the simplicity and lack of optics
in the detection part of the microscope .
the sample is placed directly
above the filter wheel assembly and the filter wheel assembly is housed
on top of the wide area pmt detector .
thus , the fluorescence in the
forward direction goes through the filter assembly and is collected
by the pmt ( figure 1a ) . the combination of
a bg39 filter ( placed to protect the pmt from direct light at 840
nm ) and an ug11 filter results in fluorescence detection from 300
to 400 nm ( magenta shaded area in figure 1b )
which is suitable for tryptophan fluorescence .
the simplicity of the
emission path of the diver results in higher sensitivity of the uv
emission and makes it especially suitable for uv detection , for both
harmonic generation and fluorescence .
( a ) the upright excitation scheme
and positioning of sample directly
on top of wide area pmt .
( b ) overlap of the transmission spectra of the filters
used , ug11 ( red line ) and bg39 ( black line ) , and the detection efficiency
of the pmt ( blue line )
. the overlapped wavelength area ( purple ) is
used for detection of tryptophan fluorescence .
the experiments to obtain three - photon
excitation fcs had three
main objectives .
the first one involved proving that three - photon
excitation can be achieved using a commercial ti
sapphire laser
in the diver . to obtain evidence of the three - photon effect , log
the slope
of the linear fit of this plot is indicative of the multiphoton effect
and a slope of one , two , or three indicates the one - photon , two - photon ,
or three - photon effect , respectively .
fluorescence
intensity traces of four different samples : n - acetyl - l - tryptophanamide ( nata ) and three tryptophan - containing protein
systems , -chymotrypsinogen a , rat liver phosphofructokinase ,
and phage f1 virus , were measured as a function of increasing laser
power and the log log plots of observed count rate versus excitation
power were constructed .
figure 2 represents
these plots , and in each of these cases the value of the slope of
the linear fit has a value close to three , determining the three - photon
excitation .
we note that the concentrations used for these experiments
were much higher than those used for the subsequent fcs experiments ,
since the goal was simply to observe the effect of excitation power
on the count rate .
excitation power dependence of ( a ) nata , ( b ) -chymotrypsinogen
a , ( c ) phosphofructokinase , and ( d ) phage f1 virus .
the slopes of
the linear fits were around 3 , demonstrating three - photon excitation
of these molecules .
once it was determined
that the fluorescence from the three - photon
excitation effect can indeed be achieved in the diver microscope the
next step was to determine the point spread function ( psf ) for the
three - photon excitation .
unlabeled polystyrene beads ( d = 100 nm ) were embedded in a gelatin matrix and excited with 1038
nm ( /3 = 1038/3 = 346 nm ) .
these beads absorb at 840 nm ( wavelength
of tryptophan excitation ) , but the gelatin matrix melts due to a local
heating effect and local motions start complicating the measurements .
to obtain the psf , z - stack images of these beads
were measured with a pixel size of 0.012 m and z steps of 0.20 m . prior to imaging
enrico gratton at the laboratory for
fluorescence dynamics at the university of california , irvine .
simfcs
was also used for the data acquisition by the diver and for further
analysis described in this study .
the size of the bead was smaller
than the psf , and thus deconvolution of the intensity image with the
size of the bead resulted in the calculation of the size of the psf ,
0.2 m in the xy and 0.5 m at the z axis .
3d reconstruction of the z stack image of polystyrene
beads embedded in gelatin matrix when excited with 1038 nm laser line .
the sizes of the images of beads were measured using the measuring
cuboid ( shown in red in the zoomed in image ) .
the next step was to obtain three - photon excitation fcs and
study
the oligomerization equilibrium of the rat liver phosphofructokinase
( pfk ) .
rat liver phosphofructokinase is believed to exist as a tetramer
in the presence of mgatp and in the absence of fructose-6-phosphate
( f6p ) . in the presence of f6p , the tetramers associate and create
larger oligomers , believed to be an octamer of tetramers .
there are ten tryptophans present in each monomeric unit of the
phosphofructokinase , and thus the tetramer and the octamer of tetramers
contain 40 and 320 tryptophan residues , respectively .
the large number of tryptophans increases the particle
brightness , defined as counts per protein ( molecule ) per second , and
makes the fluorescence easier to detect at the low concentration of
protein required to have sufficient fluctuations in the excitation / emission
volume .
the three - photon excitation fcs decays
were measured for the 20 and 7 m subunit ( i.e. , monomer ) concentration
of pfk in 20 mm mops buffer in the presence of 5 mm f6p and after
substitution of f6p with 5 mm atp . 5 mm mgcl2 was present
in all samples .
the correlation is much more prominent
than the one in the presence of atp , and the ratio of the two g(0 ) values are 6.2 .
this observation is due to
the fact that in the presence of f6p the major contributing species
to the correlation function is the octamer of tetramers . in a solution
containing just the octamer of tetramers ,
the concentration of fluorescent
species is one - eighth compared to the same solution containing only
tetramers .
the amplitude of the correlation function g(0 ) is inversely proportional to the concentration , and thus the
octamer of tetramers in the presence of f6p has a higher correlation
amplitude .
when f6p is substituted with atp , the large oligomers dissociate
and the major contributing species becomes the tetramers .
thus , the
concentration increases and the amplitude of the autocorrelation decreases .
the ratio of g(0 ) values indicates this change in
concentration .
( a ) single point fcs decays of the phosphofructokinase
( pfk ) in
the presence of fructose 6 phosphate ( f6p ) ( black ) and in the presence
of atp ( blue ) .
( b ) overlap of normalized autocorrelation functions
of pfk ( red ) , 100 nm polystyrene beads ( black ) . to compare the time scales of autocorrelation decays of biological
systems with different sizes , three - photon excitation fcs decays
the normalized autocorrelation decays of these two samples are plotted
in figure 4b , and the red and black colors
represent the decays for pfk and beads , respectively .
these correlation
decays were fitted with the following diffusion equation to obtain
characteristic diffusion time of these two samples:1where d is the characteristic
diffusion time of a biomolecule and s is the shape
factor and has a value of 2.5 .
the octamer of tetramers are the main
contributors toward the fcs decays of pfk in the presence of f6p and
have a characteristic diffusion time of 1.45 0.1 ms .
this value
is similar to that obtained for the correlation decays of the 100
nm polystyrene beads ( 1.65 0.1 ms ) .
these results demonstrate
that three - photon excitation fcs can indeed be achieved and used to
study oligomerization equilibrium using the commercially available
ti sapphire laser and the high sensitivity and forward geometry
of the diver microscope . in this study
we have shown and expanded
the idea of using three - photon
excitation fcs to look at the oligomerization equilibria of proteins
using their intrinsic tryptophan fluorescence .
we have demonstrated
that the combination of the uv sensitivity of the diver microscope
and three - photon excitation achieved by the ti
multiphoton excitation was
used before to excite tryptophan , for fcs measurements and for imaging .
the detection limits for the two - photon excitation fcs varied from
a few million ( highly unlikely in biological systems ) to a few hundred
tryptophans in each unit of the fluorescent species . in the most recent
paper involving measurement of fcs curves in proteins using tryptophan
fluorescence by sahoo et al .
, the authors
looked at aggregated proteins containing greater than 300 000
tryptophan residues . in our system , to obtain a clean correlation
decay in 5 min acquisition time , the number of tryptophan
residues in a protein complex present at submicromolar oligomer concentration
is required to be 200 .
this observation shows the extraordinary
sensitivity of diver combined with three - photon excitation can lead
to a single - point fcs measurement of proteins in buffer .
this study used extrinsic label of dna base
analogue 2-aminopurine , and the corresponding correlation decay was
very noisy and was not used by the authors to calculate diffusion .
currently we are trying to improve the sensitivity of the system and
to use this system to look at virus particles in more detail .
the
final goal is to follow the infection process of virus particles without
labeling and to monitor the real time state of infection .
the very
large number of tryptophans in viruses can be useful for the strong
signal - to - noise ratio and identification of virus capsid inside the
cells .
the three - photon excitation fcs can also be extended to study
other protein oligomerization processes , provided there are enough
tryptophan to give rise to significant signal - to - noise ratio . | three - photon excitation fluorescence
correlation spectroscopy was
used to detect oligomerization equilibria of rat liver phosphofructokinase .
the fluorescence intensity produced by the three - photon excitation
of tryptophan
was collected using the diver microscope . in this home - built
upright microscope , a large area photomultiplier , placed directly
below the sample ,
is used as the detector . the lack of optical elements
in the microscope detection path results in a significantly improved
detection efficiency in the uv region down to about 300 nm , which
encompasses the fluorescence emission from tryptophan .
the three - photon
excitation autocorrelation decays obtained for phosphofructokinase
in the presence of f6p showed the presence of large oligomers .
substitution
of f6p with atp in the buffer medium results in dissociation of the
large oligomers , which is reported by the decreased autocorrelation
amplitude .
the three - photon excitation process was verified from the
slope of the log log plot of intensity against laser power . |
the differential diagnoses of a groin mass include inguinal hernia , round ligament varicosities ( rlv ) , mesothelial cysts , lymphadenopathy , endometriosis , subcutaneous lipoma , cyst of nuck ( persistent embryonic remnants of the process vaginalis with cyst formation ) , vascular aneurysms , soft tissue malignancies , abscess , and cystic lymphangiomas [ 1 - 5 ] .
the rlv can easily be mistaken for an inguinal hernia ( especially a strangulated or incarcerated groin hernia that needs an emergency operation ) , because they share the same clinical appearance .
we report a patient with rlv during pregnancy diagnosed by doppler sonography and managed with conservative therapy ( repeated sonography and obstetric practice ) .
a 29-year - old woman presented at 36 weeks gestation with a palpable mass and pain in the right groin during her second pregnancy .
the opinion of the obstetrician was inguinal hernia , and he transferred her to a general surgeon .
the general surgeon diagnosed a right inguinal hernia , and he transferred her to our hospital . on physical examination ,
the mass ( 2 2 cm ) was palpable softly , but tender , and was reducible completely by supine position change ( fig .
gray - scale sonography showed a mass in the right groin composed of multiple , echo - free , tubular channels .
the color doppler sonography confirmed hypervascular and abundant venous flow consistent of a mass ( fig .
2 ) . there was no sonographic evidence of a herniated bowel , or thrombus , or adenopathy . based on sonographic results , we diagnosed round ligament varicosities , and performed repeated sonography and obstetric practice .
the patient had an uncomplicated vaginal delivery at 40 weeks , and by two weeks postpartum , the mass subsided .
the most common diagnosis of a female groin swelling during pregnancy is an inguinal hernia .
however , inguinal hernias during pregnancy are relatively rare , with a reported incidence of 1 in 1,000 to 3,000 pregnant women .
a thorough literature search revealed only 16 cases internationally of rlv during pregnancy reported in the last 53 years .
the rlv was first reported in 1941 by verovitz and first reported in korea in 2010 by jung et al . .
the rlv are prominent veins within the round ligament and are more common in pregnancy because pregnancy promotes increased venous return and reduced venous tone .
the varicose vein formation of the round ligament during pregnancy have several mechanisms , including progesterone - mediated venous smooth muscle relaxation , a raised cardiac output causing increased venous return from the limbs and leading to engorgement of venous tributaries , and most importantly , pelvic venous obstruction by the gravid uterus [ 1 - 3,5 ] .
the clinical manifestation of rlv usually includes swelling and tenderness in the groin region , which can be provoked by increased intra - abdominal pressure in cases of coughing or valsalva maneuver .
a clue that might suggest rlv is the coexistence of vulvar or lower limb varicosities .
our case , however , absence of vulvar or lower limb varicosities did not exclude rlv .
the characteristic sonographic findings include a prominent venous plexus with accompanying dilated draining veins and the typical " bag of worms " appearance of smaller veins .
color doppler imaging can confirm venous flow and augmentation of this flow with valsalva maneuver . according to mckenna et al .
, ultrasound criteria that may be used in the diagnosis of rlv includes : multiple dilated veins passing through the inguinal canal , absence of bowel or lymph nodes in the inguinal mass , and veins seen to drain into the inferior epigastric vein .
the differential diagnoses of rlv include inguinal hernia , lymphadenopathy , endometriosis , subcutaneous lipoma [ 1 - 5 ] .
the sonographic findings of inguinal hernia are a superficial , well - delineated echogenic mass that appears adjacent to , yet distinctly separate from , the uterine wall .
the sonographic findings of lymphadenopathy are hypoechoic round or oval masses of varying size in the region of the inguinal area .
the characteristic sonographic appearance of endometrioma is that of a well - defined unilocular or multilocular , predominately cystic mass containing diffuse homogeneous , low - level , and internal echoes .
the sonographic findings of lipoma show a homogeneous , and echogenic mass that is surrounded by a thin echogenic capsule .
after a correct diagnosis of rlv , a wait - and - see policy is justified .
after delivery , when pelvic venous obstruction by the gravid uterus is relieved , spontaneous resolution will occur in most patients [ 2 - 5,9 ] .
however , rlv requires close monitoring during pregnancy as rupture of varices and acute variceal thromboses have been reported .
rupture and thrombosis of the rlv may occur and give an intense painful swelling groin , in which case emergency surgical exploration is recommended .
this recommendation comes first of all to rule out a strangulated hernia , and secondly to reduce pain or discomfort caused by the inguinal mass .
when a pregnant patient with an inguinal mass visits our hospital , we have to know that rlv is a part of the differential diagnosis of an inguinal mass during pregnancy , and we must perform a diagnosis using duplex sonography . if the rlv is diagnosed by sonography with the absence of a herniated bowel , or thrombus , we manage it conservatively ( repeated sonography and obstetric practice ) . however , if rlv presenting with rupture of varices and variceal thromboses are observed , emergency surgical exploration is recommended . | round ligament varicosities during pregnancy are rare , and can easily be mistaken for an inguinal hernia . on physical examination ,
round ligament varicosities and groin hernia are difficult to distinguish .
the diagnosis of round ligament varicosities can be established on gray - scale and color doppler sonography .
we experienced a case of round ligament varicosities in which a 29-year - old woman presented symptoms at 36 weeks gestation .
the patient was diagnosed using doppler sonography , managed with conservative therapy , and had an uncomplicated vaginal delivery at 40 weeks .
the symptoms were resolved completely by two weeks postpartum .
we report a case of round ligament varicosities that was diagnosed at 36 weeks of gestation with a review of the literature . |
acute myocardial infarction ( ami ) results from total coronary artery occlusion , usually due to thrombus formation on complicated atherosclerotic plaque .
the patients with ami have higher mortality rates , especially during the first 30 days .
platelets play an important role in development , destabilization , and rupture of the atherosclerotic plaque , as well as in formation of platelet - fibrin plug at the complicated atherosclerotic plaque .
platelet count is associated with increased risk of ami and short- and long - term mortality after ami [ 58 ] .
lymphocytes play a dominant role in chronic inflammation of atherosclerosis , and lower lymphocyte count is associated with increased cardiovascular risk and mortality in ami .
platelet / lymphocyte ratio ( plr ) has been reported as a novel marker of long - term mortality in patients with non - st - elevated ami . in the present study , we tested the hypothesis that plr is associated with in - hospital cardiovascular mortality in patients with st - elevated ami ( stemi ) .
records of patients with ami who were admitted to the coronary care unit between january 2009 and november 2011 were evaluated retrospectively .
we consecutively evaluated 645 patients who were diagnosed with stemi and excluded 4 patients with intracranial hemorrhage because of the intra - venous thrombolytic treatment ( streptokinase , tissue plasminogen activator , or tenecteplase ) and 5 patients without eligible laboratory results .
a diagnosis of stemi was defined as > 30 minutes of continuous typical chest pain and st - segment elevation 2 mm in 2 contiguous electrocardiography leads within 12 hours of symptom onset or within up to 18 hours if there was evidence of continuing ischemia or hemodynamic instability . from medical records
, we obtained demographic information , cardiovascular history and risk factors for coronary artery disease ( cad ) , and treatment received during the in - hospital period .
patients who had been treated with antihypertensive drugs or those whose baseline blood pressure exceeded 140/90 mmhg were diagnosed with hypertension ( ht ) .
diabetes mellitus ( dm ) was defined as fasting blood sugar level above 126 mg / dl or the use of anti - diabetic medications .
the admission glomerular filtration rate ( gfr ) was estimated by the simplified mdrd ( modification of diet in renal disease ) equation .
renal failure was defined as a glomerular filtration rate ( gfr ) < 60 ml / min / m .
ventricular tachycardia or ventricular fibrillation were defined at least 24 hours after the beginning of the symptoms , advanced heart failure was defined as killip classification 2 , and cardiovascular death was defined as death due to ami , heart failure , or arrhythmia .
complete blood counts and biochemical values were evaluated retrospectively from blood samples obtained by antecubital vein puncture upon admission to the emergency department .
total and differential leukocyte counts were determined with the beckmancoulterlh 780device ( beckman coulter ireland inc .
the high plr group ( n=212 ) was defined as having values in the highest tertile ( plr > 144 ) , and the low plr group ( n=424 ) was defined as having values in the lower 2 tertiles ( plr 144 ) .
quantitative variables are expressed as the mean value sd or median ( interquartile range ) , and qualitative variables are expressed as percentages ( % ) .
a comparison of parametric values between high and low plr groups was performed using the t test or the mann - whitney u - test .
categorical variables were compared by the likelihood - ratio test or fisher s exact test . a backward stepwise multivariate logistic regression analysis , which included variables with p<0.1 ,
age 70 , female sex , renal failure ( gfr < 60 ml / min / m ) , time of chest pain , not receiving thrombolytic treatment , nlr>4.1 , and plr>144 were entered into the model .
all statistical studies were carried out with the spss program ( version 17.0 , spss , chicago , il , usa ) .
records of patients with ami who were admitted to the coronary care unit between january 2009 and november 2011 were evaluated retrospectively .
we consecutively evaluated 645 patients who were diagnosed with stemi and excluded 4 patients with intracranial hemorrhage because of the intra - venous thrombolytic treatment ( streptokinase , tissue plasminogen activator , or tenecteplase ) and 5 patients without eligible laboratory results .
a diagnosis of stemi was defined as > 30 minutes of continuous typical chest pain and st - segment elevation 2 mm in 2 contiguous electrocardiography leads within 12 hours of symptom onset or within up to 18 hours if there was evidence of continuing ischemia or hemodynamic instability . from medical records
, we obtained demographic information , cardiovascular history and risk factors for coronary artery disease ( cad ) , and treatment received during the in - hospital period .
patients who had been treated with antihypertensive drugs or those whose baseline blood pressure exceeded 140/90 mmhg were diagnosed with hypertension ( ht ) .
diabetes mellitus ( dm ) was defined as fasting blood sugar level above 126 mg / dl or the use of anti - diabetic medications .
the admission glomerular filtration rate ( gfr ) was estimated by the simplified mdrd ( modification of diet in renal disease ) equation .
renal failure was defined as a glomerular filtration rate ( gfr ) < 60 ml / min / m .
ventricular tachycardia or ventricular fibrillation were defined at least 24 hours after the beginning of the symptoms , advanced heart failure was defined as killip classification 2 , and cardiovascular death was defined as death due to ami , heart failure , or arrhythmia .
complete blood counts and biochemical values were evaluated retrospectively from blood samples obtained by antecubital vein puncture upon admission to the emergency department .
total and differential leukocyte counts were determined with the beckmancoulterlh 780device ( beckman coulter ireland inc .
the high plr group ( n=212 ) was defined as having values in the highest tertile ( plr > 144 ) , and the low plr group ( n=424 ) was defined as having values in the lower 2 tertiles ( plr 144 ) .
quantitative variables are expressed as the mean value sd or median ( interquartile range ) , and qualitative variables are expressed as percentages ( % ) .
a comparison of parametric values between high and low plr groups was performed using the t test or the mann - whitney u - test .
categorical variables were compared by the likelihood - ratio test or fisher s exact test . a backward stepwise multivariate logistic regression analysis , which included variables with p<0.1 , was performed to identify independent predictors of in - hospital cardiovascular mortality .
age 70 , female sex , renal failure ( gfr < 60 ml / min / m ) , time of chest pain , not receiving thrombolytic treatment , nlr>4.1 , and plr>144 were entered into the model .
all statistical studies were carried out with the spss program ( version 17.0 , spss , chicago , il , usa ) .
a total of 636 patients ( 505 men and 131 women ) were enrolled into the present study .
no differences were found between the groups regarding thrombolytic and other treatments received during the in - hospital period .
tobacco use , ht , dm , and chd history were not significantly different between the groups .
the rate of male patients ratio was lower in the high plr group ( 73% vs. 82.8% , p=0.004 ) .
the patients in the high plr group were older ( 63.712.1 vs. 61.412.0 , p=0.022 ) .
baseline platelet and neutrophil levels were significantly higher in the high plr group than in the low plr group ( 9.6 [ 3.225.1]10/mm
vs. 7.3 [ 2.218.4]10/mm , p<0.001 ; 245 [ 121779]10/mm
vs. 215 [ 43419]10/mm , p=0.001 ) , whereas the baseline lymphocyte level was significantly lower in the high plr group than in the low plr group ( 1 , 2 [ 0.43.7]10/mm
vs. 2.4 [ 0.69.1]10/mm , p=0.001 ) .
neutrophil - to - lymphocyte ratio ( nlr ) was higher in high plr group ( p<0.001 ) ( table 2 ) .
the high plr group had a significantly higher incidence of in - hospital cardiovascular mortality than the low plr group ( 12.7% vs. 5.9% , p=0.003 ) .
advanced heart failure ( killip class 2 ) was more frequent in patients with high plr values ( 18.9% vs. 12.3% , p=0.026 ) .
independent predictors of in - hospital cardiovascular mortality were determined by backward stepwise multivariate logistic regression . chest pain duration more than 6 hours , female sex ,
not receiving thrombolytic treatment , renal failure , age 70 years , nlr > 4.1 , and plr > 144 were found to be associated with increased in - hospital cardiovascular mortality in a logistic regression analysis ( table 4 ) .
