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cytochrome p450 enzymes
( p450s ) are heme - containing monooxygenases
that catalyze a variety of oxidative transformations in primary and
secondary metabolism .
p450 enzymes are
widely distributed in natural product biosynthetic pathways from bacteria ,
fungi , and plants .
these enzymes play a key role in the generation
of structural complexity , including hydroxylation by activation of
c h bonds , epoxidation , c c bond cleavage , and functional group rearrangement / migration .
in addition , p450s from secondary metabolism
have also been shown to catalyze less common reactions , including
recent examples of heterocycle formation and cationic terpene cyclization .
an
increasing number of oxidative enzymes have been shown to catalyze
multistep oxidation at distinct carbon atoms of substrates , resulting
in drastic structural transformations ( figure 1 ) .
for example , during lovastatin biosynthesis , lova
catalyzes consecutive oxidations at opposite sides of the decalin
ring to yield the precursor monacolin j , while tami catalyzes successive hydroxylation and epoxidation reactions
to afford the bicyclic ketal system in tirandamycin . considering the central roles of such p450s in biosynthetic
pathways , as well as the potential applications of these enzymes as
c
h activation biocatalysts , discovery of novel multifunctional
p450s from nature is of both fundamental mechanistic significance
and practical importance
. examples of natural products of which multifunctional
p450s play
important roles in the biosynthesis .
fumagillin ( 1 ) from aspergillus fumigatus is a meroterpenoid that has numerous biological activities .
compound 1 and its derivatives have been intensely studied for their
potential use in the treatment of amebiasis , microsporidiosis , rheumatoid arthritis , and for their antiangiogenic properties by the
irreversible inhibition of human type 2 methionine aminopeptidase
( metap2 ) .
structurally , 1 consists of
a highly oxygenated , rearranged sesquiterpene esterified to a polyketide - derived
tetraenoic diacid . the terpenoid portion , fumagillol ( 2 ) is derived by cleavage and multiple oxidation of the bicyclic sesquiterpene
hydrocarbon intermediate -trans - bergamotene
( 3 ) ( figure 2 ) .
the transformation of 3 to 2 involves
a dramatic skeletal rearrangement in which the strained cyclobutane
bridge is opened to yield the 1,8-bisepoxide - containing cyclohexanediol
with six contiguous stereocenters . therefore , given the unusual c
c
cleavage and the requisite pair of c h activation reactions
starting from 3 , it appeared likely that a p450 might
be centrally involved in the biosynthesis of 2 and 1 .
this fma cluster
contains the first reported membrane
bound class i terpene cyclase ( fma - tc ) that catalyzes
the formation of 3 from farnesyl pyrophosphate ( fpp ) .
the fma cluster also encodes a polyketide synthase
( fma - pks ) that synthesizes a dodecapentaenoate precursor
that is trans - esterified to 2 by the acyltransferase
fma - at to yield the intermediate prefumagillin 4 .
it was proposed that the polyene portion of 4 would be oxidatively cleaved to yield 1 . following
our discovery , wiemann et al .
reported that the fma gene cluster is embedded within a supercluster on chromosome 8 of a. fumigatus that also encodes the biosynthetic pathways
for fumitremorgin and pseurotin .
the fma gene cluster ( figure 3a ) contains
four oxygenases that are most likely responsible for the oxidative
tailoring steps that transform 3 to 1 , including af470 ( antibiotic biosynthesis monoxygenase superfamily
monooxygenase ) , af480 ( nonheme iron - dependent dioxygenase ) , af510 ( cytochrome p450 monooxygenase ) and af440 ( flavin - binding monooxygenase / methyltransferase ) .
an additional
redox enzyme in the fma gene cluster is the partial
pks ( af490 ) , in which only the dehydratase ( dh ) and
ketoreductase ( kr ) domains are present .
the mechanistic role of each
of these enzymes , especially that of the p450 ( encoded by af510 ) , which is hypothesized to play a central role in
the conversion of 3 to 2 , has remained unknown .
here we describe the complete characterization of the biosynthetic
pathway of fumagillin ( 1 ) .
using a combination of genetic
knockout , chemical complementation , heterologous reconstitution in saccharomyces cerevisiae , and in vitro biochemical assays ,
we have identified the role of each of the required pathway enzymes
and biosynthetic intermediates .