plr > 144 was found to be an independent predictor of in - hospital cardiovascular mortality in multivariate analyses ( hazard ratio : 2.16 , 95% confidence interval : 1.164.0 , p=0.014 ) .
an plr value of 144 was determined as an effective cut - off point in stemi of in - hospital mortality , with a sensitivity of 51% and a specificity of 69% ( area under the curve=0.59 , 95% confidence interval 0.500.67 ) ( figure 1 ) .
in this study we show that high plr at admission to hospital is an independent predictor of early cardiovascular mortality in patients with ami .
older age , renal failure , and not treated with thrombolytic treatment are other independent predictors of mortality . to our knowledge
this is the first study to investigate the relationship between plr and mortality in stemi .
atherosclerotic cad is still the most common cause of mortality and morbidity in developed countries .
activated platelets precipitate to produce inflammatory substances from endothelial cells and leucocytes that cause monocyte adhesion and transmigration , and thereby increase the inflammatory process and progression of atherosclerotic plaque .
furthermore , these activated adhesion molecules and chemokines increase the activation of leucocytes , and produce reactive oxygen molecules and matrix metalloproteinase that cause plaque destabilization in atherosclerotic plaque .
ami usually occurs as a result of coronary artery occlusion due to complication of an atherosclerotic plaque .
platelets play a dominant role in pathogenesis of acute coronary syndromes by formation of platelet - fibrin complexes .
platelet count was found to be associated with development of ami and presence of cad .
gary et al . concluded that higher platelet volume may change blood viscosity and increase inflammation .
healy et al . reported that expression of cd-49 and plasma myeloid protein in platelets is increased in patients with st - elevated mi .
in addition , it was found that platelet count is associated with short- and long - term mortality in patients with st - elevated and non - st - elevated ami , and unstable angina pectoris [ 2022 ] .
showed a relationship between platelet count and mortality in patients with recurrent ami in the first year after primary percutaneous intervention .
in addition , fibrinogen levels and platelet counts are positively correlated and related to inflammation in ami patients .
these findings suggest that platelets are one of the most important components of cad and cardiovascular events .
lymphocytes are an important part of chronic inflammation in the atherosclerotic process . in ami , lymphocytes infiltrate to the ischemic and reperfused myocardium and express interleukin-10 , which may play a significant role in transmigration of mononuclear cells , and induce the expression of tissue inhibitor of metalloproteinase-1 .
recent studies showed that higher plr is related to presence of cad and is correlated with c - reactive protein and fibrinogen levels .
reported that plr above 170 is an independent predictor of long - term mortality in non - st - elevated ami patients . in our study , we found that high plr ( above 144 ) is an independent predictor of in - hospital mortality in patients with stemi who received thrombolytic treatment .
complications of ami , including heart failure , shock , and ventricular rupture , are more frequent in older ami patients . in the present study ,
neutrophils may cause plaque rupture as a result of stimulating the release of proteolytic enzymes , superoxide radicals , and arachidonic acid derivates , and exacerbates the inflammatory condition .
microvascular obstruction due to neutrophil - platelet plug , vasoconstriction due to thromboxanes , and vasoactive molecules may cause no - reflow and cause further myocardial injury .
these high inflammatory processes appear to be responsible for high rates of heart failure and mortality .
nlr was higher in the high plr group and was strongly related to cardiovascular mortality in our population , in line with results of previous studies . in multivariate analyses , we found that nlr was not an independent predictor and that plr is an independent predictor of mortality .
patient long - term survival and cardiac conditions could not be assessed because the patient records and the coronary angiography results of patients were not known .
anti - platelet treatment before ami is an important factor affecting in - hospital mortality , but our data were not able to demonstrate previous antiplatelet use .
ami is the most important cause of mortality ( especially in the first month ) and morbidity worldwide .
this study showed that high plr is an independent predictor of in - hospital cardiovascular mortality in patients with stemi .
complete blood count analysis is a routine and inexpensive method that may be useful for the identification of high - risk patients .
plr and other inflammatory markers and clinical findings might be helpful in identifying high - risk patients and treatment strategies . | backgroundplatelet - to - lymphocyte ratio ( plr ) is a new prognostic marker in coronary artery disease .
we aimed to evaluate the relationship between plr and in - hospital mortality in patients with st - elevated acute myocardial infarction ( ami).material / methodsthe present study included 636 patients with st - elevated ami .
the study population was divided into tertiles based on their admission plr .
patients having values in the third tertile was defined as the high plr group ( n=212 ) and those having values in the lower 2 tertiles were defined as the low plr group ( n=424).resultsrisk factors of coronary artery disease and treatments administered during the in - hospital period were similar between the groups .
male patient ratio was found to be lower in the high plr group ( 73% vs. 82.8% , p=0.004 ) . in - hospital
mortality was increased in the high plr group when compared to the low plr group ( 12.7% vs. 5.9% , p=0.004 ) .
the plr > 144 was found to be an independent predictor of in - hospital cardiovascular mortality ( hr : 2.16 , 95% ci : 1.164.0 , p=0.014).conclusionsthis study showed that plr is an independent predictor of cardiovascular mortality in patients with st - elevated ami . |
popliteal artery stenosis or occlusive disease is a common occurrence in elderly patients , diabetics , smokers and those with cardiovascular disease .
it leads to significant morbidity and mortality by reducing or completely blocking blood supply through the popliteal artery and into the lower leg .
we describe an unusual presentation of a 24-year - old male with a popliteal artery stenosis masquerading as an infective foot ulcer .
we believe this case illustrates the importance of a meticulous history and physical examination when assessing foot ulcers .
a 24-year - old male attended the department with a painful ulcer under the third metatarsal head of his left foot .
this ulcer had been present for 2 months prior to presentation following a skin laceration sustained while travelling in thailand .
the patient had received antibiotic therapy and surgical debridement on two occasions while abroad ; however , the ulcer had failed to heal . on
closer questioning the patient reported that he sustained the injury while walking close to coral reef in the sea in thailand .
there had been no history of freshwater exposure , drug misuse or of any risk - taking behaviour .
initially , the patient was treated with empirical antibiotics on instruction from our microbiology department .
antibiotic therapy was altered according to these sensitivities , and the erythema and swelling began to settle over the coming weeks .
unfortunately , the ulcer continued to enlarge and a mri scan of the left foot revealed increased uptake in the third toe and metatarsal head indicating underlying osteomyelitis .
, it proved necessary to amputate the third toe and metatarsal head as they could not be salvaged due to infective destruction . on conclusion of the procedure , the tourniquet which had been applied to the lower limb
was deflated and it was noted that the left foot was slow to reperfuse . a vascular opinion was sought .
ankle brachial pressure index ( abpi ) in the left leg was 0.46 when compared with 0.82 in the right leg , indicating a significant decrease in arterial blood flow to the left foot .
a ct femoral angiogram demonstrated a left popliteal short segment occlusion with reforming of the tibio - peroneal trunk with small vessels . on detailed probing , a history of intermittent claudication was revealed with a claudication distance of 100 m. subsequently , a left superficial femoral artery to popliteal artery bypass was performed by the vascular team using a reverse vein graft . within 6 weeks , all antibiotic therapy had been stopped and the wound from amputation on the left foot had completely healed .
stenosis of the popliteal artery is a common condition , most often seen in the context of peripheral vascular disease where an atherosclerotic pathogenesis is accountable .
however , in a cohort of younger patients , there is a much broader range of pathogenesis accounting for such stenosis .
conditions such as popliteal artery entrapment syndromes ( paes ) , popliteal artery aneurysm , cystic adventitial disease , multiple hereditary exostosis , some arteritis disorders ( e.g. takayasu 's ) and some rare congenital syndromes ( e.g. cutis marmorata telangiectatica congenital ) must be considered . in each of these conditions
, there is an underlying disease process that culminates in a reduction in the cross - sectional area in the popliteal artery and consequently , flow through the vessel is decreased in accordance with laplace 's law .
, there is an insidious process that can be unmasked by an acute insult ( e.g. trauma and embolism ) .
the presentation of congenital popliteal artery stenosis is very similar to that in peripheral vascular disease , with intermittent claudication , slow healing wounds and transitory change in skin colour and temperature . a persistent wound or ulcer in a young and
otherwise fit individual with no obvious predisposing factors is a red flag and should prompt further investigation .
a thorough history with targeted questioning of the above presenting symptoms is important , followed by rigorous physical examination .
initial bedside assessment of vascular status should be performed with abpis and doppler flow studies .
blood flow may be posturally dependent in approximately a third of paes cases ; however , this is unlikely to be the case with a fixed stenosis of the popliteal artery .
ct is a useful adjunct if external compression of the artery is a possibility ; furthermore , mri is useful in adventitial cystic disease .
management of vascular insufficiency in young patients depends on the underlying pathogenesis . in adventitial cystic disease
, symptoms may resolve spontaneously if cysts rupture ; however , recurrence is common . in congenital popliteal artery stenosis , vascular bypass
is often required as with the 24-year - old gentleman under consideration here . in conclusion
, this case emphasizes the importance of maintaining a broad differential diagnosis in young individuals presenting with persistent foot ulcers . in a young person with a persistent non healing ulcer , with unusual bacteriology and normal pulses , always consider a proximal vascular lesion . a prompt diagnosis of an unusual congenital vascular insufficiency can prevent complications and long - term morbidity for the patient .
| here , we present a case report of a young male who had been travelling in thailand .
the patient sustained a relatively minor trauma to the soft tissue under his left third metatarsal head on a piece of coral reef .
he subsequently developed an infected ulcer , which did not heal despite appropriate antibiotic therapy and surgical debridement .
unfortunately , the patient required amputation of the left third toe and metatarsal head due to osteomyelitic destruction .
it later transpired that the ulcer was initially ischaemic in nature due to previously undiagnosed and asymptomatic popliteal artery stenosis . |
laparoscopic posterior mesh rectopexy ( lpmr ) is a good surgical option for rectal prolapse with low morbidity and mortality .
specific complication like mesh erosion into the rectum following lpmr is very rare with only one such case , in which mesh got expelled via rectum , reported in literature till date .
we report a case of mesh erosion into the rectum after lpmr which was managed with laparoscopic mesh removal and review the published literature .
a 40-year - old man presented with occasional bleeding per rectum since 10 months with low grade intermittent fever for 1 week .
he had history of lpmr for complete rectal prolapse 2 years back , carried out elsewhere .
sigmoidoscopy revealed hyperemic mucosa with ulceration 15 cm from anal verge , with a part of the polypropylene mesh projecting into the rectum [ figure 1 ] .
computed tomography ( ct ) scan of abdomen showed asymmetric anterior wall thickening of rectosigmoid junction with fat stranding and minimal fluid collection .
review of his surgical notes revealed that a 15 cm 10 cm polypropylene mesh was used for rectopexy . since it was a large mesh fixed with multiple tacks and possible dense adhesions ,
transanal approach was not attempted . under general anesthesia , in lithotomy position , diagnostic laparoscopy was done .
appendix was inflamed with its tip densely adherent to the mesh [ figure 2 ] .
there was a small rectal perforation at the site of erosion , which was confirmed by air leak test .
sigmoidoscopic appearance of mesh erosion into rectum intra - operative images of ( a ) tip of appendix adherent to the mesh and ( b ) mesh eroding into rectum
laparoscopic rectopexy is presently the preferred management approach for rectal prolapse as it has better results especially in terms of less post - operative pain , shorter hospital stay , and similar recurrence rate as open rectopexy .
it may present with discharge of pus or blood per rectum , even several months or years after surgery .
it can be evaluated by doing a flexible sigmoidoscopy , which will usually reveal an ulcerated lesion at the site of erosion with visualization of mesh .
ct scan of abdomen with contrast helps in localizing the mesh in relation to rectum as well as pelvic collection , if any .
it is less common with type i mesh when compared to type ii , iii , and iv meshes due to bigger pore size with lesser chance of infection .
other factors that are found significant are poorly controlled diabetes mellitus , tobacco use , prior history of pelvic irradiation , and repeat procedures , which contribute to poor wound healing and subsequent infection , erosion or extrusion .
same causes can be considered as possible etiologies for mesh erosion after lpmr as well .
certain surgical technical errors like unrecognized rectal injury and deeper stitches through the rectum may also contribute to mesh erosion .
bigger sized mesh which can produce more folding after fixation , as in our case , may be a possible cause for erosion .
another possible cause in our case could be infection of mesh due to adhesion of inflamed appendix to it .
since , the incidence is very rare , no definite protocol regarding management is available in literature .
we preferred laparoscopic approach in our case , as it was too big mesh to remove through transanal approach .
moreover , only a part of mesh was protruding into the rectal lumen and it was fixed with multiple tacks to the sacrum .
severe inflammation and friable tissues precludes primary repair of the rectal defect where diverting ileostomy or colostomy plays an important role . in summary ,
the predisposing factors could be mesh infection , larger mesh , and unrecognized rectal injuries .
laparoscopic excision of mesh is a feasible and promising approach to manage such an unusual complication following lpmr . | laparoscopic posterior mesh rectopexy ( lpmr ) is now an accepted surgical treatment for complete rectal prolapse .
it is associated with complications such as partial mucosal prolapse , fecal impaction , constipation , and rarely recurrence .
erosion of the mesh into the rectum after lpmr is very rare .
we report herein the case of 40-year - old man who presented with mesh erosion into the rectum and managed successfully by the laparoscopic excision of mesh .
this is probably the first such case managed by the laparoscopic approach . |
its phenotype is predominantly characterized by sensorimotor polyneuropathy and/or infiltrative cardiomyopathy ( cm).1,2 moreover , ttr causes a nongenetic disease with deposition of wild - type ( wt ) ttr amyloid ( attr ) mainly in the heart of elderly males.2 in contrast to the hereditary form , no typical mutation in the ttr gene can be found in wtattr patients , and the pathogenic mechanisms are a subject of ongoing research.3 cardiac involvement is by far the most relevant predictor of outcome in both hereditary and wtattr amyloidosis .
median survival ranged between 4 and 6 years.4,5 cardiac wtattr deposition was found postmortem in 25% of patients above 85 years of age.4 a recent analysis of skeletal scintigraphy revealed a prevalence of wtattr - cm near 1.4% among males in the ninth decade of life.6 currently , causative treatment of wtattr - cm is lacking .
diuretics are capable of reducing dyspnea , but standard heart failure medication does not affect amyloid deposition itself.7 during the course of 1 year , wtattr - cm usually worsens , as indicated by an increase of the left ventricular ( lv ) wall thickness by 0.2 mm , an increase of nt - probnp by 1,487 pg / ml , and a decline of lv ejection fraction by 11%.8 recently , in vitro experiments have shown that epigallocatechin-3-gallate ( egcg ) , the most abundant catechin in green tea , inhibits fibril formation of diverse amyloidogenic proteins.911 disruption of attr fibrils was observed after a daily oral administration of 100 mg / kg egcg for 6 weeks12 using a transgenic mouse model of familial amyloidotic polyneuropathy carrying the human amyloidogenic val30met ttr variant . in a recent study , we demonstrated a decrease of lv mass in a small cohort of patients with hereditary attr - cm and wtattr - cm after a daily consumption of green tea for 12 months.13 in the present study , we report on our findings in a larger , more homogenous cohort of patients with exclusively wtattr - cm .
twenty - five male patients ( 71 [ 64 ; 80 ] years ) were recruited at the heidelberg amyloidosis center ( heidelberg , germany ) between 2008 and 2012 .
all patients underwent myocardial biopsy and diagnosis of wtattr was confirmed by immunohistochemistry and molecular genetic testing .
patients started green tea consumption on their own initiative due to the rather widespread knowledge of the effects of egcg on the disease among patients .
two patients died during the study period , and follow - up data were not available for two further patients .
data from seven patients had already been reported in our previous study.13 all patients had stable heart failure for at least 3 months prior to study inclusion .
echocardiography , cardiac magnetic resonance imaging ( cmri ) ( n=14 ) , and laboratory testing , including total cholesterol , troponin - t , and nt - probnp , were performed before and after 12 months of daily consumption of 1,200 mg green tea extract ( gte ) containing 600 mg egcg in four capsules of praevent - loges ( dr . loges & co gmbh [ winsen , germany ] ) .
the study was conducted according to the principles expressed in the declaration of helsinki and was approved by the institutional review board of the medical faculty of the university of heidelberg , germany ( vote number s-024/2008 ) .
transthoracic echocardiograms were performed using commercially available ultrasound diagnostic systems ( vivid 7 ; ge healthcare , milwaukee , wi , usa ) .
examinations were analyzed blindfolded offline on a commercially available workstation ( centricity cardiology ca1000 2.0 ; ge medical systems , milwaukee , wi , usa ) by two experienced sonographers ( ma and rb ) who were unaware of all other clinical data .
thickness of interventricular septum ( ivs ) and posterior wall were measured in late diastole ; lv cavity diameter was scaled in end diastole and end systole .
longitudinal myocardial function was quantified by mitral ( mapse ) and tricuspid ( tapse ) annular plane systolic excursion obtained from the apical four - chamber view using m - mode imaging .
all cmri exams were performed on a 1.5 t scanner ( achieva ; philips , best , the netherlands ) with a 32-element cardiac receiver coil and analyzed by two experienced investigators ( fs and hs ) , both being unaware of the remaining clinical data .
vector electrocardiogram - gated standard steady - state free precession cine sequences were acquired in short axes covering the whole lv and in three long - axis views ( two- , three- , and four - chamber views ) .
cardiac volumes , lv ejection fraction , and myocardial mass were measured in late diastole from short - axis image stacks by manual delineation of endocardial and epicardial borders excluding papillary muscles using a commercially available workstation ( viewforum , version 3.4 ; philips ) .
all analyses were performed using spss ( version 20.0 , ibm corporation , armonk , ny , usa ) .
differences between the nonparametric , continuous parameters assessed at study inclusion and at 12-month follow - up were compared by the wilcoxon matched - pairs signed - rank test .
twenty - five male patients ( 71 [ 64 ; 80 ] years ) were recruited at the heidelberg amyloidosis center ( heidelberg , germany ) between 2008 and 2012 .
all patients underwent myocardial biopsy and diagnosis of wtattr was confirmed by immunohistochemistry and molecular genetic testing .
patients started green tea consumption on their own initiative due to the rather widespread knowledge of the effects of egcg on the disease among patients .
two patients died during the study period , and follow - up data were not available for two further patients .
data from seven patients had already been reported in our previous study.13 all patients had stable heart failure for at least 3 months prior to study inclusion .
echocardiography , cardiac magnetic resonance imaging ( cmri ) ( n=14 ) , and laboratory testing , including total cholesterol , troponin - t , and nt - probnp , were performed before and after 12 months of daily consumption of 1,200 mg green tea extract ( gte ) containing 600 mg egcg in four capsules of praevent - loges ( dr . loges & co gmbh [ winsen , germany ] ) .
the study was conducted according to the principles expressed in the declaration of helsinki and was approved by the institutional review board of the medical faculty of the university of heidelberg , germany ( vote number s-024/2008 ) .
transthoracic echocardiograms were performed using commercially available ultrasound diagnostic systems ( vivid 7 ; ge healthcare , milwaukee , wi , usa ) .
examinations were analyzed blindfolded offline on a commercially available workstation ( centricity cardiology ca1000 2.0 ; ge medical systems , milwaukee , wi , usa ) by two experienced sonographers ( ma and rb ) who were unaware of all other clinical data .
thickness of interventricular septum ( ivs ) and posterior wall were measured in late diastole ; lv cavity diameter was scaled in end diastole and end systole .
longitudinal myocardial function was quantified by mitral ( mapse ) and tricuspid ( tapse ) annular plane systolic excursion obtained from the apical four - chamber view using m - mode imaging .
all cmri exams were performed on a 1.5 t scanner ( achieva ; philips , best , the netherlands ) with a 32-element cardiac receiver coil and analyzed by two experienced investigators ( fs and hs ) , both being unaware of the remaining clinical data .
vector electrocardiogram - gated standard steady - state free precession cine sequences were acquired in short axes covering the whole lv and in three long - axis views ( two- , three- , and four - chamber views ) .
cardiac volumes , lv ejection fraction , and myocardial mass were measured in late diastole from short - axis image stacks by manual delineation of endocardial and epicardial borders excluding papillary muscles using a commercially available workstation ( viewforum , version 3.4 ; philips ) .
all analyses were performed using spss ( version 20.0 , ibm corporation , armonk , ny , usa ) .
differences between the nonparametric , continuous parameters assessed at study inclusion and at 12-month follow - up were compared by the wilcoxon matched - pairs signed - rank test .
clinical demographics of the study cohort including the subgroup of cmri are given in table 1 .
none of the patients discontinued green tea consumption and no serious side effects were reported during the study period .
no changes in new york heart association ( nyha ) class could be observed , and no significant change in surface electrocardiogram parameters ( rhythm , heart rate , pq , qrs , qtc ) occurred .
one patient died due to sepsis , another due to spontaneous retroperitoneal hematoma requiring a prolonged stay in the intensive care unit with artificial ventilation and recurrent infections , resulting in multiorgan failure and , finally , death .
follow - up over periods of 44 , 25 , and 30 months , respectively , was achieved in three patients . in 18 ( 85.7% ) patients , a significant decrease of total serum cholesterol by 8.4% was observed ( 191 [ 118 ; 267 ] vs 173 [ 106 ; 287 ] mg / dl ; p=0.006 ) during the 12-month study period . in three patients , an increase of cholesterol by 3% was observed . in 16 ( 76.2% ) patients , low - density lipoprotein ( ldl )
cholesterol decreased by 8.0% ( 110 [ 60 ; 180 ] vs 99 [ 55 ; 188 ] mg / dl ; p=0.02 ] ) . in four patients , ldl cholesterol
glomerular filtration rate calculated by the modified diet in renal disease ( mdrd ) formula ( 62 [ 31 ; 99 ] vs 57 [ 26 ; 101 ] ml / min ; p=0.23 ) , got ( 30 [ 16 ; 56 ] vs 33 [ 17 ; 74 ] u / l ; p=0.90 ) , and gpt ( 29 [ 10 ; 57 ] vs 29 [ 10 ; 74 ] u / l ; p=0.95 ) remained unchanged .
ttr plasma levels remained stable during the study period ( 0.28 [ 0.14 ; 0.90 ] vs 0.26 [ 0.18 ; 0.50 ] mg / dl ; p=0.64 ; n=16 ) .
mean thickness of ivs ( 17 [ 13 ; 28 ] vs 18 [ 14 ; 25 ] mm ; p=0.1 ) and posterior wall ( 16 [ 11 ; 24 ] vs 17 [ 12 ; 19 ] mm ; p=0.4 ) remained unchanged during the study period ( figure 2 ) . in seven ( 33.3% )
patients , an increase of either 1 ) both ivs and posterior wall thickness ( n=4 ) or 2 ) isolated posterior wall thickness or ivs thickness ( n=3 ) occurred , by an average of 1.9 mm ( 13% ) .
mapse ( 10 [ 5 ; 23 ] vs 8 [ 4 ; 13 ] mm ; p=0.3 ) , tapse ( 14 [ 8 ; 23 ] vs 15 [ 2 ; 20 ] mm ; p=0.4 ) , and passive mitral inflow / lateral early diastolic mitral annular velocity ( e / e ) ratio ( 11 [ 7 ; 28 ] vs 14 [ 4 ; 31 ] ; p=0.8 ) remained unchanged .
results of echocardiography are shown in table 3 . in the subgroup of patients assessed by cmri ( n=14 ) ( figure 3 ;
table 4 ) , in 12 ( 86% ) patients , a decrease of lv myocardial mass by 5.9% was observed during follow - up ( 196 [ 100 ; 247 ] vs 180 [ 85 ; 237 ] g ; p=0.03 ) . in the remaining two patients , an increase by 11% and by 15%
lv ejection fraction ( 53% [ 33% ; 69% ] vs 54% [ 28% ; 71% ] ; p=0.75 ) and longitudinal function ( mapse : 6.5 [ 3 ; 10 ] vs 6 [ 3;9 ] mm ; p=0.08 and tapse : 12 [ 4 ; 17 ] vs 11 [ 5 ; 16 ] mm ; p=0.28 ) remained unchanged .
lv mass of the three patients with long - term observation are shown in figure 4 .
patient 1 showed a reduction of 11 g ( 4.4% ) after 44 months , patient 2 an increase of 5 g ( 3.2% ) after 25 months , and patient 3 a reduction of 10 g ( 10% ) after 30 months .
clinical demographics of the study cohort including the subgroup of cmri are given in table 1 .
none of the patients discontinued green tea consumption and no serious side effects were reported during the study period .
no changes in new york heart association ( nyha ) class could be observed , and no significant change in surface electrocardiogram parameters ( rhythm , heart rate , pq , qrs , qtc ) occurred .
one patient died due to sepsis , another due to spontaneous retroperitoneal hematoma requiring a prolonged stay in the intensive care unit with artificial ventilation and recurrent infections , resulting in multiorgan failure and , finally , death .
follow - up over periods of 44 , 25 , and 30 months , respectively , was achieved in three patients .
in 18 ( 85.7% ) patients , a significant decrease of total serum cholesterol by 8.4% was observed ( 191 [ 118 ; 267 ] vs 173 [ 106 ; 287 ] mg / dl ; p=0.006 ) during the 12-month study period . in three patients , an increase of cholesterol by 3%
was observed . in 16 ( 76.2% ) patients , low - density lipoprotein ( ldl )
cholesterol decreased by 8.0% ( 110 [ 60 ; 180 ] vs 99 [ 55 ; 188 ] mg / dl ; p=0.02 ] ) . in four patients , ldl cholesterol
glomerular filtration rate calculated by the modified diet in renal disease ( mdrd ) formula ( 62 [ 31 ; 99 ] vs 57 [ 26 ; 101 ] ml / min ; p=0.23 ) , got ( 30 [ 16 ; 56 ] vs 33 [ 17 ; 74 ] u / l ; p=0.90 ) , and gpt ( 29 [ 10 ; 57 ] vs 29 [ 10 ; 74 ] u / l ; p=0.95 ) remained unchanged .
ttr plasma levels remained stable during the study period ( 0.28 [ 0.14 ; 0.90 ] vs 0.26 [ 0.18 ; 0.50 ] mg / dl ; p=0.64 ; n=16 )
mean thickness of ivs ( 17 [ 13 ; 28 ] vs 18 [ 14 ; 25 ] mm ; p=0.1 ) and posterior wall ( 16 [ 11 ; 24 ] vs 17 [ 12 ; 19 ] mm ; p=0.4 ) remained unchanged during the study period ( figure 2 ) . in seven ( 33.3% )
patients , an increase of either 1 ) both ivs and posterior wall thickness ( n=4 ) or 2 ) isolated posterior wall thickness or ivs thickness ( n=3 ) occurred , by an average of 1.9 mm ( 13% ) .
mapse ( 10 [ 5 ; 23 ] vs 8 [ 4 ; 13 ] mm ; p=0.3 ) , tapse ( 14 [ 8 ; 23 ] vs 15 [ 2 ; 20 ] mm ; p=0.4 ) , and passive mitral inflow / lateral early diastolic mitral annular velocity ( e / e ) ratio ( 11 [ 7 ; 28 ] vs 14 [ 4 ; 31 ] ; p=0.8 ) remained unchanged .
in the subgroup of patients assessed by cmri ( n=14 ) ( figure 3 ; table 4 ) , in 12 ( 86% ) patients , a decrease of lv myocardial mass by 5.9% was observed during follow - up ( 196 [ 100 ; 247 ] vs 180 [ 85 ; 237 ] g ; p=0.03 ) . in the remaining two patients , an increase by 11% and by 15% was observed , respectively .
lv ejection fraction ( 53% [ 33% ; 69% ] vs 54% [ 28% ; 71% ] ; p=0.75 ) and longitudinal function ( mapse : 6.5 [ 3 ; 10 ] vs 6 [ 3;9 ] mm ; p=0.08 and tapse : 12 [ 4 ; 17 ] vs 11 [ 5 ; 16 ] mm ; p=0.28 ) remained unchanged .
lv mass of the three patients with long - term observation are shown in figure 4 .
patient 1 showed a reduction of 11 g ( 4.4% ) after 44 months , patient 2 an increase of 5 g ( 3.2% ) after 25 months , and patient 3 a reduction of 10 g ( 10% ) after 30 months .
we report on a cohort of wtattr - cm patients consuming gte for at least 1 year to demonstrate potential effects on inhibition of amyloid fibril formation.10,14 a significant decrease of lv myocardial mass was observed by cmri while no change of lv wall thickness was detected by echocardiography .
a decrease of lv mass was found in two of three patients after 30 and 44 months of observation , while there was an increase by 3% in the third patient after 25 months of treatment .
no side effects were reported during the study period . within a time span of 1 year , attr - cm
was found to be associated with a significant increase of lv myocardial mass and ivs thickness as well as a reduction of lv ejection fraction.8,15 thus , we concluded that egcg may halt progression of this fatal disease .
wtattr - cm is characterized by predominantly cardiac deposition of wtattr . according to more recent data achieved by skeletal scintigraphy
, it appears to cause lv infiltration in up to 1% of patients above 60 years of age.6 without treatment , attr - cm was found to be associated with an increase of lv mass by 8% within a 1-year period15 and a decrease of lv ejection fraction by 3.2% during 6 months.8 an association between amyloid load and myocardial mass could be demonstrated in a mouse model by ogawa.16 causative treatment options are lacking . in other studies ,
prognosis of patients with attr - cm remained poor , with median survival of 46 years.5,17 diverse in vitro and in vivo strategies have been aimed to stop amyloid fibril formation by stabilization of ttr protein ( tafamidis [ vyndaqel ] , egcg ) and by rna interference using aln - ttrsc ( revusiran ) or aln - ttr02 ( patisiran ) or to dissolve amyloid fibrils by egcg .
the health benefit of green tea , in particular egcg , has been reported for many years,18 and only rarely were adverse effects ascribed to the consumption of gte.19 actually , two clinical phase iii studies using tafamidis and rna interference drugs are recruiting patients , but high costs and lack of long - term data on toxicity have to be considered as limitations so far.20 green tea is considered to positively affect cardiovascular and metabolic complications21 and to improve lipid profile.12 thus , decrease of ldl cholesterol appears to be an indicator of gte bioavailability . to assure a daily constant intake dose of egcg , and to facilitate fluid restriction in those patients with severe heart failure , we administered capsules containing egcg and vitamin c. vitamin c improves the intestinal stability of egcg.22,23 hunstein s experiment to treat his light - chain amyloidosis with green tea14 was stimulated by the experiments of bieschke j et al9 and ehrnhoefer de et al,10 who demonstrated amyloid fibril disruption incubated with egcg . in a retrospective analysis of a larger cohort of patients with light - chain amyloidosis , a significant reduction of myocardial mass and an improvement of lv ejection fraction and nyha functional class
were reported in patients consuming green tea , while no changes were observed in patients without intake of green tea.24 recently , we demonstrated by cmri an average reduction of lv myocardial mass by 12.5% in a heterogeneous cohort of patients with mutant attr ( n=8 ) and wtattr ( n=6).13 this study was focused on patients with wtattr - cm to avoid modifying effects from inherited forms of attr - cm .
different individual metabolic conditions and renal excretion might have an impact on egcg plasma concentration .
up to now , no standardized test has been validated that allows reproducible quantification of egcg plasma concentration .
we assumed that the higher inter- and intraobserver variability of echocardiography within a small sample size could be a reasonable explanation.25 cmri is considered as the gold standard for analysis of cardiac morphology and function.26,27 compared with other types of cardiac amyloidosis , wtattr - cm progresses slowly .
demonstration of a long - term benefit of egcg requires a longer follow - up beyond 1 year . according to the mechanism of action ,
it is proposed that egcg has an additional effect on amyloid fibril formation and disruption to novel drugs that have been reported to inhibit fibril formation from ttr .
tafamidis , a novel stabilizer of human ttr,28 has been shown to stop progression of attr v30 m polyneuropathy in two - thirds of patients.29,30 it was approved by the european medicines agency in december 2011 and by the japanese pharmaceuticals and medical devices agency in september 2013 for treatment of stage 1 polyneuropathy in patients with hereditary attr amyloidosis .
attr - act , a phase iii study on the impact of tafamidis on cardiac attr amyloidosis , started in december 2013.31 the well - known different binding sites of tafamidis and egcg suggest a synergistic effect .
this is an observational report on a relatively small , but well - sized , study cohort with respect to the prevalence of this disease .
as green tea is widely available , of low cost , and , importantly , appears to have only minor side effects with long - term use , the potential for a placebo - controlled clinical trial is limited .
furthermore , egcg capsules are over - the - counter products , and the broad spectrum of effects of egcg , including amyloid fibril inhibition , has been well known for many years to most attr amyloidosis patients .
thus , we were not able to recruit a control group for the present study .
we assumed that the observed reduction of myocardial mass was due to reduction of amyloid load .
further mri studies demonstrated the use of a novel technique for tissue characterization , t1 mapping , for early diagnosis3234 and prognosis35 in different forms of cardiac amyloidosis .
t1 mapping allows quantification of the extracellular volume fraction . recently , reduction of extracellular volume by t1 mapping indicative of reduction of amyloid load has been demonstrated in a small cohort of patients with attr amyloidosis.36 however , histopathological validation of this technique is lacking .
this study supports evidence for the benefit of egcg to stabilize myocardial mass in wtattr - cm patients .
a reduction of myocardial mass was observed by cmri , while echocardiographic data and cardiac biomarkers , however , remained unchanged during the study period of 1 year . | backgroundcausative treatment of patients with wild - type transthyretin amyloid cardiomyopathy ( wtattr - cm ) is lacking .
recent reports indicate the potential use of epigallocatechin-3-gallate ( egcg ) , the most abundant catechin in green tea , to inhibit amyloid fibril formation .
we sought to investigate changes of cardiac function and morphology in patients with wtattr - cm after consumption of green tea extract ( gte).methodstwenty - five male patients ( 71 [ 64 ; 80 ] years ) with wtattr - cm were submitted to clinical examination , echocardiography , cardiac magnetic resonance imaging ( cmri ) ( n=14 ) , and laboratory testing before and after daily consumption of gte capsules containing 600 mg epigallocatechin-3-gallate for at least 12 months.resultsa significant decrease of left ventricular ( lv ) myocardial mass by 6% ( 196 [ 100 ; 247 ] vs 180 [ 85 ; 237 ] g ; p=0.03 ) by cmri and total cholesterol by 8.4% ( 191 [ 118 ; 267 ] vs 173 [ 106 ; 287 ] mg / dl ; p=0.006 ) was observed after a 1-year period of gte consumption .
lv ejection fraction by cmri ( 53% [ 33% ; 69% ] vs 54% [ 28% ; 71% ] ; p=0.75 ) , lv wall thickness ( 17 [ 13 ; 21 ] vs 18 [ 14 ; 25 ] mm ; p=0.1 ) , and mitral annular plane systolic excursion ( 10 [ 5 ; 23 ] vs 8 [ 4 ; 13 ] mm ; p=0.3 ) by echocardiography remained unchanged.conclusionthis study supports lv mass stabilization in patients with wtattr - cm consuming gte potentially indicating amyloid fibril reduction . |
eligible patients were required to have histologically or cytologically proven inoperable stage iiia or iiib nsclc as defined in the tnm classification of malignant tumors ( 6th edition ) , no previous chemotherapy or radiation therapy , a performance status of 01 on the eastern cooperative oncology group scale , adequate bone marrow reserves , as well as normal liver and renal function .
patients were excluded if they had malignant pleural or pericardial effusion , supraclavicular lymphnode metastasis , active secondary cancer , or a concomitant serious illness that contraindicated chemotherapy or pbt . for staging ,
all the patients underwent computed tomography ( ct ) of the thorax and either ct or magnetic resonance imaging ( mri ) of the brain .
the dose escalation of pbt was decided based on consultation with the independent efficacy and safety evaluation committee .
the total doses were 66 gy in arm 1 as the standard dose and 74 gy ( 12% higher ) in arm 2 as the elevated dose .
the protocol was to treat three patients at a given dose of pbt and perform followup for at least 90 days , and then to escalate to the next level if no doselimiting toxicities ( dlt ) occurred .
if one patient developed a dlt , then up to three additional patients were treated at the same dose .
dose escalation was stopped if two or more dlt occurred at a given dose level among six patients , and a maximum tolerated dose and rd was determined at the previous dose level .
if a dose of 74 gy ( relative biological effectiveness ; rbe ) did not cause dlt , then the actual maximum tolerated dose was not determined ; rather , 74 gy ( rbe ) was defined as the rd .
adverse events were graded according to the national cancer institute common terminology criteria for adverse events , version 3.0 .
dlt was defined as pbtrelated toxicities occurring within 190 days from the start of radiotherapy and included grade 3 nonhematologic toxicity , excluding nausea , vomiting and esophagitis , as well as grade 4 toxicity , excluding neutropenia .
volumetric ct images at the end of the exhalation phase were obtained for treatment planning using a respiratory gating system .
external respiratory signals were obtained by monitoring abdominal wall movement throughout simulation on ct , and carried out at every treatment session .
each patient was positioned in an immobilization device in the treatment position on a flat table . the primary tumor and clinically positive lymph nodes , seen either on the planning ct ( shortaxis diameter > 1 cm ) or pretreatment positron emission tomography images , constituted the gross tumor volume .
the total planning target volume ( ptv ) included the clinical target volume plus a total margin of at least 1.0 cm .
pbt started on day 1 of the first cycle of chemotherapy and was delivered once daily for 5 days per week .
the total dose of pbt was 66 gy ( rbe ) in 33 fractions in arm 1 and 74 gy ( rbe ) in 37 fractions in arm 2 .
the volume of both lungs that received 20 gy ( rbe ) or more of pbt was kept below 30% of the total volume of the lungs , and brachial plexus doses were limited to below 66 gy ( rbe ) .
the mean dose to the esophagus and heart were optimally kept below 34 gy ( rbe ) and 40 gy ( rbe ) , respectively .
if the treatment volume is larger than the field size limit , ptv is divided into several ptv that are smaller than the maximum field size .
treatment in eligible patients began with the administration of two cycles of concurrent chemoradiotherapy and two cycles of consolidation chemotherapy .
this consisted of oral administration of s1 twice daily from day 1 to 14 , along with a 60min intravenous infusion of cddp ( 60 mg / m ) on day 1 and , thereafter , at 4week intervals . the patients received one of three fixed oral doses of s1 based on their body surface area .
the three doses administered were 40 mg ( body surface area < 1.25 m ) , 50 mg ( body surface area , 1.251.50 m ) and 60 mg ( body surface area 1.50 m ) .
the dose escalation of pbt was decided based on consultation with the independent efficacy and safety evaluation committee .
the total doses were 66 gy in arm 1 as the standard dose and 74 gy ( 12% higher ) in arm 2 as the elevated dose .
the protocol was to treat three patients at a given dose of pbt and perform followup for at least 90 days , and then to escalate to the next level if no doselimiting toxicities ( dlt ) occurred .
if one patient developed a dlt , then up to three additional patients were treated at the same dose .
dose escalation was stopped if two or more dlt occurred at a given dose level among six patients , and a maximum tolerated dose and rd was determined at the previous dose level .
if a dose of 74 gy ( relative biological effectiveness ; rbe ) did not cause dlt , then the actual maximum tolerated dose was not determined ; rather , 74 gy ( rbe ) was defined as the rd .
adverse events were graded according to the national cancer institute common terminology criteria for adverse events , version 3.0 .
dlt was defined as pbtrelated toxicities occurring within 190 days from the start of radiotherapy and included grade 3 nonhematologic toxicity , excluding nausea , vomiting and esophagitis , as well as grade 4 toxicity , excluding neutropenia .
volumetric ct images at the end of the exhalation phase were obtained for treatment planning using a respiratory gating system .
external respiratory signals were obtained by monitoring abdominal wall movement throughout simulation on ct , and carried out at every treatment session .
each patient was positioned in an immobilization device in the treatment position on a flat table . the primary tumor and clinically positive lymph nodes , seen either on the planning ct ( shortaxis diameter > 1 cm ) or pretreatment positron emission tomography images , constituted the gross tumor volume .
the total planning target volume ( ptv ) included the clinical target volume plus a total margin of at least 1.0 cm .
pbt started on day 1 of the first cycle of chemotherapy and was delivered once daily for 5 days per week .
the total dose of pbt was 66 gy ( rbe ) in 33 fractions in arm 1 and 74 gy ( rbe ) in 37 fractions in arm 2 .
the volume of both lungs that received 20 gy ( rbe ) or more of pbt was kept below 30% of the total volume of the lungs , and brachial plexus doses were limited to below 66 gy ( rbe ) .
the mean dose to the esophagus and heart were optimally kept below 34 gy ( rbe ) and 40 gy ( rbe ) , respectively .
if the treatment volume is larger than the field size limit , ptv is divided into several ptv that are smaller than the maximum field size .
treatment in eligible patients began with the administration of two cycles of concurrent chemoradiotherapy and two cycles of consolidation chemotherapy .
this consisted of oral administration of s1 twice daily from day 1 to 14 , along with a 60min intravenous infusion of cddp ( 60 mg / m ) on day 1 and , thereafter , at 4week intervals . the patients received one of three fixed oral doses of s1 based on their body surface area .
the three doses administered were 40 mg ( body surface area < 1.25 m ) , 50 mg ( body surface area , 1.251.50 m ) and 60 mg ( body surface area 1.50 m ) .
nine patients were enrolled in this study , six in arm 1 and three in arm 2 . the patient cohort consisted of five men and four women , with a median age of 72 years .
patient characteristics and outcomes ad , adenocarcinoma ; awd , alive with disease ; dod , dead on disease ; ned , no evidence of disease ( recurrence ) ; nos , no specific ; os , overall survival ; pfs , progressionfree survival ; sq , squamous cell carcinoma ; m , male ; f , female ; ps , performance status .
the median followup time was 43 months for surviving patients and the median progressionfree survival was 15 months . in arm 1 , grade 3 infection occurred in one patient who then stopped pbt at 60 gy ( rbe ) and chemotherapy at one cycle .
no other dlt occurred in arm 1 . similarly , no dlt occurred in arm 2 .
all patients without dlt in arm 1 , and all patients in arm 2 , completed their pbt up to the prescribed dose , together with four cycles of chemotherapy .
however , one patient developed grade 3 esophageal fistula 9 months after the initiation of pbt ( fig .
the maximum esophageal dose of pbt did not exceed 74 gy ( rbe ) , with the exception of the patient with esophageal fistula , who received a dose of 75.8 gy ( rbe ) .
this patient also experienced recurrence in the primary tumor site , but not in the lymph nodes , and secondline chemotherapy ( docetaxel ) had started 1 month before the appearance of esophageal fistula .
grade 3 esophageal fistula occurring in a patient 9 months after the initiation of pbt .
upper middle : ( b ) ct image at 2 months after the initiation of pbt .
upper right : ( c ) ct image at 9 months after the initiation of pbt .
lower middle : ( e ) upper gi endoscopy at 9 months after the initiation of pbt .
lower right : ( f ) bronchial endoscopy at 9 months after the initiation of pbt .
overall toxicities experienced by patients following therapy to explain our results , we compared the pbt plans with those of 3dimensional conformal photon radiotherapy ( 3dcrt ) using the same targets and prescribed doses . for all patients , planning images and targets used in pbt
were registered to the pinnacle treatment planning system ( phillips , milpitas , ca , usa ) .