we show that af510
indeed encodes a multifunctional p450 with a spectacular range of
catalytic prowess , including hydroxylation of 3 , oxidative
cleavage of the bridging cyclobutane ring , and two epoxidation reactions .
in addition to the on - pathway intermediates , we also demonstrate the
range of off - pathway compounds that can be obtained using p450 starting
from 3 .
another notable finding includes the functional
assignment of af490 as a stereoselective ketoreductase
in the biosynthesis of 2 .
we first set
out to determine the roles and timing of the several
redox enzymes in the fma gene cluster .
we individually
deleted five genes , af510 , af490 , af480 , af470 , and af440
in a. fumigatus cea17 akub strain ( pyrg89 , akub ) , which is deficient in nonhomologous
end joining ( supporting information ) . in comparison to the isogenic control strain ,
the metabolic profile of af510 , af480 and af470 showed complete
abolishment of the formation of 1 ( figure 3b ) , while af440 retained production
of 1 .
the af490 strain showed
decreased titers of 1 ( 86% decreasing in the culture
of 4 days using cya medium ) .
the nonessential role of af440 is in accordance with that reported by wiemann et al . , in which af440 ( which corresponds to psof )
however in contrast to the wiemann report , in the analysis of the
af470 profile , we found the accumulation of 4 with m / z 471 [ m+na ] ( figure 3b ) , therefore suggesting
a role for af470 in the oxidative cleavage of the
terminal alkene of the dodecapentaenoate side chain into the carboxylic
acid present in 1 . on the other hand ,
deletion of af480 led to the accumulation of a polyene - containing compound 5 , with maximum uv absorbance at 333 nm and m / z 451 [ m+na ] ( figure 3b ) .
compound 5 was isolated from a 6-day liquid
culture of a. fumigatus af480 mutant strain and structurally characterized . on the basis of
the h , c and 2d nmr analyses ,
5 was elucidated as 6-demethoxyfumagillin , in contrast to an aldehyde
shunt product reported by wiemann et al .
the structure of 5 revealed that the nonheme iron - dependent
oxygenase encoded by af480 catalyzes hydroxylation
of c6 in the biosynthesis of 1 .
( b ) hplc analysis of metabolites extracted
from isogenic control and af510 , af480 , and af470 strain showing loss of 1 and accumulation of 5 from af480 and 4 from af470 strain ,
respectively .
( c ) gc - fid analysis of the af510 strain showing the accumulation of 3 .
the only remaining unassigned oxygenase in the
gene cluster was
therefore the p450 gene , af510 , which shows the strongest
sequence homology to the multifunctional p450 orda in aflatoxin biosynthesis ( 47% protein identity ) .
wiemann
et al . reported that deletion of af510 in a. fumigatus led to no detectable accumulation of any intermediates .
while the same phenotype was observed here using lc
ms assay
conditions , a more careful extraction with hexane and analysis of
the hydrocarbon extract by gc - fid revealed the presence of -trans - bergamotene ( 3 ) ( figure 3c ) . in the isogenic control ,
this result suggested that the initial oxidation of 3 is catalyzed by the enzyme encoded by af510 .
the enzyme encoded by af470 ( fma - abm ) was initially revealed to harbor a duf4188
conserved domain of unknown function by ncbi conserved domain search .
a further homology modeling search with phyre2 showed , however , that fma - abm appears to be related to
the cofactor - independent abm superfamily monoxygenases with a ferrodoxin - like
fold .
to further test the role of fma - abm ,
we cloned the intron - less af470 ( for reconstruction
of the gene from mrna , see supporting information ) into a yeast 2 m expression vector which was transformed
into s. cerevisiae strain bj5464-npga . upon supplementing 4 to the yeast culture expressing af470 ,
on the basis of protein structure prediction
( supporting information , figure s7a ) , fma - abm was predicted to be a membrane - bound protein . upon
purification of the microsomal fractions of the yeast strain expressing
fma - abm and incubation with 4 , the same
conversion to 1
these results confirmed the role of fma - abm in the oxidative
cleavage of 4 to 1 .
verification of the function
of fma - abm , fma - c6h , and fma - mt .