all plans were normalized such that 95% of the ptv received the same dose of pbt .
the lung and heart doses are summarized in table 3 , and the esophageal doses for each patient are listed in table 4 .
lung and heart doses for proton and photon radiotherapy lung v5 , relative lung volume irradiated more than 5 gy ; lung v20 , relative lung volume irradiated more than 20 gy ; mhd , mean heart dose ; mld , mean lung dose ; rbe , relative biological effectiveness .
esophageal proton dose ( rbe ) and volume ( cc ) patient # 9 experienced esophageal fistula .
rbe , relative biological effectiveness ; v66 , v70 and v74 , the esophageal volume irradiated more than 66 gy ( rbe ) , 70 gy ( rbe ) and v74 ( rbe ) , respectively .
proton beam therapy has an advantage over other radiation therapies in that the dose drops off and it is , therefore , utilized to reduce the risk to surrounding structures , particularly the lowdose regions in critical structures such as the heart , lungs and esophagus.7 indeed , only radiation pneumonitis below grade 2 occurred in this pbt trial .
in general , sparing of normal tissues in the lowtomoderate dose range is superior with proton therapy compared to photon therapy .
however , we noted a case with severe esophageal toxicity resulting in a thoracoesophageal fistula without local recurrence in this lesion .
first , the esophageal dose may have been too high to avoid a late toxicity probably because the esophagus was within the ptv of this patient , and was , hence , irradiated more than the prescribed dose .
moreover , an adaptive planning strategy generally improves sparing of the esophagus;8 however , this was not performed in our study .
a second potential reason for the observed toxicity is that combination with concurrent cddp and s1 chemotherapy was not feasible with highdose radiotherapy . a previous phase i study demonstrated that 74 gy photon radiotherapy , together with cddp and s1 , was feasible and promising.6 however , other reports regarding highdose thoracic radiotherapy adopted carboplatin and paclitaxel chemotherapy.4 , 5 , 9 , 10 . a third reason for the toxicity could be the secondline chemotherapy started 1 month before the esophageal fistula , which may have caused a recall phenomenon in the esophageal mucosa.11 after accounting for all possible factors , it is most likely that the high esophageal pbt dose is the main reason for this late esophageal toxicity . in fact , the esophageal dose in other patients without esophageal late toxicities did not exceed 74 gy ( rbe ) . for stage iii nsclc , lymphnodes are located in the mediastinum , which may lead to maximum esophagus doses to prescribed dose or more and it appears to be at a greater risk of severe esophageal toxicity . for the dose to serial organs in the mediatinum such as the esophagus and the bronchus , is a limiting factor in dose escalation
. actually , a recent study comparing 74gy and 60gy photon radiotherapy showed no advantage in both local recurrence and overall survival.10 we assumed that overall toxicities were unacceptably high by photon 74 gy irradiation . in this study ,
the predefined dlt were toxicities occurring up to 90 days after proton beam therapy . when this study was planned , we thought that longer observation periods in the 66gy arm were not necessary to assess the need for dose escalation because 66 gy is considered a standard and safe dose .
however , if we had set a period longer than 90 days for dlt observation , we could have detected late toxicity in arm 2 .
future study should have a longer observation period so that the need for dose escalation can be properly assessed . in conclusion ,
considering these issues , we maintain that pbt at a dose of 66 gy ( rbe ) is more appropriate to reduce overall toxicity and improve therapeutic ratios , and identified this as the rd for future clinical trials .
| the purpose of this study is to determine the recommended dose ( rd ) of proton beam therapy ( pbt ) for inoperable stage iii nonsmall cell lung cancer ( nsclc ) .
we tested two prescribed doses of pbt : 66 gy ( relative biological effectiveness [ rbe ] ) in 33 fractions and 74 gy ( rbe ) in 37 fractions in arms 1 and 2 , respectively .
the planning target volume ( ptv ) included the primary tumor and metastatic lymph nodes with adequate margins .
concurrent chemotherapy included intravenous cisplatin ( 60 mg / m2 , day 1 ) and oral s1 ( 80 , 100 or 120 mg based on body surface area , days 114 ) , repeated as four cycles every 4 weeks .
doselimiting toxicity ( dlt ) was defined as grade 3 or severe toxicities related to pbt during days 190 .
each dose level was performed in three patients , and then escalated to the next level if no dlt occurred .
when one patient developed a dlt , three additional patients were enrolled .
overall , nine patients ( five men , four women ; median age , 72 years ) were enrolled , including six in arm 1 and three in arm 2 .
the median followup time was 43 months , and the median progressionfree survival was 15 months . in arm 1 , grade 3 infection occurred in one of six patients , but no other dlt was reported . similarly , no dlt occurred in arm 2 .
however , one patient in arm 2 developed grade 3 esophageal fistula at 9 months after the initiation of pbt . therefore , we determined that 66 gy ( rbe ) is the rd from a clinical viewpoints .
( clinical trial registration no .
umin000005585 ) |
root canal preparation is performed with the use of endodontic instruments and auxiliary chemical substances , seeking to achieve cleaning , shaping , and disinfection , in order to fill the root canal later .
the use of rotary nickel titanium ( niti ) instruments has increased considerably due to their safe use , when used in accordance with the recommendations of their manufacturers , enabling the canal to be prepared more quickly , when compared with manual instruments , and even with better quality , in case of canals with severe curvatures .
cutting dentin is an important step during root canal preparation , as it is necessary to remove contaminated dentin and shape the canal to create conditions for it to be filled . due to elasticity
, one could suppose that the cutting efficiency of niti instruments would be lower than that of steel instruments , because they undergo deflection or bend during contact with the dentinal surface .
however , studies have shown that niti instruments have a more efficient cutting capacity when compared with steel instruments .
although there have been significant advancements in rotary instrumentation , the influence of the rotary cutting instrument blade design is still controversial , with respect to the efficiency of their cleaning capacity .
their cutting capacity results from a complex inter - relationship between different parameters , such as , the sectional design of the instrument , radial lands or active cutting blades , metallurgical properties,[911 ] and treatment of instrument surfaces with the incorporation of ions .
the aim of this study is to evaluate the cutting efficiency of different rotary niti instruments ; that is , k3 , niti tee , profile , and quantec .
forty instruments with taper size .04/25 were tested and divided into groups according to their brands ( n = 10 ) : k3 ( sybron - endo , orange county , ca , usa ) , niti tee ( sjding sendoline , kista , sweden ) , profile ( dentsply maillefer , ballaigues , switzerland ) , and quantec ( analytic endodontics , mexico ) . the characteristic of each instrument is shown in table 1 .
different instrument designs forty acrylic resin blocks ( dentsply maillefer , petrpolis , rj , brazil ) , with simulated canals were used in this study , and for each block , a rotary instrument was used .
the canals initially had a diameter of a caliber 10 instrument , having been manually prepared in this manner , up to a caliber 20 instrument , to their full extent , using the crown - down technique , under irrigation with 5 ml distilled and deionized water , at every change of instrument . before any intervention
, the blocks were washed in an ultrasonic vat , with distilled water and detergent solution , in the proportion of 10 : 1 for 20 minutes , dried with air jets , and placed in an oven at 60c for 48 hours .
instrumentation of each block with the analyzed instruments was performed by the same operator using a tc 3.000 engine ( nouvag , tcm endo , goldach , switzerland ) at a constant speed of 300 rpm .
the instruments were introduced into the canal , inundated with 5 ml distilled and deionized water , until the working length was reached , which was 0.5 mm short of the total working length of the simulated canal .
each instrument remained at the working length for 5 seconds , and was then removed , while it was still running .
the procedure of washing and drying the acrylic resin blocks was performed before the instruments were used , was repeated after instrumentation , to determine the final weight ( fw ) , and also performed before the use of 04/25 instruments .
the cutting efficiency of each instrument was measured by means of measuring the acrylic resin block mass before and after using the .04/25 instruments ( fw - iw ) , with the aid of a precision digital analytic balance ( bel engineering , italy ) , which was due to the quantity of material removed by instrumentation . after obtaining the results ,
having verified the normality of the sample , the analysis of variance ( p < 0.05 ) was performed .
forty instruments with taper size .04/25 were tested and divided into groups according to their brands ( n = 10 ) : k3 ( sybron - endo , orange county , ca , usa ) , niti tee ( sjding sendoline , kista , sweden ) , profile ( dentsply maillefer , ballaigues , switzerland ) , and quantec ( analytic endodontics , mexico ) . the characteristic of each instrument is shown in table 1 .
forty acrylic resin blocks ( dentsply maillefer , petrpolis , rj , brazil ) , with simulated canals were used in this study , and for each block , a rotary instrument was used .
the canals initially had a diameter of a caliber 10 instrument , having been manually prepared in this manner , up to a caliber 20 instrument , to their full extent , using the crown - down technique , under irrigation with 5 ml distilled and deionized water , at every change of instrument . before any intervention
, the blocks were washed in an ultrasonic vat , with distilled water and detergent solution , in the proportion of 10 : 1 for 20 minutes , dried with air jets , and placed in an oven at 60c for 48 hours .
instrumentation of each block with the analyzed instruments was performed by the same operator using a tc 3.000 engine ( nouvag , tcm endo , goldach , switzerland ) at a constant speed of 300 rpm .
the instruments were introduced into the canal , inundated with 5 ml distilled and deionized water , until the working length was reached , which was 0.5 mm short of the total working length of the simulated canal .
each instrument remained at the working length for 5 seconds , and was then removed , while it was still running .
the procedure of washing and drying the acrylic resin blocks was performed before the instruments were used , was repeated after instrumentation , to determine the final weight ( fw ) , and also performed before the use of 04/25 instruments .
the cutting efficiency of each instrument was measured by means of measuring the acrylic resin block mass before and after using the .04/25 instruments ( fw - iw ) , with the aid of a precision digital analytic balance ( bel engineering , italy ) , which was due to the quantity of material removed by instrumentation . after obtaining the results ,
having verified the normality of the sample , the analysis of variance ( p < 0.05 ) was performed .
the means and standard deviations of mass loss from the acrylic resin blocks during instrumentation are shown in table 2 .
the results test showed that the acrylic resin blocks prepared with k3 , niti tee , and profile instruments presented the greatest mass loss , showing no statistically significant difference among them ( p < 0.05 ) .
the lowest mass loss was found in the blocks prepared with quantec instruments ( p < 0.05 ) .
biomechanical techniques for preparing root canals have evolved from manual methods to rotary techniques employing nickel - titanium instruments . using niti rotary instruments for root canal instrumentation
has enabled clinicians to create consistently tapered preparations more predictably and efficiently , while minimizing procedural mishaps , especially in curved canals .
the greater cutting capacity of k3 instruments , as compared to quantec , is possibly due to the unique cross - section design of k3 , which has a positive angle of inclination for greater cutting efficiencies and ample radial lands and relief at the posterior extremity of the blade , to reduce friction . unlike quantec , which has two cutting blades
thus , the k3 instruments present excellent cutting capacity , as the debris resulting from its cutting action is easily displaced from the working area , and removed by the unique helical angle of this file .
schafer and florek conducted a study in which they compared the efficiency of the k3 system and k - flexofile files for smear layer removal .
the authors obtained excellent results with the k3 system , although complete root canal cleaning had not been obtained .
the niti tee instruments showed a cutting efficiency similar to that of the k3 and profile instruments , and superior to that of the quantec instruments .
these instruments had a rounded , non - cutting tip and a positive cutting angle , without the presence of radial land .
in addition , they had two 90 cutting blades that caused grooves ( scratches ) on the canal wall , which allowed the removal ( drainage ) of dentin scrapings and other debris resulting from instrumentation .
jodway and hlsmann , comparing k3 and niti tee instruments , observed that they were safe as regards their use , and had similar root canal cleaning and shaping capacities .
the good cutting capacity of profile instruments could be attributed to the u - shaped , cross - sectional design , with radial lands and central parallel core . with a neutral and negative angle of inclination
thus , the debris is transported up to the coronal portion and effectively removed from the root canals .
some studies have indicated that the positive cutting angle improves the cutting efficiency of the instrument . nevertheless , this is not the only determining factor for the cutting capacity of instruments , as the profile instruments , which have a negative cutting angle , presented the same efficiency as the k3 and niti tee instruments and greater efficiency than the quantec instruments , fitted with a positive cutting angle .
quantec instruments have two cutting blades and a helical angle combined with spaces created in the instrument , which progressively increase as they withdraw from the cutting edge , creating a larger area of escape and allowing rapid debris removal from the canal .
thus , when activated , the instrument does not make a thread ; therefore it does not stick to the canal walls , diminishing the risk of instrument fracture , because it has a less aggressive cut , which possibly reduces its cutting power , as it presents the lowest cutting efficiency among the studied instruments .
the cutting capacity of endodontic instruments is an important factor to evaluate , as the preparation of dentinal walls , by eliminating contamination and shaping endodontic space , is an important step in successful endodontic therapy .
however , after debridement , in some oval and flattened root canals , the walls may not be prepared , irrespective of the debridement technique , leaving a smear layer , debris , and unprepared root canal walls . this complex anatomy may be considered as one of the main challenges in controlling the root canal infection , because the pulp tissue as well as the root dentin may conceal toxins and microorganisms , compromising the final result of endodontic treatment .
the use of endodontic irrigant solutions and ultrasound agitation , or even the association of endodontic instruments are ways that may improve the cleaning of roots that have a complex anatomy .
from the results of the present study it can be concluded that k3 , niti tee , and profile instruments present a better cutting efficiency than the quantec instruments . | aim : the aim of this study was to evaluate the cutting efficiency of rotary nickel - titanium ( niti ) instruments k3 , niti tee , profile , and quantec with taper size 04/25.materials and methods : the number of samples was 10 for each group ( n = 10 )
. the cutting efficiency was measured by the mass loss from each acrylic resin block after instrumentation of a simulated canal using the crown - down technique.results:the analysis of variance ( anova ) showed that there was a statistically significant difference among the studied groups .
the tukey 's test showed that the acrylic resin blocks prepared with instruments k3 ( 0.00369 0.00022 ) , niti tee ( 0.00368 0.00023 ) , and profile ( 0.00351 0.00026 ) presented the greatest mass loss , showing no statistically significant difference among them ( p < 0.05 ) .
the lowest mass loss was found in the blocks prepared with quantec instruments ( 0.00311 0.0003 ) ( p < 0.05).conclusions : it could be concluded that the k3 , niti tee , and profile instruments presented a greater cutting efficiency than the quantec instruments . |
duchenne muscular dystrophy ( dmd ) is a recessive x - chromosome - linked disease that affects ~1/36006000 liveborn males .
the muscular weakness is progressive , causing loss of ambulation by early adolescence ( between 9 and 12 years of age ) .
dmd is caused by mutations in the dmd gene that code for dystrophin , a sarcolemmal cytoskeletal protein [ 24 ] .
dmd gene mutations that result in complete loss of dystrophin interrupt their translation , giving rise to dmd .
mutations that conserve the open reading frame produce reduced quantities of dystrophin or a dysfunctional or truncated form of dystrophy , resulting in becker muscular dystrophy ( bmd ) , a less severe phenotype .
dystrophin is an element of the dystrophin - glycoprotein complex ( dgc ) that provides a mechanical link between the extracellular matrix and cytoskeleton of muscle cells , allowing the plasma membrane of the muscle fiber to resist the mechanical process of the muscle during muscle contraction [ 6 , 7 ] .
it has been suggested that the dgc also participates in important cell - signalling processes , functioning as a binding platform for certain ligands such as neuronal nitric acid synthase , which stimulates glucose transport .
skeletal muscle is responsible for > 80% of insulin - stimulated glucose uptake in the body .
therefore , destabilization in dgc or their components may generate abnormal signalling of insulin in muscle fibers and lead to alterations in functionality .
in fact , it was reported that the disturbance within the surface dgc may contribute to insulin resistance ( ir ) and abnormalities characteristic of the skeletal muscle of diabetic goto - kakizaki rats , because an abnormal subcellular location of glucose transporter 4 ( glut4 ) vesicles in muscle fiber was observed in these rats . however , information on this regard from human studies is lacking .
this information led us to propose that dgc alterations may generate changes in glucose metabolism such as ir in skeletal muscle of patients with dmd or bmd .
dgc modifications provoke an imbalance in plasma membranes permeability , inducing myofibers through deterioration - regeneration cycles until exhausting their repair capacity .
this poses muscle fibers susceptible to necrotic development and to be replaced by both fibrous connective tissue and adipose tissue [ 1 , 13 ] , increasing adiposity .
previous body composition studies have showed that dmd patients present greater body fat mass than similarly aged healthy subjects [ 13 , 14 ] .
in addition , other investigators have observed that patients with dmd develop overweight or obesity from the age of 7 , reaching a frequency of > 50% at 13 years of age .
in addition to the alteration of dgc , overweight and obesity are also risk for ir , which increases the risk for other severe morbidities such as cardiovascular disease and type 2 diabetes in dmd patients
. however , the concomitant effect of alterations in dgc and obesity on the risk for insulin resistance has not been properly addressed .
thus , the purposes of this investigation were to determine the frequency of ir in patients with dmd / bmd and to evaluate the association of deletions in specific regions of the dmd gene with obesity and ir , as well as exploring molecular mechanisms likely leading to ir , by evaluating molecules involved in glucose metabolism such as insulin receptor , insulin receptor substrate , and glut4 localization in muscle biopsies of dmd / bmd patients .
the institutional ( instituto mexicano del seguro social ) ethics committee approved the study prior to the start of patient recruitment . all dmd / bmd patients seen at the outpatient electrodiagnostic muscular dystrophy service at the national institute of rehabilitation were recruited for the cross - sectional study between january 2011 and december 2013 ; 117 patients ( aged 4 years to < 18 years ) were included .
subjects with a clinical diagnosis of dmd / bmd were candidates to participate in the study , and confirmatory molecular diagnosis of dystrophy was carried out .
patients were eligible for inclusion in this study if they had a deletion in the dmd gene analyzed by multiplex polymerase chain reaction ( mpcr ) .
parents and patients received an explanation of study fundamentals , procedures , benefits , right to confidentiality , and the right to withdraw from the study if they wished .
all parents provided written informed consent in adherence with the human subjects ' guidelines of the institutional ethics committee . on the day of the study ,
a peripheral blood sample was collected in vacutainer with and without anticoagulant in a fasting state for genomic dna extraction from leukocytes to determine glucose and insulin , respectively .
body composition to measure adiposity was evaluated by dual - energy x - ray absorptiometry ( dexa ) .
trained personnel carried out measurements of body weight ( kg ) and height ( mts ) . for subjects who were able to stand erect ,
height was measured with a wall - mounted stadiometer ( model 208 , seca ) . for subjects unable to stand
subjects ' weight was measured ( model bwb-700 , tanita , for ambulatory patients and model 954 seca for wheelchair - bound patients ) wearing light clothing and without shoes .
diagnosis of overweight and obesity was obtained using body mass index ( bmi ) , expressed as percentiles .
children with bmi 5th percentile were classified as underweight , those with bmi > 5th but < 85th percentile as normal weight , those with bmi 85th but < 95th percentile as overweight , and those with bmi 95th percentile as obese , in accordance with criteria established by the centers for disease control and prevention ( cdc ) in 2009 about bmi for children and teens ( http://www.cdc.gov/healthyweight/assessing/bmi/childrens_bmi/about_childrens_bmi.html ) .
body composition measurements were carried out by dexa ( lunar prodigy , ge medical systems , madison , wi ) and encore software , v. 2004 ( lunar corporation ) , was used to analyze whole - body dexa scans .
body fat mass was considered high according to the classification as described previously that used dexa to predict % body fat mass corresponding to the bmi cutoff in male children and adolescents ( 318 years ) as overweight ( range : 18%23% ) and obesity ( range : 2436% ) .
one hundred seventeen patients were screened for deletions and duplications by mpcr with the primer sets of chamberlain et al . and
beggs et al . using the mpcr kit for human dmd / bmd set i + ii ( maxim biotech , san francisco , ca ) according to the protocol recommended by the manufacturer . serum insulin ( u / ml ) was quantified utilizing a commercial kit ( linco research , st .
louis , mo ) based on radioimmunoanalysis . a value > 12 u / ml was considered as hyperinsulinemia as was previously reported .
serum glucose ( mg / dl ) concentration was measured by the glucose - oxidase method ( glucose - lq , spinreact , s.a . ,
ir was calculated from insulin and glucose data using the homeostasis model assessment - insulin resistance ( homa - ir ) method ( homa - ir = [ fasting insulin , u / ml ] [ fasting glucose , mmol / l])/22.5 .
a portion was used for immunofluorescence analysis . in this study , we had availability for tissues from only five patients with dmd / bmd of the entire population studied .
we also used five biopsies of gastrocnemius muscle from five healthy individuals as controls ( age 4455 years ) to compare cellular localization of dystrophin , insulin receptor , insulin receptor substrate , and glut4 .
we analyzed skeletal muscle biopsy from older controls because we have not access to biopsies of muscle from healthy subjects of the same age . nevertheless , older controls allowed the main purpose to evaluate the cellular localization of these molecules in human healthy muscle .
specific mouse monoclonal primary antibodies used were glut4 , insulin receptor ( ire ) ( santa cruz biotechnology , santa cruz , ca ) , and dystrophin n - terminus ( dys - n ) and c - terminus ( dys - c ) ( vector laboratories , burlingame , ca ) .
polyclonal antibody against insulin receptor substrate ( irs ) ( santa cruz biotechnology ) was also used .
afterwards , 7 m cryosections were prepared and added to the coverslip covered with poly - l - lysine .
next , nonspecific binding was blocked by incubations of the cryosections with 5% bovine serum albumin in phosphate - buffered solution ( pbs ) for 60 min at 25c .
samples were incubated with cy3-secondary antibody ( goat anti - mouse , jackson immunoresearch laboratories , west grove , pa ) for 60 min at 25c in the dark .
subsequently , samples were washed with pbs and mounted with dapi ( labeling of nuclei ) and vectashield ( vector laboratories ) .
tissues were observed under an olympus bx60 fluorescence microscope ( olympus , tokyo , japan ) .
a subjective value was assigned ( normal + + + , decreased + + , considerably decreased + , and absent ) to describe staining , indicating the presence of dystrophin .
statistical analysis was performed using the minitab statistical software ( minitab 14 , state college , pa ) .
results are presented as median ( minimum , maximum ) ; p value 0.05 was considered significant .
pearson correlation analyses were used to evaluate associations between the most frequently deleted exons and fasting insulin , homa - ir , bmi , and body composition .
comparisons among nutritional status groups were conducted with one - way anova and dunnett 's method as post hoc test , considering the normal nutritional status group as control .
logistic regression models were carried out to identify risk factors for ir , introducing each exon deletion as a predictor and taking into account adiposity .
we are reporting the results of 66 subjects identified as carriers in at least one exon of the two deletion - prone regions in the dmd gene .
most alterations ( 73.7% ) were clustered in exons 4360 in the major hot spot .
deletions of exons 19 , 45 , 47 , 48 , 50 , and 51 were more frequently detected .
patient ages were 8.96 ( 4.61 , 17.75 ) years , median ( minimum , maximum ) , and 53% of the population were between 7 and 10 years of age .
a low percentage of patients ( 9.1% ) were between 15 and 18 years of age .
fasting insulin concentration ranged from 5.2 to 59.9 u / ml , but ~50% present hyperinsulinemia .
homa - ir values range from 1.04 to 12.9 , and 36% of the patients had ir ( table 1 ) .
most boys presented normal nutritional status ( 59% ) , but 22.7% were overweight / obese and 18.2% were underweight . according to the classification of taylor et al .
( see subjects and methods ) , a higher ( 68% ) prevalence of overweight / obesity was observed in contrast to using bmi ( 22.7% ) .
thus , we show group data according to nutritional status from dmd / bmd patients as underweight , normal , and overweight / obese ( table 2 ) .
we observed significant differences in insulin concentrations and homa - ir values among nutritional status groups , but no differences in age were detected .
overweight / obese boys presented higher glucose , insulin , and homa - ir values as compared to normal bmi group .
that group exhibited also a higher proportion ( 80% ) of subjects with ir determined by homa - ir > 3.16 ( : 11.48 , p = 0.004 ) compared to normal nutritional status .
interestingly , an important percentage of underweight patients presented hyperinsulinemia and ir ( 27.3% ) .
body fat mass was higher in overweight / obese and lower in underweight than in patients with normal nutritional status ( table 2 ) .
both fasting insulin and homa - ir were positively correlated with all anthropometric and body composition parameters .
however , the highest correlation was found between homa - ir and body fat mass ( figure 2 ) .
carriers of deletions in exon 45 ( or = 9.32 ; 95% ci 1.1674.69 ; p = 0.036 ) or exon 50 ( or = 8.73 ; ci95 = 1.1765.10 ; p = 0.035 ) were associated with the risk for ir , even adjusting for adiposity ( figure 3 ) .
staining of glut4 vesicles was observed as cytoplasmic aggregates in biopsies from patients 3 , 5 , and 7 ( figure 4 ) , although in patients 3 and 7 there are a smaller number of these aggregates ; the three patients presented hyperinsulinemia and ir ( table 3 ) and patient 5 showed hyperglycemia .
staining for ire and irs was similar to control tissue muscle in myofiber sections from five patients .
dystrophin staining intensities in myofibers from five patients ( figure 3 ) were lower than those observed in normal biopsy specimens .
hyperinsulinemia and ir observed in our patients are important risk factors for developing pathologies such as cardiovascular disease or type 2 diabetes .
the frequency of these metabolic alterations increases ( 80% ) importantly when dmd / bmd patients are overweight / obese , with a rate higher than in nondystrophic obese boys reported in our country ( ~50% in children between 3 and 18 years ) .
the increased intramuscular body fat mass deposition in dmd patients and hyperinsulinemia and ir are strong indicators of muscle metabolic defects ; this deposition is present in dgc - related muscular dystrophy .
but it is important to consider that almost a third ( 27.3% ) of underweight patients presented hyperinsulinemia and ir which indicates that there is not an intramuscular body fat mass deposition in those patients .
therefore , ir in dmd / bmd may be an independent result of fat mass content where alterations of components of dgc such as dystrophin may be involved . to our knowledge , there are no studies that clearly demonstrate whether patients with dmd or bmd present ir or alterations in glucose metabolism .
freidenberg and olefsky reported that serum glucose and insulin concentrations after oral glucose administration showed an abnormal increase in the area under their respective curves , suggesting alterations in glucose metabolism from dmd patients .
abnormal subcellular accumulation of glut4 vesicles was observed in nonobese , type 2 diabetic skeletal muscle fibers of rats .
accordingly , we also observed abnormal cytoplasmic aggregates of glut4 in myofibers from dmd / bmd patients , suggesting a possible alteration of glucose incorporation into the muscle , leading to hyperglycemia .
furthermore , we observed that deletions of exons 45 or 50 increase the risk ( ~9.0 times ) for developing ir . in this sense ,
these exons encode an acting - binding domain and maybe the alteration between f - actin and dystrophin link may disturb some cell - signalling process as binding platform ligands arising in metabolic alteration . anyway , our data suggest that the result of the mutation in the dmd gene may possibly be associated with a metabolic alteration in dmd / bmd patients .
the presence of these metabolic alterations in underweight subjects suggests a possible relationship with dmd / bmd .
because we included a low number of muscle biopsies from dmd / bmd patients with different grades of damage in the muscle fiber , we detected abnormal cytoplasmic aggregates of glut4 in myofibers only in three patients . at any rate , this information opens an interesting field of study to explore , in muscle biopsies from dmd / bmd patients , cellular signalling pathways involved in glucose metabolism and possibly related to muscle morphology .
another limitation of this study is the lack of distinction between dmd and bmd and the lack of appropriate control subjects of the same age .
anyway , the main strength of this study is that we demonstrated that those patients develop metabolic alterations , which is essential information for caregivers and physicians in order to prevent other pathologies .
we identified differences in nutritional status of dmd / bmd patients according to bmi ( percentiles ) .
prevalence of overweight or obesity in these patients was lower ( 22.7% ) than that reported by other authors where ~50% of boys with dmd / bmd were obese by 13 years of age according to bmi [ 13 , 15 ] .
for the first time , we present information about the prevalence of obesity in dmd without effect of steroid treatment .
the higher total body fat mass observed in boys with dmd is mostly due to increased intramuscular body fat mass deposition in both the central and peripheral regions . in agreement with the classification of taylor et al . , 2002 , body fat mass was higher ( ~68% ) than in healthy boys and in boys with dmd [ 1315 , 25 ] . in conclusion , to our knowledge , these novel results present evidence regarding the presence of metabolic alterations in dmd / bmd patients such as obesity , hyperinsulinemia , and ir , without the effects of steroid treatment .
ir had a high frequency and increased significantly when dmd / bmd patients are overweight / obese .
deletion of exons 45 or 50 increases the risk ( ~9 times ) for developing ir .
abnormal cytoplasmic aggregates of glut4 in myofibers from dmd / bmd patients suggest a possible alteration of glucose incorporation into the muscle . | aim . our aim was ( 1 ) to determine the frequency of insulin resistance ( ir ) in patients with duchenne / becker muscular dystrophy ( dmd / bmd ) , ( 2 ) to identify deleted exons of dmd gene associated with obesity and ir , and ( 3 ) to explore some likely molecular mechanisms leading to ir .
materials and methods . in 66 patients with dmd / bmd without corticosteroids treatment , ir , obesity , and body fat mass were evaluated .
molecules involved in glucose metabolism were analyzed in muscle biopsies .
results show that 18.3% , 22.7% , and 68% were underweight , overweight , or obese , and with high adiposity , respectively ; 48.5% and 36.4% presented hyperinsulinemia and ir , respectively .
underweight patients ( 27.3% ) exhibited hyperinsulinemia and ir .
carriers of deletions in exons 45 ( or = 9.32 ; 95% ci = 1.1674.69 ) and 50 ( or = 8.73 ; 95% ci = 1.1765.10 ) from dmd gene presented higher risk for ir than noncarriers .
we observed a greater staining of cytoplasmic aggregates for glut4 in muscle biopsies than healthy muscle tissue .
conclusion .
obesity , hyperinsulinemia , and ir were observed in dmd / bmd patients and are independent of corticosteroids treatment .
carriers of deletion in exons 45 or 50 from dmd gene are at risk for developing ir .
it is suggested that alteration in glut4 in muscle fibers from dmd patients could be involved in ir . |
scleroderma , or systemic sclerosis , is a chronic connective tissue disease generally
classified as one of the autoimmune rheumatic diseases
a 58-year - old woman was informed of her scleroderma , while receiving oral prosthetic
rehabilitation .
the psychological load of the disease itself , along with dental treatment
cessation , resulted in patient s disappointment and social separation .
three hypnosis
sessions were conducted for normalizing patient s psychological status primarily , and
for the facilitation of dental treatment , as well . the range of mouth opening changed
from 3.7 to 6.2 cm , allowing for easier entrance of dental appliances into the mouth
for completion of prosthetic rehabilitation . with the tooth prostheses in place ,
the
patient gained more social confidence and started sharing her experience with other
patients with the same condition .
this case illustrates that hypnosis therapy may prove beneficial for patients with
limited mouth opening secondary to scleroderma , facilitating the prosthetic
rehabilitation programs for both dental specialists and patients , with a secondary
important impact on the patient s social perception of himself .
scleroderma , or systemic sclerosis , is a chronic connective tissue disease generally
classified as one of the autoimmune rheumatic diseases . the word scleroderma comes from
two greek words : sclero meaning hard , and derma
hardening of
the skin is one of the most visible manifestations of the disease .
the nose appears pinched and
thinned and the opening of the mouth might be decreased in size ( microstomia or small
mouth ) .
the development of dilated
small blood vessels , called telangiectasia , affects the face and other parts of the body .
the appearance of the eyes can also alter , all of which can negatively impact the patient s
appearance ( 4 ) .
the general tightening of the
skin over the face makes lip and mouth movements , as well as oral hygiene , difficult .
microstomia may make it difficult to open the mouth wide enough for dental procedures or
even oral hygiene .
therefore , preventative dental care through regular flossing and
brushing of the teeth and gums after each meal is of utmost importance ( 5 ) .
the best approach to management is by means
of facial grimacing and mouth stretching exercises , performed daily .
floss holders , pump toothpaste tubes , and
built - up handles on toothbrushes can help people with hand impairment , while an electric
toothbrush is strongly advised ( 6 ) . from the psychological aspect , a common reaction to being told that one has a disease such
as scleroderma is ,
it is not clear why only several people develop the disease
and others do not .
one does not bring scleroderma upon him or herself , and therefore ,
should not feel guilty or responsible for the illness . a person newly diagnosed with
scleroderma may feel alone and uncertain about where to turn for help ( 7 ) .
psychopatologically speaking , the majority of
the sclerodermatic patients may manifest mild anxiety due to apprehension and vigilance
related to the perceived threat from the illness and the uncertainties over etiology ,
prognosis and outcome .
he , or
she , may experience a number of other feelings and emotional reactions from time to time ,
including initial shock or disbelief , fear , anger , denial , self - blame , guilt , grief ,
sadness , somatization , profound feelings of generalized hostility , and interpersonal
sensitivity , which may be due to the gradual disfigurement resulted from skin thickening .
the severity of illness , and consequent disability , could be regarded as the central
factors contributing to the development of the psychiatric symptomatology .
paranoid
ideation and psychoticism of the sclerodermatic patients probably reflect a vulnerability
towards the manifestation of more serious , psychotic , psychopathology .
medication should
not be regarded as indifferent in this cumulus of psychological anxiety triggers ( 7 , 8) .
feelings , in themselves , are neither good
nor bad ; one simply has them . sharing them with family members and friends , or with
others who have had the same experiences
other
factors , such as social support , may also play significant roles ( 8) .
professional counseling can also help people with scleroderma
and their family members who are having difficulty coping with their feelings . instead of
scleroderma patient , the term person with scleroderma
the person
with scleroderma may be a patient in the doctor s office , hospital or clinic , but he or
she is much more than that .
thinking of oneself as a total person , with a full life , may
help to cope with scleroderma and enable one to maintain a positive , and also realistic
insight ( 9 ) .
hypnosis is defined as an attentive , receptive , focal concentration , with diminished
peripheral awareness .
hypnotizability , expectancies , motivation , absorptive
capacity / fantasy proneness , and attitudes towards hypnosis are five psychological factors
most frequently mentioned as important .
the rapport ( also referred to as resonance and
harmony ) and social context are identified as social factors .
evidence supports the usefulness of hypnotic treatments , to address biological ,
psychological and social factors , and their interactions in any medical condition .
the
biopsychosocial model of hypnosis can help in understanding complex issues , such as
scleroderma .
importantly , this model allows the possibility for multiple factors to play a
role and contribute to hypnotic responding ( 10 ) .
while receiving dental services for prosthetic rehabilitation , our 58-year - old patient
referred to dermatologist just because of limitation of mouth opening , which is essential
for the optimal performance of dental treatment . a comprehensive battery of tests revealed
that she was suffering from scleroderma .
this was a major turning point , because both the
oral prosthetic service , and also several other aspects of her and her family life , got
affected .
therefore , we changed our plan from a regular somatic to a more comprehensive
biopsychological approach .
the first measure implied in the management of this case was to
conducting a team work , including an endodontist who has been trained as an advanced
hypnotherapist , a restorative dentistand also prosthodontist for dental treatments .
although , at first , five sessions were preplanned to complete whole levels of treatment ,
fortunately , prior to commencing the fourth session , treatment finished convincingly with
optimal results .
as a first step , a simple interview was performed to see if there is the
potential for a hypnosis intervention . simultaneously , and also during pre - talk , several
myths in which the patients believed concerning hypnosis , were corrected .
the spiegel
biological scale of the patient scored as 3 out of 5 . at the first one - hour session , all
activity had been targeted just for relaxation , anxiety management , attitude correction
about the new encountered unusual somatic condition , correction of coping skills for new
life , increasing self - confidence , and , eventually , conditioning with light folkloric music
and also , her very kind mother s name .
two - week intervals were considered between all three
hypnosis sessions . during the second hypnosis session , which lasted 50 minutes ,
she was
fallen more easily and more deeply into trance , using the conditioning keys and suggested
specially for her dental treatment procedure , particularly useful cooperation with the
dentist to gain suitable results .
meanwhile , her attitude about her new facial appearance
and the others reactions were repaired .
surprisingly , less pain and discomfort would have
been felt thereafter , due to any joint movement throughout the body , especially in case of
the shoulder and temporomandibular joints .
the third session , lasting for 40 minutes , was
spent for reinforcement of previous inductions and conditions , and a new induction was
performed for increasing the intention to help the other persons newly and unexpectedly
faced with disturbing conditions , such as scleroderma , or any other .
prior to the fourth
session , she obtained satisfactory psychological capabilities again , and hence , no need was
felt for continuing hypnotic interventions urgently .
all the pre - desired dental treatments
were delivered to the subject , and she was also asked to use hypnosis any time needed .
three follow up visits showed no relapse , although the disease itself had its ongoing
nature .
nevertheless , it was not detrimental psychologically , since the subject had learnt
new copping skills for such circumstances , emphasizing that the hypnotic inductions and
treatments must be promising and real , as well
. additionally , from that time on , the
subject herself did explain the benefits of hypnosis for the others in the society .
participating actively in one s own healthcare is of prime importance to the person with
scleroderma .
it is equally important to cooperate and communicate effectively with the
doctor who is managing the disease .
while these two the person with scleroderma and the
doctor , are the focal point of the management team , multiple other individuals and
resources can be enlisted to form a health and support network ( 2 ) .
several skin conditions are well appreciated to resonate with the individual s mental
climate . among them , there is scleroderma , which is sensitive to psychological pressures
and possessing recognized immunological components . using hypnosis , both somatic and
psychological elements of scleroderma can be covered and influenced by appropriate
suggestions ( 4 ) . regarding the facts mentioned
, our hypnotic intervention could effectively cover the
complex issues related to the patient with scleroderma : somatically , to provide a suitable
situation for conduction of dental treatments , and psychologically , to return the patient
to an acceptable level of expectation , and acceptation .
this case report hopefully
lightened the missed , yet necessary chain of actions required the in treatment of any
psychosomatic impairment . | introduction : scleroderma , or systemic sclerosis , is a chronic connective tissue disease generally
classified as one of the autoimmune rheumatic diseasescase presentation : a 58-year - old woman was informed of her scleroderma , while receiving oral prosthetic
rehabilitation .
mouth opening limitation had unfortunately led to treatment
interruption .
the psychological load of the disease itself , along with dental treatment
cessation , resulted in patient s disappointment and social separation .
three hypnosis
sessions were conducted for normalizing patient s psychological status primarily , and
for the facilitation of dental treatment , as well . the range of mouth opening changed
from 3.7 to 6.2 cm , allowing for easier entrance of dental appliances into the mouth
for completion of prosthetic rehabilitation . with the tooth prostheses in place ,
the
patient gained more social confidence and started sharing her experience with other
patients with the same condition.discussion:this case illustrates that hypnosis therapy may prove beneficial for patients with
limited mouth opening secondary to scleroderma , facilitating the prosthetic
rehabilitation programs for both dental specialists and patients , with a secondary
important impact on the patient s social perception of himself . |
bloodstream infection is a leading cause of morbidity and mortality in neonatal intensive care units .
the microbiological diagnosis of sepsis in newborns ( nbs ) is a difficult task , because blood culture ( bc ) , which is currently the gold standard for diagnosing nbs ( 1 ) has the disadvantage of producing an elevated percentage of false negatives ( 2 ) .
previous studies have shown that 47.1% of nbs who begin antimicrobial treatment have negative culture results , and some analysis concluded that sepsis is diagnosed excessively ( 3 ) .
in addition , diagnoses based on clinical criteria and experience favor overdiagnosis and antibiotic abuse ( 4 , 5 ) . many evaluations of the available diagnostic tests have attempted to solve the problem described above , but their success has been limited ( 6 ) .
unfortunately , the decision to delay treatment until obvious signs and symptoms of sepsis occur , leads to the risk of preventable mortality ( 7 ) .
however , few studies have addressed the clinical impact of the use of lc - sf ( a technique of molecular biology for rapid detection of bacterial and fungal infections ) in nbs .
in the present study , we aimed to evaluate the accuracy of the lc - sf assay for detection of bacterial and fungal agents compared to bc and crp , using lc sf like a routine clinical practice in five hospitals .
we evaluated the impact of used lc - sf on some outcome variables of nbs with ns .
first , the study was performed prospectively in patients admitted to the central south hospital of petroleos mexicanos , the gynecological - obstetrics hospital number 4 of the mexican institute of social security ; the dalinde hospital ( private institution ) ; and the monterrey nuevo leon university hospital , and national institute of perinatology .
all patients with clinical sepsis suspiction and those presenting a score of > 8 points on the nosep-1 scale were included in the study .
nosep-1 scale is composed by c - reactive protein > 14 mg / dl ( 5 points ) , neutrophils > 50% ( 3 points ) , thrombocytopenia < 150 10/l ( 5 points ) , parenteral nutrition > 14 days ( 6 points ) , fever > 38.2c ( 5 points ) ( 8) ; it was previously validated for our population ( 9 ) .
after the nosep-1 punctuation criterion was satisfied , two blood samples were collected from each patient prior to antimicrobial treatment .
only one episode per patient was considered ; we did not exclude nbs who had previously used prophylactic or antimicrobial treatment ( figure 1 ) .
for the lc - sf test , dna was extracted ( using sterile technique ) from a 1-ml blood sample in a tube containing edta as an anticoagulant .
the sample was mechanically lysed using the magna lyser instrument ( roche diagnostics gmbh , germany ) .
the septifast prep mgrade kit was used to purify the dna , and the samples were incubated with a protease and a lysis buffer at 56 c to liberate the dna and protect it from dnases .
subsequently , the binding buffer was added , and the solution was transferred to a fibreglass column in which the human , bacterial and fungal genomic dna was retained while the remainder of the material was removed by two washes .
finally , the nucleic acids were eluted in 300 l of the solution and used as templates for the amplification reaction .
the lightcycler septifast test mgrade kit was used for amplification reactions containing taq polymerase and primers / specific probes designed to detect gram - positive bacteria , gram - negative bacteria and yeasts .
the purified dna samples and the other reaction components were added to the capillaries ( 100 l ) of the lightcycler mgrade , and the pcr amplification was performed with the lightcycler 2.0 equipment ( roche diagnostics gmbh , germany ) .
a melting curve was then generated for the controls and the amplified samples and was analysed automatically by specific identification software ( septifast identification software , roche diagnostics gmbh , germany ) .
each 1-ml sample was inoculated in a bact / alert bottle ( biomerieux sa ) and incubated using an automated system made by the same company .
the blood cultures were requested as part of the protocol to study children with suspected sepsis .
additionally , the impact of lc - sf results on hospitalization days , mortality , mechanical ventilation days and days of antibiotics use were analyzed retrospectively .
we compared 86 nbs with result of lc - sf vs 86 nbs collected from clinical records with nosocomial sepsis suspiction with bc results .
the demographic characteristics of the patients were analyzed using descriptive statistics , mainly averages and medians .
the efficacy of septifast for microbial identification was analyzed by calculating its sensitivity , specificity and predictive values using the blood culture like gold standard .
the concordance between the two types of microbial identification tests , blood culture and lc - sf was analyzed by calculating the kappa coefficient .
the demographic characteristics of the patients were analyzed using descriptive statistics , mainly averages and medians .
the efficacy of septifast for microbial identification was analyzed by calculating its sensitivity , specificity and predictive values using the blood culture like gold standard .
the concordance between the two types of microbial identification tests , blood culture and lc - sf was analyzed by calculating the kappa coefficient .
a total of 86 samples from 86 nbs with suspiction of nosocomial sepsis were included in the study .
fifty nine ( 68.6% ) patients were male and 27 ( 31.3% ) female . among the patients , 43.7% weighed > 1500 g , and 56.3% weighed < 1500 g. a gestational age of more than 35 weeks
was reached by 43.7% of the nbs , whereas 56.3% were born at or before 35th week .
results of 86 episodes ( paired blood samples tested by blood culture and septifast ) were evaluated .