( a )
lc ms detection of 1 upon expression of fma - abm
in s. cerevisiae bj5464-npga with supplement of 4
( b ) lc ms analysis of in vitro assays of yeast
microsomes containing fma - abm toward 4 .
( c ) lc ms
analysis of in vitro assays of fma - c6h with 6 ; and combined
fma - c6h and fma - mt with 6 , respectively . to confirm the role of the enzyme
encoded by af480 ( fma - c6h ) ,
the
6xhis - tagged enzyme was solubly
expressed from e. coli bl21 ( de3 ) and purified to
homogeneity ( supporting information , figure
s5 ) .
bioinformatics analysis showed that fma - c6h shares a similar
sequence with the members of the phytanoyl - coa dioxygenase ( phyh )
superfamily , thereby requiring fe ( ii ) and -ketoglutarate ( -kg )
for its activity .
incubation of recombinant fma - c6h
with -ketoglutarate , sodium ascorbate , and substrate 5 , however , did not lead to the formation of any hydroxylated
product . we therefore reasoned that fma - c6h may function
upstream in the pathway prior to attachment of the polyene portion .
to obtain such a substrate , the hydrolysis of 5 under
basic condition was performed to afford 6-demethoxyfumagillol ( 6 ) ( supporting information ) .
when
recombinant fma - c6h and 6 were incubated
under the same condition as above , complete conversion of 6 to 6-hydroxylfumagillol ( 7 ) was obtained ( figure 4c ) .
furthermore , coincubation of fma - c6h with the recombinant methyltransferase ( fma - mt ) encoded by af390400 in the presence
of the necessary cofactors ( -ketoglutarate , sodium ascorbate ,
and sam ) and 6 resulted in the formation of the expected 2 ( figure 4c ) . collectively , these
studies confirmed the function of af480 and revealed
that 6 ( or a related compound ) may be a key intermediate
in the biosynthetic pathway of 1 . to test this hypothesis ,
feeding of 6 to
a. fumigatus blocked
in bergamotene formation ( deletion of fma - tc , af520 ) restored biosynthesis of 1 as well as
the production of 5 ( see compilation of chemical complementation
traces in figure 7 ) . with this information
in hand , we then turned to investigation of the possible routes of
oxidative modification of 3 into 6 . the transformation of 3 into 6 requires at least three oxidation steps
to introduce the one c5 hydroxyl group and the two epoxide functionalities .
in addition , the cleavage of the c c bond between c5 and c8
must also take place .
several proposals for this mechanistically unresolved
transformation have been advanced , including desaturation of the c5c6
bond followed by concomitant cleavage of the c5c8 bond . in light of the functional assignment of af440 , af470 , and
af480
described above , it appeared likely that the p450 encoded by af510 ( fma - p450 ) is a multifunctional p450 that is responsible
for most , if not all , of the oxidative steps between 3 and an advanced intermediate such as 6 .
we previously
showed that expression of fma - tc alone in s. cerevisiae can lead to accumulation of -trans - bergamotene
( 3 ) . to investigate the
activity of fma - p450 toward 3 , we cloned
the intron - less af510 into a yeast 2 m expression
vector for microsomal expression . to equip fma - p450
with the optimal redox partner
, we also cloned the a. fumigatus cytochrome p450 oxidoreductase ( afcpr ) for coexpression .
all three genes ( encoding fma - tc , fma - p450 , and afcpr ) were placed under the adh2 promoter and transformed into bj5464-npga . after four
days of culturing followed by extraction with hexanes / etoac ( 1:1 ) ,
we observed a series of sesquiterpene compounds ( 6 , 819 ) that are derived from 3 ( figure5a ) .
of these compounds , 10 , 11 , 14 , and 17 were the
major products ( 0.61.1 mg / l ) ; 8 and 12 were minor products ( 0.30.5 mg /
l ) ; and the remaining ( 6 , 9 , 13 , 15 , 16 , 18 , and 19 ) were present at
less amounts ( < 0.3 mg / l ) .
the same set of metabolites was also
observed when 3 was directly supplied to the yeast culture
expressing only fma - p450 and afcpr ( figure 5b ) . in the absence of 3 or fma - p450 ,
to elucidate the structures of these products , large scale
( 12 l ) fermentation of the triply transformed yeast strain was performed ,
followed by isolation and characterization of each compound ( figure 5d and supporting information ) .
compounds 6 and 9 were identified as 6-demethoxyfumagillol and cordycol , respectively ,
based on ms and nmr comparison to standards .