13/86 ( 15.1% ) blood cultures were positive , and 34/86 ( 39.5% ) were positive with septifast tests ( table 1 ) .
the sensitivity of septifast test compared with blood culture was 0.69 , specificity 0.65 , positive predictive value 0.69 and the negative predictive value 0.65 .
results for isolates from all episodes are shown in figure 2 , compared with blood culture , septifast showed similar or higher percentages of isolates for all the studied microorganisms , with differences : e. coli ( 20% ) , k. pneumoniae ( 42.8% ) , scn ( 44.4% ) , pneumococcus ( 0% ) , s. bovis ( 0% ) , candida spp , enterobacter spp , e. faecalis and pseudomonas spp ( 100% ) .
polymicrobial infection rates were found in 4.6% . with septifast and 0% with blood culture ( table 2 ) .
kappa index concordance between lc - sf and bc was 0.21 ; while kappa index concordance between lc - sf and crp was 0.17 ( table 3 ) .
the response time was 6 - 8 hours using the lc - sf test and from 4 to 7 days using blood culture detection .
hospitalization days , days of antimicrobial use , and number of subjects died diminished meaningful ( table 4 ) .
negative results in both , septifast and blood cultures are not considered in the table ( n = 48 ) . the sensitivity of septifast test compared with blood culture was 0.69 , specificity of 0.65 , positive predictive value was 0.69 and the negative predictive value was 0.65 .
e coli , escherichia coli ; k pneumon , klebsiella pneumonia ; cns , coagulase - negative staphylococcus ; s bovis , streptococcus bovis ; e faecalis , enterococcus faecalis . using blood culture ,
8 gram - positive and 5 gram - negative bacteria were isolated , whereas dna sequences from 15 gram - positive bacteria , 18 gram - negative bacteria and 5 yeasts were identified using lc - sf . in two cases ,
lc - sf identified streptococcus pneumoniae , which is a rare infectious agent in our neonatal population . in both cases ,
the blood culture results confirmed the presence of s. pneumoniae five days after its detection with molecular biology .
abbreviations : bc , blood culture ; cf , concordance factor ; ea , expected average ; ki , kappa index;. oa , observed average .
the understanding of neonatal sepsis as a disease with a critical public health impact is based on the identification of factors that magnify the problem by favoring long hospital stays and increasing the use of diagnostic and treatment resources ( 10 ) .
the high degree of variation in clinical judgement adds to the difficulty of sepsis microbial identification and is a key reason behind overdiagnosis and the indiscriminate use of antimicrobials ( 11 ) . as the golden standard validation technique
, bc has poor sensitivity , favors confusion and contributes to the lack of diagnostic specificity that is characteristic of the systemic inflammatory response syndrome ( sirs ) in nbs .
the disadvantages of relying on bc to diagnose sepsis are exemplified by the adoption of terminology to justify antibiotic treatment , such as the concept of sepsis without an isolated germ applied to a child who fulfills the criteria for sirs but lacks an isolated microorganism .
the main consequence of this variability is the abuse of antibiotics in an effort to prevent mortality due to sepsis ( 12 ) .
lc - sf is a multiplex real - time pcr assay based on specific probes that detect and identify dna sequences from 25 microorganisms ( primarily gram - positive bacteria , gram - negative bacteria and yeasts ) that represent 90% of the species isolated from septic patients .
detection of virus or mycobacterias is not possible with lc - sf ; and the microbial sensitivity to antibiotics and antibiogram is not available .
this technique has led to improved rates of detection of the causative bacteria for diseases such as infectious endocarditis , sepsis in neutropenic patients with hematological malignancies , bacteremias and fungemias in specific groups ( 13 - 16 ) .
the reported sensitivity levels of 79 - 90.5% , specificity of 87% , negative predictive value of 83% and positive predictive value of 83% compared with blood culture ( 17 - 19 ) are higher than the values from the present study . a meta - analysis by chang et al .
concluded that the septifast assay had a sensitivity of 75% and a specificity of 92% for detecting bacteremia or fungemia ( 20 ) .
nonetheless , the identification capacity of septifast was greater than that of blood culture both in the overall data analysis and in the analyses of individual microorganisms , especially yeasts and pseudomonas .
the early detection of microorganisms using a technique based on the identification of nucleic acids will promote the prompt and appropriate use of antimicrobials , which decreases mortality .
the prompt detection of the causative agent is crucial for the proper treatment of an infection
first , we did not examine the possibility of contamination during the identification process , a factor that impacts the efficacy of most assays according to previous studies ( 21 ) , because the conditions for inclusion in our study ensured that none of the isolates were considered contaminated .
coagulase - negative staphylococci were considered possible agents of sepsis in these children because these bacteria are frequently isolated in mexican population ( 22 )
. additional support for the absence of contamination in the present work is found in another study that evaluated lc - sf and reported no cases of contamination ; those researchers applied the standardised criteria for sirs developed by a consensus of the american college of chest physicians / society of critical care medicine ( accp / sccm ) , which ensures compliance with practices that minimise contamination ( 17 ) .
previously an algorithm for interpretation of bc and lc - sf results was reported by lucignano b et al .
there were criteries to consider a pathogen and a contaminant ; these criteries were the same as in our research ( 23 ) .
second , some authors , such as lodes ( 17 ) have reported that the septifast test detects polymicrobial infection at rates as high as 11.4% , a higher ( 4.6% ) rate than that was found in our study .
this discrepancy might be explained by the lodes study s examination of surgical patients from an intensive care unit , where the polymicrobial infection rate is high .
unlike other studies , our findings influenced the treatments administered to our subjects , which substantiates our results .
as a prime example , the identification of positive fungal lc - sf results led to specific patient treatments , which resulted in clinical improvement . therefore , analyzing the follow - up care of our subjects enabled a more complete evaluation of the usefulness of the lc - sf test in terms of the length of hospitalisation , use of antimicrobials and duration of recovery . microbial identification in neonates is a relatively understudied topic that has generated some controversy because of possible incorrect decision making due to false positives .
therefore , the present work provides valuable support for the use of multiplex pcr as an identification method that can be performed together with bc , has the advantage of a 6 hour response time and allows the use of more targeted antimicrobial therapy .
similar to scenarios involving patients with negative blood culture results , when patients have negative lc - sf results but solid clinical data and a high suspicion of sirs , the clinical criteria will continue to be the mainstay for diagnosis and treatment . the initiation of antimicrobial treatment based on suspicion will most likely not decrease .
we predict that the only difference associated with lc - sf - based diagnosis will be a higher percentage of positive test results ( compared with bc ) and a more rapid response time leading to decision - making based on more solid evidence , which is the ultimate objective of auxiliary discriminatory tools .
it is highly unlikely that septifast ( or another biomarker ) will replace clinical criteria for diagnosis . | background : nosocomial sepsis ( ns ) in newborns ( nbs ) is associated with high mortality rates and low microbial recovery rates . to overcome the latter problem , new techniques in molecular biology
are being used.objectives:to evaluate the diagnostic efficacy of septifast test for the diagnosis of nosocomial sepsis in the newborn.materials and methods:86 blood specimens of nbs with suspected ns ( nosep-1 test > 8 points ) were analyzed using light cycler septifast ( lc - sf ) a real - time multiplex pcr instrument .
the results were analyzed with the roche septifast identification software .
another blood sample was collected to carry out a blood culture ( bc).results : sensitivity ( sn ) and specificity ( sp ) of 0.69 and 0.65 respectively , compared with blood culture ( bc ) were obtained for lc - sf .
kappa index concordance between lc - sf and bc was 0.21 .
thirteen ( 15.11% ) samples were bc positive and 34 ( 31.39% ) were positive with lc - sf tests.conclusions:compared with bc , lc - sf allows the detection of a greater number of pathogenic species in a small blood sample ( 1 ml ) with a shorter response time . |
adenoid cystic carcinoma ( acc ) is a malignant tumor characterized by perineural invasion , slow growth , and insidious destruction of surrounding tissues .
it usually arises in the major salivary gland , but also in minor salivary gland occasionally .
acc arising in the pterygopalatine fossa is extremely rare , only 3 cases have been reported . in these cases , 1 patient presented visual deficit because of neural invasion .
owing to its proximity to the superior orbital fissure and optic canal , neoplasms arising in the pterygopalatine fossa may cause optic neuropathy by direct invasion or oncothlipsis .
current literature has described possible visual recovery following excision of anterior clinoid mucocele , but few authors reported whether the visual deficit could be resolved after the resection of the tumor arising in the pterygopalatine fossa . in current study
, we described 1 patient with visual dysfunction induced by acc arising in the pterygopalatine fossa .
the zhengzhou university institutional research committee approved our study , and the participant signed an informed consent agreement .
a 44-year - old chinese man presented a mass in the palate for 7 months , there was no ulcer or pain .
one month ago , the visual acuity of the right eye dropped gradually and then there was no vision at diagnosis .
preoperative radiographs presented a huge mass in the pterygopalatine fossa and infratemporal fossa ( figure 1a ) , but we could not determine whether the optic nerve was invaded ( figure 1b ) . after explaining the possible risks including total blindness to the patient , a surgical plan was formulated .
and the patient reported the visual acuity had returned to normal at 10 days after the operation . at 2 months after discharge from the hospital
, there was no recurrence of the tumor ( figure 1c ) , and the patient was satisfied with the appearance and the functional results .
( a ) huge space - occupying lesion in the pterygopalatine fossa and infratemporal fossa was noted .
( b ) the tumor involved the orbital apex , but the association between the optic nerve and the tumor could not be justified clearly .
( c ) at 2 months after discharge from the hospital , there was no recurrence of the tumor .
the most distinctive biological behavior of acc was perineural spread . in previous case reports ,
all these findings supported the above - mentioned viewpoint . in current study , optic dysfunction was also noted , but it would be contributed by external compression rather than direct tumor invasion . various surgical approaches for the pterygopalatine fossa and infratemporal fossa have been advocated . it could be divided into 3 groups inferior approach , lateral approach , and anterior approach .
inferior approach could not provide adequate exposure of the retromaxillary and the skull base , and it was usually suggested for the tumor restricted in the parapharyngeal space .
lateral approach was limited for tumors extending medial to the pterygomaxillary fissure and especially those stretching across the medline .
the maxillary swing approach was first introduced by wei et al for recurrent nasopharyngeal carcinoma as a novel anterior approach . nowadays
, the route has been the preferable approach for tumors arising in the pterygopalatine fossa and infratemporal fossa .
compared to the facial translocation approach , the maxillary swing approach did not require neurorraphy and could avoid the development of a free facial graft by keeping the anterior maxilla and translocated hard palate attached to the cheek flap . in current study ,
possible postoperative complications such as palatal fistula , epiphora , and trismus have been reported .
the most interesting finding in such case was complete recovery of visual disorder following surgical resection of the disease .
suri et al have studied the visual outcome in patients with suprasellar tumors who experienced preoperative blindness , and the authors found male sex , shorter duration of blindness , operative evidence of hemorrhage in tumor , and soft tumor consistency were significantly associated with the visual outcome ; another research conducted by mathiesen et al presented early optic nerve decompression and primary tumors seemed to predict better visual prognosis .
similarly , kitano et al compared postoperative improvement of visual function among different approaches in treating suprasellar meningiomas , and the authors found extended transsphenoidal approach might resulted in improvements in visual acuity .
other associated factors , such as the size of mass , the pathology type , tumor consistency and , so on , have also been proven in the literature .
the same principle might also be compliant to the present case . in summary , acc arising in the pterygopalatine fossa
visual loss contributed by the tumor in the pterygopalatine fossa could recover in selected patients . | abstractadenoid cystic carcinoma ( acc ) arising in the pterygopalatine fossa was rare , only 3 cases have been reported . in previous literature , few authors reported whether the visual deficit could be resolved following the resection of the tumor.one patient with visual dysfunction induced by acc arising in the pterygopalatine fossa was reported.complete visual recovery was achieved following the operation . and the patient was satisfied with the appearance and the functional results in the follow-up.visual loss contributed by the tumor in the pterygopalatine fossa could recover in selected patients . |
lead poisoning has been recognized as a major public health risk , particularly in developing countries .
the release of lead into the environment from automobile emissions , lead - based paint or kitchenware , incineration of waste containing lead additives , and the burning of coal and its subsequent bioaccumulation has had profound public health implications .
air , dust , soil , drinking water , cosmetics , dietary and herbal supplements , and soiled parental work clothing are additional potential sources of exposure to lead for children and for all populations .
it has been shown that adulteration of lead opioids could result in severe lead toxicities .
lead affects major organ systems in the body including hematopoietic , gastrointestinal , respiratory , renal , nervous , and cardiovascular systems mainly through increased oxidative stress , ionic mechanisms , and apoptosis .
lead exposure occurs mainly through the respiratory and gastrointestinal systems , and it is stored in soft tissues as well as bones .
autopsy studies have suggested that 33% of the absorbed lead is stored in the liver .
ingestion of lead is one of the primary causes of its hepatotoxic effects .
currently , there are very few case reports , which have described lead induced hepatotoxicity in humans .
mokhtarifar and colleagues , reported the first case from mashhad last year , in which intrahepatic cholestasis caused by lead toxicity was described .
although , acute abdominal pain of unclear origin , caused by lead poisoning in patients with opium addiction , has been reported , we report a case with environmental lead toxicity with severe abdominal pain whose pain responded only to intravenous + intramuscular chelator therapy at a lead level that was lower than recommended level for parenteral chelator therapy in various references . according to toxicology consult , the patient was treated only with oral chelator agents as it was mentioned in the toxicology references for the lead level of our patient the low potent oral agent could not resolve the pain and as a result additional unnecessary .
a 19-years - old single woman presented to the emergency surgery department of ghaem hospital in mashhad , northeast iran with abdominal pain , icterus , and anemia .
there was no significant disease in her medical history but in her social history she had addiction to hookah .
she was a pilotage student and was working several hours of a day in the airport .
there was no history of drug consumption in the recent months to justify related abnormal liver function tests .
the patient had nausea and vomiting intermittently and also had anorexia but without significant weight loss .
the patient s physical examination revealed stable hemodynamic , and mildly icteric sclera . in abdominal examination ,
tenderness of llq ( left lower quadrant ) in deep palpation without rigidity , or rebound tenderness was revealed .
the other examinations such as chest and bowel sounds and also digital rectal examination were normal .
abbreviations : wbc : white blood cells ; rbc : red blood cells ; hb : hemoglobin ; hct : hematocrit ; mcv : mean corpuscular volume fbs : fasting blood sugar cr : creatinine ; ast : aspartate transaminase ; alt : alanine transaminase ; alp : alkaline phosphatase ; total bil : total bilirubin ; direct bil : direct bilirubin ; esr : erythrocyte sedimentation rate ; u / a : urine analysis ; hbs ag : hepatitis b surface antigen ; anti - hcv : antibody hepatitis c virus ; anti - hav : antibody hepatitis a virus ; ana : antinuclear antibodies ; asma : anti - smooth muscle antibodies ; ama : anti - mitochondrial antibodies ;
anti - lkm1 : anti - liver kidney microsome 1 antibodies ; p - anca : perinuclear anti - neutrophil cytoplasmic antibodies ; spep : serum protein electrophoresis ; nl : normal ; afp : alpha feto protein
plain abdominal radiography was normal . ultrasound study of the abdomen and pelvis revealed several lymphadenopathies in the right lower quadrant and anterior to right psoas muscle .
abdominal pain subsided with conservative management in the emergency surgery department , and the patient was referred to gastroenterologists for more evaluations because of abnormal liver function tests .
other potential causes of icter and abnormal aspartate transaminase and alanine transaminase including sepsis , viral hepatitis , autoimmune diseases , primary biliary cirrhosis , and primary sclerosing cholangitis were ruled out with proper laboratory and imaging modalities ( table 1 ) .
a new abdominopelvic sonography revealed normal liver and biliary tract except large amounts of sludge in the gallbladder .
terminal ileum was normal and biopsy of ileum revealed chronic inflammation , no villous atrophy , and no intraepithelial lymphocytosis .
according to hookah consumption and an unreliable history for addiction to opioids ,
the serum lead level was 415/6 g / l ( 41/5 g / dl ) with normal range of < 150 g / l , thus hematology and toxicology consult was performed .
causes of anemia including iron deficiency and hemolytic anemia were excluded by checking serum iron , total iron binding capacity ( tibc ) , ldh(lactate dehydrogenase ) , and reticulocyte count levels ( table 1 ) .
morphology of rbc in peripheral blood smear showed anisocytosis(+ ) , microcytosis(+ ) , hypochromia(+ ) , ovalocytosis(+ ) , and ostomatocytosis ( + ) .
basophilic stippling was also seen in the peripheral smear , which can be seen in lead poisoning ( figure1 ) .
toxicology consult was done , and serum lead level was rechecked , which revealed 537 g / l ( 53/7 mg / dl ) . because of the serum lead level that was between 40 to 70 g / dl and having considered the toxicology references only oral chelator ( succimer ) was recommended for the patient.the recommended dose was 10 mg / kg three times a day for five days , followed by 10 mg / kg twice a day for two weeks ( maximum 500 mg / dose ) .
despite oral chelator consumption
the pain continued , for which we decided to perform more evaluation for the patient .
we found mild serum amylase and lipase elevation in the repeated lab tests ( table 2 ) . and according to the presence of sludge in the abdominal sonography of gallbladder
the report read : there were multiple hyperechoic lesions and one small stone with posterior shadow inside the distal part of cbd .
because of these findings and the presence of severe abdominal pain , endoscopic retrograde cholangiopancreaticography ( ercp ) was done .
ercp revealed no dilation of cbd , so sphincterotomy was done and sludges were extracted .
despite these therapeutic managements , the abdominal pain deteriorated during the admission , and
gradually did not respond to ordinary analgesics , and only opioid analgesics could relief the pain in the last nights of admission in the gi ward .
surgical consultation was done , by which laparoscopy was recommended to approach to the unknown origin of continuous pain and also to the lymphadenopathies , which were noted in the abdominal sonographies .
its report read : small intestine from proximal to distal was investigated which was normal but some lymphadenopathies was seen in the mesenter .
stomach , liver , uterus , ovaries , were normal.cholecystectomy was done and biopsies obtained from the lymphnodes , liver and peritoneum .
pathological evaluation of the specimens revealed : 1-cholecystectomy : acute on chronic cholecystitis , 2-mesenteric lymph node : reactive , 3- wedge liver biopsy : without significant pathologic changes , 4-labeled as peritoneal biopsy : fibromuscular tissue .
conservative management was done after surgery , but the pain continued . because of the patient s job in the airport , consultation with occupational medicine specialist was done , who recommended re - evaluation for lead poisoning considering her work place that is a source of environmental lead poisoning and also history of substance abuse ( hookah addiction ) and unreliable history for probably opioid addiction . therefore the patient was admitted to toxicology ward .
at the toxicology ward ,
bll was rechecked , which was 54 g / dl . because of no response to previous oral chelator therapy in the same bll , and the severe suspicious to lead poisoning as the cause of the abdominal pain , intravenous ( iv ) and intramuscular ( i m ) chelator therapy were recommended .
dimercaprol or bal ( also referred to as british antilewisite ) in conjunction with calcium edta was started for the patient .
the recommended dose for bal was 4 mg / kg to be injected deep i m every 4 hours for 3 days , in conjunction with edetate calcium disodium with the second dimercaprol dose ; 25 - 50 mg / kg / day , iv for 5 days .
after 3 days of the treatment , clinical findings including nausea , vomiting , and abdominal pain subsided and finally the pain relieved dramatically .
the patient was discharged after 5days of bal prescription with maintenance therapy with oral succimer ( 10 mg / kg twice a day for two weeks ) .
the patient was recommended to avoid substance abuse ( hookah consumption ) , and also she was referred to occupational and environmental medicine specialists to evaluate the relationship between lead toxicity and her working environment .
she was recommended for monthly follow - up visits for six months ( table 2 ) .
abbreviations : alt , alanine amino transferase ; alp , alkaline phosphatase ; ast , aspartate amino transferase
this report is another piece of evidence showing the relationship between lead poisoning and acute abdominal pain .
ab - dominal pain and anemia have also been reported , particularly in areas where opioid use or air lead contamination is common .
despite of resolving the acute abdominal pain with oral chelators in previous case reports
, our case did not respond to oral chelator agents , hence underwent some unnecessary invasive evaluations .
but our patient responded dramatically to only 5 days iv+im chelators therapy in second toxicology consult .
despite of some references ( for example , goldfrank textbook of toxicology ) which recommend treatment for lead poisoning in upper lead levels ; we recommend more liberal chelator therapy in symptomatic patients with lead poisoning as is noted in the new version of up to date .
we also recommend parenteral chelator therapy , which is more potent than oral chelators in patients with severe signs and symptoms of lead poisoning , as it is mentioned in up to date .
there are several guidelines and recommendations for chelation therapy in lead poisoning , for excample ;
goldfrank , s textbook of toxicology emergencies recommends no treatment for patients with lead level below 70 mg / dl .
pranay et al recommended iv therapy is preferable for persons with blls of 70 g / dl or higher .18 use the combination of dimercaprol and cana2 edta with blls of 70 g / dl or higher and in the presence of lead encephalopathy . in adults , consider chelation therapy for patients with blls of 70 g / dl or higher .
also consider chelation therapy in symptomatic adults with blls exceeding 50 g / dl .
as mentioned above some authors recommend chelation therapy in higher bll , while internal medicine reference up to date recommends ; in general , chelation should be initiated for individuals with blls greater than 100 g / dl ( 4/83mol / l ) , and should also be considered for levels between 80 to 100 g / dl in asymptomatic individuals and for blls between 50 to 80 g / dl ( 2/41 to 3/86
in addition , sometimes consider chelation in persons with even lower blls [ eg , 40 g / dl ( 1/93mol / l ) ] if they have continued symptoms and elevated blls after two weeks of removal from exposure .
there are four major chelator agents that are used for treatment of lead poisoning :
succimer , or meso 2,3-dimercaptosuccinic acid ( dmsa ) , is an analogue of dimercaprol used in lead poisoning .
it has high sensitivity for lead , but its ability to chelate essential trace metals is low .
its main indication is for persons whose blls are 45 g / dl or higher .
rogan and colleagues noted that succimer was a water - soluble , oral chelating agent that was appropriate for use with blls higher than 45 g / dl .
d - penicillamine is a second - line oral chelating agent , although it is not approved by the us food and drug administration ( fda ) for use in lead poisoning .
it should never be used as the sole agent in patients manifesting with lead encephalopathy , because it does not cross the blood - brain barrier and can potentially lead to exacerbation of lead encephalopathy .
dimercaprol ( bal ) , which does cross the blood - brain barrier should be administered first .
dimercaprol ( bal ) , which is also referred to as british antilewisite is another parenteral chelating agent recommended as an agent of first choice for patients with lead encephalopathy . with high blls ( ie > 100 g / dl ) , it is used in conjunction with cana2 edta .
we noticed a significant difference in the blls in which the texts and guidelines recommend chelating therapy . despite the internal medicine reference up to date,20 which consider chelation in persons with even lower blls ( eg , 40 g / dl ) in some situations,20 toxicology and occupational and environmental medicine textbooks recommend chelation in higher blls .2,3,17,18
according to our experience on our case
, we finally recommend more liberal lead poisoning therapy in symptomatic patients as is noted in the new version of up to date and some guidelines .18,20,versus other texts that recommend treatment in higher lead levels .2,3,17
we also recommend parenteral chelator therapy in lower blls , instead of oral chelators in patients with severe signs and symptoms of lead poisoning , especially when oral chelators can not resolve the signs and symptoms .
| a 19-year - old woman was referred to the emergency surgery department with severe abdominal pain , icterus , and anemia .
the patient s clinical and paraclinical findings in addition to her occupational and social history , convinced us to assay blood lead level ( bll ) , which was 41/5 g / dl .
therefore toxicology consult was performed to treat lead toxicity .
recheck of the bll showed the level as 53/7 g / dl .
so oral chelator with succimer was started . despite consumption of oral chelator , there was no response and the pain continued . because our repeated evaluations were negative
, we decided to re - treat lead poisoning by intravenous and intramuscular chelators .
dimercaprol ( bal ) + calcium edta was started , and after 5 days , the pain relieved dramatically and the patient was discharged .
we recommend more liberal lead poisoning therapy in symptomatic patients , and also suggest parenteral chelator therapy , which is more potent , instead of oral chelators in patients with severe symptoms . |
papillary thyroid carcinoma ( ptc ) is the most common form of thyroid gland carcinoma which accounts for 1% of all human malignant tumors .
it was reported that the prognosis of ptc is excellent with a 10-year survival exceeding 90% .
however , approximately 2050% of ptc patients were found to have central lymph node metastases ( clnms ) [ 2 , 3 ] .
there is still a recurrence rate of 825% after primary surgical treatment , and many scholars hold the opinions that routine central lymph node dissection ( clnd ) may decrease the rate of recurrence .
this retrospective study was approved by department of breast - thyroid - vascular surgery at shanghai general hospital of nanjing medical university . from january 2013 to february 2015 , a total of 543 ptc patients who underwent radical operation of thyroid were enrolled in this study .
total thyroidectomy and ipsilateral clnd might be performed for unilateral ptc patients who met one or more of the following conditions according to the nccn guidelines : radiation history , known distance metastases , extrathyroidal extension , and tumor size > 4 cm in diameter .
other unilateral ptc patients underwent unilateral and contralateral partial resection of thyroid gland or hemithyroidectomy with ipsilateral clnd .
its upper bound was to the thyroid cartilage , and its lower bound was to thymus , and both sides were to the inner side of the central bilateral carotid sheath , including lymph nodes prior to cricothyroid membrane , lymph nodes surrounding thyroid , and paratracheal and prelaryngeal lymph node .
intraoperative frozen section ( fs ) is a popular method for doctors to determine surgical extent in surgery .
it was reported that the accuracy rates for fs in thyroid surgery was 97.8% . and
preoperative ultrasonography , preoperative thyroid function tests , and postoperative pathologic examination were performed in all patients .
based on the nccn guidelines for ptc , all of the 543 patients with clinically lymph node that was negative according to the preoperative physical examination and ultrasound underwent the surgical treatment .
79 patients underwent unilateral and contralateral partial resection of thyroid gland with bilateral / unilateral clnd .
six patients performed secondary surgical intervention because they were diagnosed as having benign lesions according to intraoperative fs , but the postoperative pathological diagnosis of the first surgery was ptc . in this study ,
the preoperative fine needle aspiration was carried out on 337 patients , and 322 cases were diagnosed with ptc . from a total of 543 patients ,
537 cases were diagnosed with ptc by the intraoperative fs , and 6 cases were diagnosed postoperatively after the histopathological examination of thyroid gland .
lt4 was given to all the patients as an alternative treatment besides the inhibition of tsh therapy .
chi - square test or fisher 's exact test was used to analyze the significance of differences of the categorical variables and logistic regression was performed to analyze the multiplicity .
all the data were processed using statistic package for social science ( spss 22.0 ) and p < 0.05 was considered as a statistically significant difference .
410 ( 75.5% ) females and 133 ( 24.5% ) males were included in this study .
the average age of all the patients was 47 years , ranging from 16 to 78 years .
the mean size of the tumors was 0.95 0.73 cm ( ranging from 0.1 cm to 5 cm ) .
132 ( 24.3% ) patients had bilateral lesions , 180 ( 33.1% ) cases had multifocal lesions , 517 ( 95.2% ) cases had tumor 0.25 cm in size , and 36 ( 6.6% ) cases had ptc with extrathyroidal extension . 190 ( 35.0% ) cases had ptc with hashimoto 's thyroiditis ( ht ) and 78 ( 14.4% ) cases had abnormal thyroid function .
we analyzed these factors including the gender , age , bilaterality , multifocality , tumor size , extrathyroidal extension , ht , and abnormal thyroid function to find the risk factors for ptc patients with clnms . in the univariate analysis ,
male gender ( p = 0.012 ) , age < 45 years ( p < 0.001 ) , bilateral lesions ( p = 0.042 ) , tumor size 0.25 cm ( p = 0.003 ) , and external invasion ( p = 0.003 ) were significantly associated with clnms .
multifocal lesions , ht , and abnormal thyroid function were not significant for clnms ( table 1 ) .
the multivariate analysis ( table 2 ) showed that the male gender ( p < 0.001 , or : 1.984 ) , age < 45 years ( p < 0.001 , or : 1.934 ) , bilateral lesions ( p = 0.006 , or : 1.585 ) , tumor size 0.25 cm ( p = 0.001 , or : 7.579 ) , and external invasion ( p = 0.002 , or : 2.370 ) were independent risk factors for clnms in ptc patients .
we grouped all the ptc patients into five groups based on the size of tumors : < 0.25 cm , 0.25 and < 0.5 cm , 0.5 and < 0.75 cm , 0.75 and < 1 cm , 1 cm . and the rate of clnms increased as the tumor size increased .
there were significant differences for these groups ( p < 0.001 ) ( table 3 ) . among the 543 cn0
table 4 showed that no clnms were found in all 7 males and 21 patients with unilateral lesion in cn0 ptc patients with tumor size < 0.25 cm .
the percentages of patients with clnms whose conditions met only zero , one , two , three , four , or all of the five risk factors were 0 ( 0/6 ) , 22.5% ( 39/173 ) , 43.7% ( 104/238 ) , 46.3% ( 50/108 ) , 77.8% ( 14/18 ) , and 0 . among the 6 patients without these five risk factors , 0% ( 0/6 )
papillary thyroid carcinoma which is considered to have relatively good prognosis still has at least 10% risk of recurrence in long - term follow - up [ 79 ] .
a large study found that the mortality of ptc patients with clnms was much higher than that of patients without clnms .
pellegriti et al . kept the option that the development of distant metastases was associated with the presence of lymph node metastases at presentation .
more and more scholars recommend that clnd is necessary for ptc patients because of the greater rate of clnms .
wang et al . reported that 44.1% cn0 ptc patients were found to have clnms .
considered that clnms were associated with local recurrence and distant metastasis but did not impair survival .
therefore , it is essential to investigate the indications for cn0 ptc patients . obviously , male gender , age < 45 years old , bilaterality , tumor size 0.25 cm , and external invasion were independent risk factors for cn0 ptc patients .
several studies demonstrated that male ptc patients have higher significant risk of clnms [ 15 , 16 ] .
however , jiang et al . found that the gender had no association with clnms . in our study , the proportion of the male gender with clnms was significantly higher than that of female gender ( 47.4% versus 35.1% , p < 0.001 , or : 1.984 ) . age is often used to judge the stage of the differentiated thyroid carcinoma .
we found that younger ptc patients ( < 45 years old ) were at higher risk of occurring clnms ( 46.6% versus 23.9% , p < 0.001 , or : 1.934 ) .
analyzed 916 cn0 ptc patients and had similar finding that the rate of clnms was considerably higher in the cases of younger patients ( p < 0.001 ; or : 2.357 ) .
jiang et al . believed that age 35 years was a good prognostic factor for ptc patients with clnms . in agreement with pellegriti et al . and vasileiadis et al .
, we found that bilateral lesions made a meaningful difference with a higher percentage to develop clnms from the unilaterality ( 45.5% versus 35.7% , p = 0.006 , or : 1.585 ) .
chang et al . and zhang et al . have demonstrated that larger tumor size ( > 0.5 cm , > 0.6 cm ) was associated with clnms . in our study ,
ptc patients with tumor size 0.25 cm were more prone to develop clnms ( 39.7% versus 7.7% , p = 0.001 , or : 7.579 ) .
many studies have demonstrated that external invasion is a risk factor of clnms for ptc patients [ 4 , 13 , 15 , 18 ] .
our study showed that nearly 61.1% percent of ptc patients with external invasion was found to have clnms and the multivariate analysis indicated that external invasion was an independent risk factor for clnms ( 61.1% versus 36.5% , p = 0.002 , or : 2.370 ) . with the development of ultrasound , palpation , fine needle aspiration ( fna ) , and intraoperative frozen section , increasing small malignant thyroid nodules
were checked out . before the dispute regarding [ 20 , 21 ] the operation for ptc patients with tumor size 1 cm , studies shall need to advance . in this study ,
the rate of tumor size with clnms increased , obviously , with the increase of tumor size in a certain range ( < 0.25 cm : 7.6% , 0.25 and < 0.5 cm : 22.7% , 0.5 and < 0.75 cm : 30.1% , 0.75 and < 1 cm : 45.5% , 1 cm : 53.4% ) .
7.6% of ptc patients with tumor size < 0.25 cm were confirmed to have metastases in central lymph node , while about 39.7% of ptc patients whose tumor size was 0.25 cm were confirmed to have clnms .
the incidence of clnms in ptc patients with tumor size 0.25 and < 0.5 cm was 22.7% . and
the incidence of clnms in ptc patients with tumor size < 0.5 cm was reported to be 20.626.2% [ 13 , 15 , 17 ] .
therefore , we think that clnd should be performed in ptc patients with tumor size 0.25 cm . in this study ,
similar studies in cn0 ptc patients reported that the incidence of clnms was 35.043.6% [ 15 , 16 , 22 ] .
the clinic risk factors for ptc patients with clnms were analyzed above , and we also evaluated the incidence of clnms with every conceivable combination to further analyze the indication of clnd . eliminating tumor size 0.25 cm , no clnms were found in male patients and patients with unilateral lesion . in the present studies ,
clnd is necessary to be performed in the ptc patients because clnms are associated with a higher risk of recurrence [ 23 , 24 ] .
hence , clnd should be performed in the patients with risk factors , and we recommend that , in cn0 ptc patients with tumor size < 0.25 cm , male patients and patients with unilateral lesion should be considered safe . in this study , some limitations should be considered .
secondly , all patients are required to have long - term follow - up to analyze the significance of clnd for cn0 ptc patients .
in summary , the incidence of ptc patients with clnms is 38.1% in our study .
male gender , age < 45 years , bilaterality , tumor size 0.25 cm , and external extension were considered as independent risk factors for ptc patients . on this account
, we think precautionary clnd should be considered for cn0 ptc patients with risk factors . and we believe that , in cn0 ptc patients with tumor size < 0.25 cm , male patients and patients with unilateral lesion could avoid clnd . | background . papillary thyroid carcinoma ( ptc ) with central lymph node metastases ( clnms ) is common .
the objective of this study was to investigate the incidence and risk factors of lymph node metastasis patients with ptc .
patients and methods . between january 2013 and february 2015 ,
a retrospective study of 543 patients with ptc undergoing hemithyroidectomy or total thyroidectomy with routine central lymph node dissection ( clnd ) was analyzed .
clinicopathologic risk factors for clnm were studied using univariate and multivariate analysis by spss 22.0 software .
results .
the incidence of clnms in ptc patients was 38.1% ( 207/543 ) . in the multivariate analysis , male gender ( p < 0.001 , or : 1.984 ) , age < 45 years (
p < 0.001 , or : 1.934 ) , bilaterality ( p = 0.006 , or : 1.585 ) , tumor size 0.25 cm ( p = 0.001 , or : 7.655 ) , and external extension ( p = 0.001 , or : 7.579 ) were independent risk factors of clnms .
furthermore , in ptc patients with tumor size < 0.25 cm , all 7 males and 21 patients with unilaterality were not found to have clnms .
conclusions .
clnms are prevalent in the ptc patients with the following risk factors : male gender , age < 45 years , bilaterality , tumor size 0.25 cm , and external extension .
ptc patients with tumor size < 0.25 cm , male patients , and patients with unilateral lesion could be considered safe from clnms . |
fasting during ramadan , the 9th month in the islamic calendar , is not mandatory for patients with diabetes mellitus ( dm ) , but many insist on fasting .
glucose - lowering therapies are cornerstone for treating all type 2 dm patients to ensure tight glycemic control to prevent acute complications like hyperosmolar nonketotic coma and chronic complications such as the micro- and macrovascular complications .
hypoglycemia is the most serious and fatal complication for fasting and for many treatment options for diabetes , such as insulin and some of the oral glucose - lowering therapies , including sulfonylurea ( su ) and meglitinides [ 2 , 3 ] . in the last decade
new classes of glucose - lowering therapies associated with reduced risk of inducing hypoglycemia have been introduced .
these include incretin mimetics , such as dipeptidyl peptidase-4 ( dpp-4 ) inhibitors , glucagon - like peptide-1 receptor agonist ( glp-1 ra ) , and the sodium - glucose cotransporter-2 ( sglt-2 ) inhibitors [ 4 , 5 ] .
there have been few review studies of the use of these new glucose - lowering therapies during ramadan .
most focus only on one class of glucose - lowering therapies [ 6 , 7 ] .
one review discussed the benefits and drawbacks for many classes of newer glucose - lowering therapies but did not include information about sglt-2 inhibitors .
furthermore , that study did not provide a conclusion on which medication is the best to be used during ramadan by patients with type 2 dm .
this study reviews the safety and efficacy of newer glucose - lowering therapies in order to identify those that are most suitable for patients with dm during the fasting month of ramadan .
this study was accomplished during september 2015 through a careful literature search using ( pubmed , pubmed central , and google scholar ) for studies from 2005 to 2015 with the one or more of following keywords in english language : diabetes dpp-4 inhibitor ( alogliptin , linagliptin , saxagliptin , sitagliptin , and vildagliptin ) , glp-1 ra ( exenatide , liraglutide , albiglutide , and lixisenatide ) , and sglt-2 inhibitors ( canagliflozin , dapagliflozin , ipragliflozin , and empagliflozin ) , in combination with the essential keyword ( ramadan ) .
all study types ( prospective observational , randomized blinded clinical trials and randomized open - label trials ) that examined the efficacy and side effects of these classes of glucose - lowering therapy on patients with type 2 dm during the fasting month of ramadan were included .
information from these studies were summarized in relation to study design , duration of study , number of participating patients , medications used , assessment criteria for medication safety and effectiveness , and final conclusions .
full text was obtained in nine studies , abstract in four studies , and posters in three studies .
eight studies were randomized clinical trials ( rct ) and eight were prospective observational studies .
information about each class of glucose - lowering therapies was summarized according to the medication used in each class and whether this medication was studied as monotherapy or as add - on therapy to other glucose - lowering therapies .
the dipeptidyl peptidase-4 ( dpp-4 ) inhibitors are a new class of oral glucose - lowering therapies for type 2 dm treatment .
they act by inhibiting the breakdown of glp-1 , increasing its systemic concentration which leads to a significant increase in endogenous insulin secretion and a decrease in glucagon secretion .
they have a glucose dependent mechanism of action , leading to a lower incidence of hypoglycemia .
in clinical practice dpp-4 inhibitors are associated with 0.60.7% reductions in glycosylated hemoglobin ( hba1c ) without causing weight gain [ 9 , 10 ] .
the first dpp-4 inhibitor , sitagliptin , was approved 10 years ago by united states government 's food and drug administration ( fda ) . since
then many dpp-4 inhibitors such as vildagliptin , saxagliptin , linagliptin , and alogliptin have been approved and are available in the pharmaceutical market .
vildagliptin and sitagliptin are the most frequently studied dpp-4 inhibitors for control of type 2 dm during ramadan . unfortunately vildagliptin is not available in usa .
there are only few studies that assess the benefits of vildagliptin as monotherapy for type 2 dm patients maybe because of its unlicensed use as monotherapy
. in nonfasting patients vildagliptin has nearly similar effectiveness to su in lowering hba1c but with less risk to induce hypoglycemia . in ramadan - fasting type 2
one of these studies was a small scale , multicenter , open - label , 4-week , observational study for 97 indian patients with type 2 dm who were fasting during ramadan .
patients were divided into two groups at which in one group 55 patients were given vildagliptin and in the other 42 patients were given su .
the incidence of hypoglycemia ( defined as blood glucose level less than 70 mg / dl ; 3.9 mmol / l ) , which was assessed depending on patient symptoms and confirmed by measuring blood glucose level , was lower in the vildagliptin group than in su group ( 0% versus 4.8% ; p = 0.104 ) .
hba1c was decreased in vildagliptin group while there was a slight increase in su group ( 0.43% versus 0.01% ; p < 0.05 ) .
more patients in the vildagliptin group achieved hba1c < 7.0% than in the su treated group ( 16.4% versus 4.8% ; p = 0.055 ) .
patients in the vildagliptin group lost an average of 1.2 kg while those in su group lost an average of 0.03 kg ( p < 0.001 ) .
although vildagliptin was shown to be safer than su in this study , this superior safety was lacking statistical significance , perhaps due to the small sample size . in another large , multiregional ,
observational study that was conducted in asia and the middle east , 1315 type 2 diabetic muslim patients were divided into two groups where 684 patients had received treatment with vildagliptin and 631 patients received su ( glibenclamide , glimepiride , gliclazide , or glipizide ) as monotherapy or as add - on to metformin .
vildagliptin was significantly more effective in reducing hba1c than su ( 0.24% versus 0.02% ; p < 0.05 ) .
also , vildagliptin was associated with significantly fewer episodes of hypoglycemic events ( defined as patient reported symptoms and/or blood glucose level less than 70 mg / dl ; 3.9 mmol / l ) in comparison with the su therapy ( 5.4% versus 19.8% ; p < 0.05 ) .
this large study confirmed that vildagliptin had significantly higher effectiveness and safety when compared to su .
these two studies showed that the risk of hypoglycemia in patients using vildagliptin is around one - third to that in patients using su . in summary ,
the use of vildagliptin 50 mg twice daily as monotherapy for ramadan fasting patients is more effective than su to control blood glucose level ( through hba1c reduction ) and body weight .
there are many studies examining vildagliptin as add - on therapy for both fasting and nonfasting patients . in nonfasting patients it was found that vildagliptin when used as add - on therapy to metformin has comparable efficacy to different su ( glimepiride and gliclazide ) but with less risk to induce hypoglycemia [ 15 , 16 ] . in ramadan fasting patients , three studies assess vildagliptin in ramadan fasting type 2 dm patients .
the 1st study in this regard was a small scale study that was conducted in london and included 52 patients with type 2 dm who were already using metformin ( 2 g / day ) ; these patients were randomized equally into two groups where half of them were given vildagliptin 50 mg daily and the other half were given gliclazide 160 mg twice daily in addition to their primary therapy .
hypoglycemic events ( defined as blood glucose < 63 mg / dl ; 3.5 mmol / l with or without symptoms ) significantly occurred less frequently for patients in the vildagliptin group than in the gliclazide group ( 7.7% versus 61.5% ; p 0.001 ) .
the lack of significant difference in the effectiveness between vildagliptin and gliclazide in this study may be due to short period ( 24 days ) of follow - up besides the small sample size which further compromises statistical significance .
another small prospective , 16-week , multicenter study was conducted in uk for patients who were using either vildagliptin 50 mg twice a day ( 30 patients ) or gliclazide ( 41 patients ) as add - on therapy to metformin during ramadan fasting .
there were no hypoglycemic events ( defined as blood glucose level less than 70 mg / dl ; 3.9 mmol / l ) for patients in the vildagliptin group while 44.4% of the patients in the su group suffered from hypoglycemic events .
there was significantly greater hba1c reduction for those taking vildagliptin ; however this higher effectiveness may be attributed to higher adherence rate by patients using vildagliptin than for those using gliclazide . on the other hand the high missing rate among patients using gliclazide
is a confirmation to the finding of this study in that vildagliptin despite regular usage resulted in fewer hypoglycemic events . in 2014
a randomized , open - label , clinical trial was done on 69 patients who were on a combination of metformin and su ( glimepiride or gliclazide ) .
patients were divided into two groups : a control group in which patients were maintained on their usual treatment regimen with dose adjustment for the fasting period and a study group at which patients were switched from su to vildagliptin 50 mg twice daily in combination with metformin .
there was no difference in the effect of vildagliptin and su on the calculated change in hba1c .
the incidence of hypoglycemia , which was confirmed by measuring blood glucose level , during ramadan was higher in the su group ( 26 episodes versus 19 episodes ; p = 0.334 ) . the number of patients who had medication noncompliance because of fasting discomfort was higher in the su than in the vildagliptin group .
all the previously discussed small scale studies [ 17 , 18 , 20 ] concluded that vildagliptin as add - on therapy to metformin was at least as effective as su with a lower risk to induce hypoglycemia for type 2 dm patients during ramadan .
another large study was a prospective , observational , 14-week study design for 198 stable patients on dual oral therapy for 2 months and with hba1c 8.0% .