9 is the
monoepoxide relative of 6 and may therefore be an immediate
precursor of 6 .
formation of these compounds does indicate
that fma - p450 alone is sufficient to transform 3 into 6 .
however , both compounds are found in
very minute quantities ( figure 5a ) .
surprisingly ,
the 5-epimeric forms of 6 and 9 , which are
5-epi - demethoxyfumagillol ( 11 ) and 5-epi - cordycol ( 10 ) , respectively , were present
as major products .
the opposite stereochemistry of the c5 hydroxyl
groups was readily established through the coupling patterns of h-5
in h nmr ( 6 and 9 : br.s ; 10 and 11 : br.t , j = 9.4 and
10.5 hz , respectively ) . to identify which of these compounds are on - pathway
intermediates in the biosynthesis of 1 ,
each compound
was supplied to the af520 blocked mutant at
40 g / ml .
as expected , only 6 and 9 restored production of 1 , even though each
strain also produced the fumagillin analogues 5 and 20 , respectively ( figure 7a and supporting information , figure s11 ) , through
the direct esterification with the polyketide side chain ( more on
this below ) . in contrast , neither 10 nor 11 restored biosynthesis of 1 ( figure 7a and supporting information , figure
s11 ) , thereby confirming that they are shunt products in s.
cerevisiae , and no further epimerization of the c5 hydroxyl
can take place in a. fumigatus .
this result also
provides insight into the intrinsic stereoselectivity of the fma - at which prefers the natural r - oh group
at c5 .
( a ) lc ms metabolic profiles of coexpression
of fma - tc ( encoded by af520 ) , fma - p450 ( encoded by af510 ) , and afcpr .
compounds 13 , 15 , 16 , 18 , and 19 are present at trace quantities and are not shown .
( b )
lc ms analysis of coexpression of fma - p450
and afcpr in s. cerevisiae bj5464-npga with supplement
of 3 ; ( c ) same as trace b but with supplement of 8 .
compound 8 contains
one additional oxygen atom compared to 3 , and was found
to be 5r - hydroxyl--trans - bergamotene
( supporting information , table s5/figures
s21s26 ) .
8 is therefore the product of direct
oxidation of c5 of 3 by fma - p450 .
nmr characterization revealed the structures of 14 and 15 to be 5,9-dihydroxyl--trans - bergamotene and 5,10-dihydroxyl - trans - bergamotene ,
respectively ( supporting information , tables
s11s12/figures s48s57 ) .
all three compounds retained
the trans - bergamotene scaffold found in 3 , and therefore represent possible early intermediates in the pathway
prior to the c5c8 bond cleavage step .
interestingly , only
the feeding of 8 to the af520
blocked mutant restored production of 1 , while addition
of either 14 or 15 failed to do so ( figure 7a and supporting information , figure s11 ) .
therefore 14 and 15 are
shunt products of fma - p450 activity in yeast . in
particular ,
the high levels of 14 in the yeast culture
extract hints that c9 is the site of the second p450 oxidation and
may be subject to rapid hydroxylation ( see discussion below ) . to prove the multifunctional nature of fma - p450
no restoration of 1 was observed , as would be expected for any additional role
of fma - p450 in the pathway .
finally , addition of
purified 8 to the yeast culture expressing fma - p450 and
afcpr similarly led to the formation of more oxidized products such
as 11 and 14 ( figure 5c ) . among the
products
isolated , 5-keto - isocordycol ( 12 ) contains a ketone functionality at c5 and an allylic alcohol resulting
from prototopic ring - opening / rearrangement of the c8c9 epoxide
( supporting information , table s9/figure
s40s41 ) .
although this compound is only present in minor quantities ,
the unexpected cyclohexanone shunt product suggests that the c5 hydroxyl
( in 8 , for example ) may serve as the source of electrons
in the oxidative cleavage of the cyclobutane ring , thereby transforming
the bicyclic trans - bergamotene scaffold into an 2,5,7-trisubstituted
cyclohexanone .