83 patients were in the metformin - sulfonylurea / glinide ( is ) cohort and 115 patients were in the metformin - vildagliptin cohort .
mmol / l ) were confirmed in 30.8% of the is cohort and 23.5% in the vildagliptin cohort ( p > 0.05 ) , while severe hypoglycemia and/or unscheduled medical visit due to hypoglycemia occurred in 10.4% of the is cohort and in 2.6% of vildagliptin cohort ( p = 0.0029 ) .
compliance with fasting was higher , as well as adherence to drug therapy in vildagliptin cohort , with 5 missed doses for 15.4% of is , compared to 8.5% only in patients using vildagliptin . in this
study the nonsignificant difference in the total hypoglycemic episodes between vildagliptin and su / glinide which was different from the finding of previous studies at which vildagliptin was less likely to induce hypoglycemia than su , maybe due to the usage of glinide in some patients but unfortunately their number was vague which keeps the final conclusion about the hypoglycemic risk of glinide in comparison with vildagliptin unknown .
another study , virtue study was a multicenter , prospective , 16-week observational study that enrolled 244 pakistani patients with type 2 dm .
all included patients were already treated with vildagliptin ( n = 121 ) or su ( n = 121 ; 67% on glimepiride , 14% on gliclazide , 18% on glibenclamide , and 1% on glipizide ) as add - on to metformin or as monotherapy for at least 4 weeks .
patients in the vildagliptin group experienced at least one episode of hypoglycemia ( defined as blood glucose measurement 70.2 mg / dl ; 3.9 mmol / l ) less frequently than patients in the su group ( 5.8% versus 14.2% ; p < 0.033 ) .
the reduction in hba1c was greater with vildagliptin than in su ( 0.3% versus 0.1% ; p < 0.054 ) .
a reduction of 0.3 kg in body weight was seen with vildagliptin treatment versus 0.2 kg weight gain in the su group .
overall adverse events ( hypoglycemia , nausea , vomiting , abdominal pain , and abdominal discomfort ) were less frequently reported in vildagliptin cohort than in the su group ( 15.7% versus 17.4% ; p = 0.729 ) .
hypoglycemic events were significantly less common in vildagliptin than in su group ( 5% versus 13.2% ; p = 0.024 ) .
git side effects including abdominal pain , nausea , and vomiting are more common in vildagliptin than in su group ( 10.8% versus 2.5% ; p < 0.05 ) ; these side effects may be symptoms for acute pancreatitis which is a rare but serious side effect of vildagliptin therapy .
however , large , multiregional , randomized studies are required to confirm the safety of vildagliptin on git system and/or pancreatitis when used as add - on therapy to metformin for ramadan fasting patients since this study was just an observational study on a limited number of pakistani patients only .
more recently a steadfast study was a multiregional , randomized , double - blinded study for 557 patients with type 2 dm who were previously treated with metformin and any su were randomized to receive vildagliptin 50 mg twice daily or gliclazide plus metformin .
the percent of patients reporting confirmed hypoglycemia ( blood glucose less than < 70.2 mg / dl ; 3.9
mmol / l ) were lower with vildagliptin than the gliclazide ( 3% versus 7.0% ; p = 0.039 ) .
there was a nonsignificant difference in effectiveness of vildagliptin versus gliclazide according to the adjusted mean change in hba1c ( p = 0.165 ) .
also there was a nonsignificant difference between vildagliptin and gliclazide on body weight change ( p = 0.987 ) .
overall safety ( measured by adverse effects on all body organs ) was similar between the treatments .
the randomized design of this study and its large sample size lead to a less biased conclusions and a thus form a strong evidence for the lower risk of hypoglycemia with comparable safety of vildagliptin when compared to gliclazide .
this finding occurs in contrast to the findings of virtue study which assume that git upset is higher by vildagliptin than in su ( the majority of patients were on glimepiride ) while in steadfast study vildagliptin has similar incidence of git side effects to gliclazide and since the incidence of git side effects is higher by glimepiride than gliclazide ; then it can be concluded that vildagliptin has at least comparable safety to su . in summary ,
the usage of vildagliptin in fasting patients as add - on therapy to metformin has comparable safety and effectiveness to su but with a lower tendency to induce hypoglycemia .
there is a complete absence for studies that evaluate the effect of sitagliptin as monotherapy for patients with type 2 dm during ramadan but there are many studies on the usage of sitagliptin as add - on therapy to other glucose - lowering therapies , specifically metformin . in a pilot prospective study involving 15 patients ,
a combination of sitagliptin with metformin was safe ( not associated with hypoglycemic events ) for ramadan fasting patients with type 2 dm ; however , the small sample size and the funding of this study by a pharmaceutical company that manufactures sitagliptin make it difficult to draw an accurate conclusion regarding the benefits of using sitagliptin during ramadan .
sitagliptin had been also studied in large , randomized , open - label multinational study that was done on 1021 type 2 dm patients who intended to fast during ramadan and were already treated with stable doses of su ( 35% glibenclamide , 35% glimepiride , or 30% gliclazide ) and metformin for at least 3 months before screening .
patients were randomized to either receive sitagliptin 100 mg / day plus metformin ( n = 507 ) or remain on their prestudy treatment ( n = 514 ) .
there was a nonsignificant difference in the incidence of symptomatic hypoglycemia ( based on patient reported symptoms and confirmed by blood glucose level less than 70 mg / dl ; 3.9 mmol / l ) for patient in the gliclazide group when compared with those in the sitagliptin ( 6.6% versus 6.7% ; p > 0.05 ) group , while a significant difference in the incidence of hypoglycemia occurs between patients using sitagliptin and those using glibenclamide and glimepiride .
furthermore , sitagliptin appeared to induce side effects to a lesser extent than su ( 3 versus 9 patients , resp . )
; all the side effects in sitagliptin group were not serious and include constipation , vomiting , or hyperglycemia , while three patients in su group developed serious problems like ischemic stroke , acute pancreatitis , and urinary tract infection . in this study
although there was a nonsignificant difference in the incidence of hypoglycemia between gliclazide and sitagliptin , gliclazide was associated with lower risk of hypoglycemia when compared to sitagliptin ; unfortunately authors in that study did not classify patients who are using gliclazide according to the used dosage form ( sustained and immediate release ) , so it is difficult to conclude that whether this lower incidence of hypoglycemia by gliclazide when compared to sitagliptin is related to specific dosage form of gliclazide or due to gliclazide itself .
another multicenter , randomized study is involving 848 ramadan fasting patients with type 2 dm , who were already treated with a stable dose of su ( 65% glimepiride , 22% glibenclamide , and 13% gliclazide ) with or without metformin ( 86% and 14% , resp . ) for 3 months and had hba1c 10% .
patients were divided into two groups : 421 patients were switched from su to sitagliptin 100 mg once daily and 427 patients remained on su .
the proportion of patients who recorded 1 symptomatic hypoglycemic event during ramadan was lower for patients in sitagliptin than in su group ( 3.8% versus 7.3% ; p = 0.028 ) .
the incidence of symptomatic hypoglycemia was the lowest in patients using gliclazide ( 1.8% ) , then sitagliptin ( 3.8% ) , then glibenclamide ( 5.2% ) , and finally glimepiride ( 9.1% ) .
the proportion of patients experiencing adverse effects other than hypoglycemia was 10.0% versus 7% in the sitagliptin and su group , respectively .
the major limitation for this study was the absence of medication efficacy assessment through measuring glycemic control and body weight .
the assumption of this study in that sitagliptin is safer than su in regard to hypoglycemia was not accurate since incidence of hypoglycemia is lower in gliclazide than in sitagliptin .
the finding of this study may provide confirmatory evidence to the finding of al sifri et al . which found a less likely risk of hypoglycemia by gliclazide when compared to sitagliptin .
furthermore , failure to assess the effect of sitagliptin versus su on glycemic control and weight for fasting patients in these studies can be considered as main limitation in drawing a reliable conclusion regarding the usage of sitagliptin during ramadan [ 2628 ] . in summary ,
the studies that evaluate the usage of sitagliptin for patients with diabetes during ramadan found that it is associated with reduced risk of hypoglycemia when used as add - on therapy to metformin compared to two sus ( glimepiride and glibenclamide ) and a slightly higher risk of hypoglycemia than gliclazide ; this difference may be attributed to the higher degree of selectivity in pancreatic receptor stimulation by gliclazide while in nonfasting patients the risk of hypoglycemia was lower in sitagliptin than su .
other rare side effects including gastrointestinal ( git ) side effects ( vomiting , constipation , and abdominal pain ) and central nervous system ( cns ) side effects ( headache , dizziness , and decreased concentration ) appeared to occur at higher percent in sitagliptin than in su treated patients .
one of the major limitations in all of these studies about the usage of sitagliptin during ramadan is that they did not focus on sitagliptin effect to control blood glucose level , so further studies are needed in this regard to find out whether sitagliptin benefit is limited to less risk of hypoglycemia or extends beyond that to include a better glycemic control than su for patients with type 2 dm during ramadan .
till the time of collecting the data for this review there are no any study ( 20052015 ) that had been evaluating the effect and side effects of using dpp-4 other than vildagliptin and sitagliptin like linagliptin , saxagliptin , and alogliptin during ramadan .
it seems that the differences are negligible regarding efficacy and incidence of hypoglycemia among all dpp-4 inhibitors .
so it may be reasonable for researchers to investigate the benefits of other new dpp-4 inhibitors in countries at which vildagliptin and sitagliptin are not available .
glucagon - like peptide-1 receptor agonists ( glp-1 ra ) act by binding to and activating the glp-1 receptor , resulting in glucose dependent increase in insulin secretion and decrease secretion of glucagon , so they are effective in decreasing blood glucose levels associated with reduced risk of hypoglycemia .
they also act to delay gastric emptying and increase satiety ; thereby they are effective in reducing body weight for diabetic patients .
the approved agents in this class include exenatide , liraglutide , albiglutide , and lixisenatide .
all of these agents are administered by subcutaneous injections with mainly git side effects such as nausea , vomiting , and diarrhea in addition to injection - site reaction [ 31 , 32 ] .
exenatide and liraglutide are the most frequently studied glp-1 ras for patients with diabetes during ramadan .
although there are many studies that examine the effect of exenatide as add - on therapy in nonfasting patients [ 33 , 34 ] , which showed that exenatide was effective to lower hba1c with less hypoglycemic risk , only few studies examined exenatide usage for ramadan fasting patients .
one pilot study for 34 patients with type 2 dm who are using different pharmacological treatment ( insulin and oral glucose - lowering therapies ) and are wishing to fast during ramadan was observed .
, it was found that neither of the two patients had experienced a hypoglycemic event ( defined as blood glucose less than 70 mg / dl ; 3.9 mmol / l ) even without exenatide dose adjustment .
however , it is so difficult to ascertain this result because of the limited sample size . in another study ,
exenatide when used as add - on therapy to metformin was associated with reduced risk of hypoglycemia when compared to a combination of metformin and gliclazide for patients with type 2 dm who fast during ramadan .
one limitation of this study is inability of the author to retrieve the full article . in above 2 studies regular exenatide was assessed while the usage of sustained release exenatide during ramadan was not assessed . in summary ,
regular exenatide is not associated with hypoglycemia when used for type 2 dm patients during ramadan ; however it is difficult to recommended the use of exenatide for fasting patients until further studies performed because the current studies are small scaled studies , with major focus on hypoglycemic side effects without focusing on the efficacy of exenatide to control blood glucose level during ramadan .
liraglutide was shown to have comparable efficacy to su in lowering hba1c but with less risk of hypoglycemia when used in nonfasting patients [ 37 , 38 ] . during ramadan
many studies assess the benefits and drawbacks for liraglutide usage ; one of the earliest studies in this regard was the treat 4 ramadan trial , which was a randomized , controlled clinical trial comparing liraglutide to su ( gliclazide 88% , glimepiride 10% , or glibenclamide 2% ) as add - on therapy to metformin in 99 adult patients with type 2 dm in uk .
after 12 weeks , patients in the liraglutide group and not those in the su group had a reduction in hba1c ( 0.3% versus 0.02% ; p = 0.06 ) .
liraglutide resulted in greater and significant reductions in both weight and diastolic blood pressure ( bp ) for patients with dm than su .
self - recorded episodes of hypoglycemia ( blood glucose 70.2 mg / dl ; 3.9 mmol / l ) were significantly lower with liraglutide ( p < 0.0001 ) .
the major limitation in this study was the reliability of the method that was used to calculate hypoglycemia ( patient self - record method ) .
lira study was an open - label , multinational randomized clinical trial involving 343 people ( 172 on liraglutide and 171 on su ) for a 33-week duration .
this study included type 2 dm patients with an intent to fast during ramadan , with hba1c 710% , and being treated with a combination of metformin and su ( at maximum tolerated dose ) .
study participants were randomized to either switch from su to liraglutide 1.8 mg once daily or continue pretrial su .
7% with no confirmed hypoglycemic events ( defined as blood glucose less than 70 mg / dl ; 3.9 mmol / l ) compared with su ( 53.9% versus 23.5% ; p < 0.0001 ) .
moreover , people treated with liraglutide experienced significantly greater weight loss ( p < 0.0001 ) and greater improvements in hba1c ( 1.24% versus 0.65% ; p < 0.0001 ) than those treated with su .
the incidence of patients experiencing adverse events ( ae ) during ramadan was similar in the liraglutide and su groups ( 23.7% versus 20.9% ; p > 0.05 ) ; meanwhile gastrointestinal side effects ( nausea , diarrhea , vomiting , abdominal pain , and abdominal distension ) occurred more commonly with liraglutide treatment ( 10.5% versus 3.7% ) . the results of this study were more reliable and add a confirmation to the findings of treat 4 trial because of its randomized design with larger sample size at which in both studies liraglutide showed a significantly better efficacy than su with a lower incidence of hypoglycemia . in summary , liraglutide usage during ramadan for patients with type 2 dm may be reasonable because it is associated with better glycemic control , improved body weight , and less hypoglycemic episodes when compared with su ; however , it should be used with caution because of its git side effects , which may negatively affect a fasting patient , since git problems may occur more frequently during ramadan .
studies for other glp-1 ra like albiglutide and lixisenatide during ramadan were lacking which may be attributed to their recent approval by fda and it may be possible to find such trials in the near future .
sodium - glucose cotransporter-2 ( sglt-2 ) inhibitors are the most recent class of oral glucose - lowering therapies that are used for treating patient with type 2 dm .
these drugs act to lower blood glucose level by decreasing renal glucose threshold through their effect to induce a competitive inhibition on the sglt-2 in the kidney which is responsible for reabsorption of 90% of filtered glucose by the kidneys and thus block the reabsorption of glucose .
the risk of inducing hypoglycemia is low with sglt-2 inhibitors because of their insulin - independent action and hence forms an attractive class for managing patients with type 2 dm during ramadan .
however , caution is recommended while using these medications because of their ability to cause dehydration , especially in the setting of absence of fluid intake , which occurs during fasting hours of ramadan [ 42 , 43 ] .
recently fda added many warning on such class of medication because of their risk to induce ketoacidosis and increase risk of foot and leg amputation , serious urinary tract infections , acute renal failure , and osteoporosis .
there are some studies that assess dapagliflozin as add - on therapy in nonfasting patients ; in all of these studies dapagliflozin was shown to have comparable efficacy to su with lower hypoglycemic risk .
to date , there is only one study that evaluates dapagliflozin during ramadan which was a 12-week , randomized , open - label study for 110 patients with type 2 dm who were already using metformin and su .
patients were divided into two groups : in the 1st group 58 patients were switched from su to dapagliflozin 10 mg once daily while in the 2nd group 52 patients were remained on their pretrial treatment .
dehydration was defined as a loss of 1.8% of body weight/13 hours of fasting daily .
dehydration was further assessed by using urine and blood tests , with physical examination and a specific set of questions to the patients about their medical history while using this medication .
there was no significant difference in the incidence of dehydration between dapagliflozin and su ( 73.1% versus 81.6% ; p = 0.258 ) ; this may be because already most of ramadan fasting persons without regard to their disease or medication status suffer from dehydration due to long period ( 1222 hour ) of abstinence from foods and water during ramadan .
there were significantly more patients in the dapagliflozin group ( 43.1% versus 23.1% ; p = 0.026 ) than in su group complained from thirst sensation .
additionally , there was a significantly higher mean for haematocrit level ( p = 0.009 ) , urine osmolarity ( p = 0.001 ) , and blood ketone ( p = 0.002 ) in dapagliflozin group ; however , there was a lack of information regarding the development of ketoacidosis in participated patients of this study .
furthermore , there was a significantly lower mean of urinary sodium ( p < 0.005 ) in dapagliflozin group when compared to su group ; however authors of that study postulated that dapagliflozin does not pose a higher risk of dehydration during ramadan .
assessment of dapagliflozin effectiveness to reduce hba1c and its risk to induce hypoglycemia was the major limitation in this study which may be because the main aim of this study was to assess the safety of dapagliflozin on excessive water excretion . in summary
, there are a limited number of trials regarding dapagliflozin and other sglt-2 inhibitors in type 2 dm patients during ramadan .
the available data showed that although dapagliflozin is not associated with increased risk of dehydration , it increases thirst sensation for patients with type 2 dm during ramadan , which may negatively affect patient compliance to continue dapagliflozin usage during ramadan .
so it is recommended not to use sglt-2 inhibitors for fasting patients with type 2 dm until performing further studies that compare the effect of dapagliflozin or other sglt-2 inhibitors with other oral antidiabetic medications on controlling blood glucose level , their risk of inducing hypoglycemia , and patient compliance .
there are many limitations in this review like the usage of free search engines for literature search and the inability to fully retrieve some articles .
although many glucose - lowering therapies with noninsulin dependent mechanisms of action have been approved recently , only few of them ( vildagliptin , sitagliptin , exenatide , liraglutide , and dapagliflozin ) have been studied during ramadan .
the hypoglycemic risk was assessed for all of the above medications except dapagliflozin ; nearly all of the assessed medications were associated with reduced risk of hypoglycemia when compared with su when used during ramadan .
dpp-4 inhibitors such as vildagliptin and sitagliptin may form a suitable glucose - lowering therapy option for ramadan fasting patients , since they are unlike liraglutide less likely to cause git upset , and unlike sglt-2 inhibitors are not associated with increased thirst sensation .
effectiveness of sitagliptin to control blood glucose level for ramadan fasting patients was not assessed in any study in contrast to vildagliptin which was shown to be effective to control blood glucose level and body weight when used as monotherapy or even as add - on therapy to metformin ; accordingly vildagliptin seems to be the most suitable glucose - lowering therapy choice for diabetic patients who are wishing to fast during ramadan .
further studies on the use of other new glucose - lowering therapies during ramadan are recommended ; furthermore it is recommended to do studies that directly compare the advantages and disadvantages between these new glucose - lowering therapies . | hypoglycemia is the most common side effects for most glucose - lowering therapies .
it constitutes a serious risk that faces diabetic patients who fast during ramadan ( the 9th month in the islamic calendar ) .
new glucose - lowering classes like dipeptidyl peptidase-4 ( dpp-4 ) inhibitors , glucagon - like peptide 1 receptor agonist ( glp-1 ra ) , and sodium - glucose cotransporter-2 ( sglt-2 ) inhibitors are efficacious in controlling blood glucose level with less tendency to induce hypoglycemia and thus may constitute a good choice for diabetic patients during ramadan .
this study reviews the safety and efficacy of newer glucose - lowering therapies during ramadan .
this study was accomplished through a careful literature search about studies that assess the benefit and side effects of these new glucose - lowering therapies during ramadan during september 2015 .
vildagliptin , sitagliptin , liraglutide , exenatide , and dapagliflozin were the only studied glucose - lowering therapies .
all of the studied newer glucose - lowering therapies except dapagliflozin were associated with reduced risk to induce hypoglycemia .
gastrointestinal upset was common with the usage of liraglutide while increased thirst sensation was common with dapagliflozin . in conclusion
dpp-4 inhibitors such as vildagliptin and sitagliptin may form a suitable glucose - lowering therapy option for ramadan fasting patients . |
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