the corresponding c5-reduced derivative , epi - isocordycol ( 13 ) , was also found in the extract with
the 5s - hydroxyl ( supporting information , table s10/figures s42s47 ) .
the other four compounds 1619 were all found to be structurally
related , but clearly off - pathway products .
compound 16 is particularly interesting with its trisubstituted sesquifenchyl
core ( supporting information , table s13/figures
s58s62 ) .
the 5-hydroxy--cis - bergamotene
( 17 ) , 5,10-dihydroxyl--cis - bergamotene
( 18 ) , and 19 all have the unexpected -cis - bergamotene scaffold and are presumably formed by isomerization
of reactive intermediates derived from 8 ( supporting information , tables s14s16/figures
s63s80 ) .
17 is a major product from the yeast
expression of fma - p450 , while 18 and 19 are further oxidized products of 17 .
the feeding
of 17 to the af520 blocked mutant
did not restore production of 1 ( figure 7a ) .
possible mechanisms of formation of 1619 are shown in figure 10 and will
be discussed below .
the array of products recovered
from s. cerevisiae clearly showcased the multifunctional
capability of fma - p450 ( hydroxylation , epoxidation ,
rearrangement ) in the oxidation of -trans - bergamotene
( 3 ) . the large number of shunt products , however , may
be a result of the weak expression of fungal membrane - bound p450 in
yeast . as a result of the low concentration of the p450 ,
reactive
intermediates may be readily intercepted or hydrolyzed to yield off
pathway products such as 14 and 17 .
therefore ,
to analyze the function of fma - p450 under more controlled conditions ,
we prepared microsomal fractions from the yeast strain that overexpressed
fma - p450 and afcpr for in vitro assay .
ms
analysis of in vitro assays of yeast microsomes containing af510 and afcpr toward ( a ) 3 and ( b ) 9 .
when 3 was incubated with 10 mg / ml of microsomal
fractions
and nadph overnight , we detected 11 as essentially the
single product in the extract ( figure 6a ) .
other major compounds observed in vivo , such as 10 , 14 , and 17 , etc .
are only present at very low
levels when searched for using selective ion monitoring . similarly ,
when the assay is repeated using 8 as the substrate ,
near complete conversion of 8 to 11 was
observed with nearly no side products ( supporting
information , figure s8 ) . to analyze one of the individual steps
catalyzed by the multifunctional p450 , we assayed the epoxidation
of 9 to 6 using the same microsomal extract .
as shown in figure 6b , we could observe incomplete
conversion of 9 to 6 only in the presence
of fma - p450 , thereby confirming the ability of this enzyme to catalyze
the specific formation of epoxide .
collectively , these in vivo
and vitro results confirmed the multifunctional
role of fma - p450 , including c5 hydroxylation , coupled
or sequential 4e - oxidative cleavage , and rearrangement
to yield epoxycyclohexanone , and the tandem epoxidation . at the same
time , it is also evident that fma - p450 alone is insufficient
to generate the correct stereoisomer of 5r - demethoxyfumagillol 6 , instead the 5s diastereomer 11 being formed in yeast or using yeast microsomes .
therefore , given
that a ketone intermediate 12 could be isolated , the
transformation of 3 most likely involves a c5 ketone
intermediate that is stereoselectively reduced into the 5r isomer . several ketoreductases ( krs ) such as the 3-kr in sterol
biosynthesis
and 3-kr for long - chain fatty acid synthase have been reported in s. cerevisiae , hence it is
possible that an endogenous yeast kr could catalyze the reduction
of the c-5 ketone to form the 5s - hydroxy moiety observed
in compounds 10 , 11 , and 13 . to test this hypothesis , we prepared both soluble and microsomal
extracts of the untransformed host bj5464-npga .
we first tested if
the isolated ketone 12 could be selectively reduced to 13 .
as shown in supporting information , figure s9 , complete reduction of 12 to 13 was observed .
the r - isomer of 13 may
be present at a very low concentrations as suggested by ion - monitoring ,
but the amount was insufficient for purification and characterization .
to further test whether this endogenous yeast kr activity is responsible
for formation of the other 5s - containing products
shown in figure 5d , we chemically prepared
the three ketone substrates , 5-keto - cordycol ( 21 ) , 5-keto - demethoxyfumagillol ( 22 ) , and 5-keto - fumagillol ( 23 ) ( figure 7d , spectroscopic data in supporting
information , tables s17s19 and figures s81s86 ) .
these compounds were synthesized from 10 , 11 , and 2 by pcc oxidation , respectively .
when either 21 or 22 was added to the control yeast extract
( soluble or microsomal ) , we observed the corresponding ketoreduction
to 10 or 11 ( supporting
information , figure s10 ) , respectively , thereby validating
the involvement of endogenous yeast krs in the formation of compounds
with the 5s hydroxyl functional group .
compounds are supplemented at 40
g / ml to ( a ) af520 ; ( b ) af510 ; ( c ) af480 .
( d ) structures
of 2123 . with the ketones 2123 in
hand ,
we performed chemical complementation experiments by feeding each
of these compounds individually to the af520-blocked
mutant .
in each case , complete restoration of biosynthesis of 1 was observed ( figure 7a ) .
therefore , the c5-ketone moiety in each of the
compounds can be processed correctly .
furthermore , chemical complementation
of af510 mutant by 22 restored
fumagillin production , indicating that fma - p450 is
not involved in the biosynthetic pathway beyond 22 and
is not essential for reduction of the c5 ketone .
lastly , complementation
of 23 to the af480 mutant efficiently
restored fumagillin production , hinting that reduction of c5 may occur
as the last step in the formation of 2 .
we hypothesized
that a kr encoded in the fma gene cluster might
be responsible for the stereospecific 5r reduction
of the ketone during the biosynthesis of 1 .
although
no standalone kr is found in the fma cluster shown in figure 3a , af490 encodes a partial pks
in which only the dh - kr domains are present . upon initial discovery
of the fma cluster
, the pseudo / partial pks was assumed
to be an inactive enzyme that had no likely role in the biosynthetic
pathway .
although deletion of af490 did not completely
abolish the production of 1 , we did notice an 86% decrease
in the titer of fumagillin ( 1 ) ( figure 3b ) .
the incomplete abolishment of the biosynthesis of 1 may be due to the presence of endogenous krs in a. fumigatus that have overlapping functions to that encoded
by the pathway enzyme af490 ( fma - kr ) . to investigate the role of fma - kr
, the 110
kda protein was therefore expressed as a 6xhis tag fusion and purified
from s. cerevisiae ( supporting
information , figure s6 ) . upon individual incubation of each
of the ketone derivatives 21 , 22 , or 23 with recombinant fma - kr , we observed complete
conversion to the corresponding 5r - hydroxy - containing
compounds , 9 , 6 , or 2 , respectively
( figure 8a c ) .
lastly , to examine
the product profile of fma - p450 in the presence of
fma - kr , we repeated the biotransformation of 3 in s. cerevisiae by coexpression of fma - p450 , fma - kr , and afcpr .
as shown in figure 8d , the product profile is altered considerably to
reflect the presence of the dedicated c5 reductase .
the 5r - hydroxy isomers 6 and 9 are now clearly
major products of the assay , while the 5s - hydroxy
diastereomers 10 and 11 have essentially
disappeared .
the same results were obtained in the in vitro assay
when fma - p450-containing yeast microsomes were coincubated
with purified fma - kr .
whereas 11 was
formed as the single product in the previous assay shown in figure 6a , inclusion fma - kr led to the
production of the natural diastereomer 6 ( figure 8e ) .
( a c ) lc ms analysis of in vitro assays of fma - kr with 2123 .
( d ) lc ms
analysis of coexpression of fma - kr , fma - p450 , and afcpr in s. cerevisiae bj5464-npga with
supplement of 3 .
( e ) lc ms analysis of in vitro
assay of fma - kr , and yeast microsomes containing af510 and afcpr plus 8 . on the
basis of the new enzymes characterized and the new compounds isolated
and identified in this work
, we can now deduce a complete biosynthetic
pathway for fumagillin ( 1 ) , as shown in figure 9 .
the pathway begins with the conversion of fpp
to -trans - bergamotene ( 3 ) by
a membrane - bound fma - tc .
the initial
oxidation of 3 by fma - p450 involves c h hydroxylation
at the bridgehead c5 position to yield 8 . subsequently
,
a four electron oxidation initiated at c-9 coupled to cleavage of
the cyclobutane c5c8 bond of the bicyclo[3.1.1 ] core yields
the on - pathway intermediate epoxyketone 21 . an additional
epoxidation reaction also catalyzed by fma - p450 then furnishes the
characteristic bisepoxide ketone 22 . a possible mechanism
for the fma - p450-catalyzed conversion of 3 to 22
the diepoxyketone 22 is then subjected
to successive c-6 hydroxylation and o - methylation
by fma - c6h and fma - mt , respectively , to yield 23 , which is then stereoselectively reduced by fma - kr to 5r - hydroxy - seco - sesquiterpene 2 .
acylation catalyzed by the fma - at with the dodecapentaenoyl group on fma - pks to yield 4 has been previously demonstrated .
finally , oxidative cleavage of 4 by the oxygenase ( fma - abm )
encoded by af470 arrives at 1 .
we have also shown that fma - kr can stereospecifically
reduce each of the three potential ketone intermediates 2123 into the corresponding 5r - hydroxyl compounds , thereby implying that this ketoreduction step
may occur at different stages during the biosynthesis of 2 . in the proposed pathway ,
however , we have assigned the enzyme to
catalyze reduction of the most advanced intermediate 23 .
we have shown that compounds 6 and 9 ,
which can be produced by reduction of 22 and 21 , respectively , can each be acylated efficiently by fma - at to yield the analogues 5 and 20 .
for
example , feeding 6 to the af520
strain resulted in the production of both 5 and 1 ( figure 7a ) , with a relative ratio
around 4:1 .
this indicated that c-6 hydroxylation by fma - c6h may be slower than the c-5 acylation catalyzed by fma - at .
the
formation of 5 and 20 also demonstrates
that fma - at has significant tolerance toward the
substitution pattern of the terpene substrate , and can intercept these 5r - reduced products in vivo .
in contrast , we have established
that fma - c6h is highly specific toward the unacylated
intermediate 6 .
therefore , 5 and 20 can not be oxidized to 1 . however , no trace of 5 or 20 is recovered from a. fumigatus , thereby suggesting that neither 6 nor 9 is an intermediate in vivo .
hence , we proposed that reduction of
the c5 ketone takes place after the 6-methoxyl function has been installed
in 23 , thereby preventing premature c5 acylation of the
terpenoid by fma - at .
in
this study , we have identified all the enzymes in the fumagillin
pathway that transform the bicyclo[3.1.1 ] sesquiterpene 3 into the richly decorated pharmacophore 2 , which acts
by binding to type 1 and 2 metap .
the
highly oxidized cyclohexane portion of 2 , which is also
present in the metabolite ovalicin from pseudeurotium
ovalis , has been the
subject of intensive synthetic efforts ( see review in yamagushi et
al . and references therein ) .
considering
the number of oxidative steps required to modify 3 into 2 , it is remarkable that only two oxygenases are responsible .
most impressively , fma - p450 encoded by af510 alone
catalyzes the eight electron oxidation of 3 into the
intermediate 22 ( figure 9 ) .
the
same set of reactions must therefore also be involved in the conversion
of 3 into ovalicin , which is structurally identical to 23 except for the additional hydroxylation at c7 ( figure 8) .
the biosynthesis of ovalicin has previously been
studied using labeled samples of mevalonate and of 3 . from these investigations , several mechanisms and intermediates
were ruled out , including the involvement 8,9-didehydrobergamotene
that had been suggested by birch ( figure 9 ) .
the pathway deduced from our genetic and biochemical
experiments is fully consistent with these earlier findings .
the multifunctional fma - p450 is the central player
in the consecutive series of chemically and mechanistically distinct
catalytic processes .
this enzyme initiates the oxidative transformation
on 3 by performing c-5 hydroxylation , followed by oxidative
ring - opening coupled to epoxidation and a second tandem epoxidation
to generate the key diepoxyketone intermediate 22 that
serves as the substrate for later hydroxylation , methylation , ketoreduction ,
and acylation catalyzed by other pathway enzymes from the fma cluster .
when fma - p450 was reconstituted in s. cerevesaie , we observed numerous oxidized products derived
from the bioconversion of 3 . on the basis of the structures
of these compounds , we can propose a detailed mechanism for fma - p450 .
either radical or cationic rearrangement mechanisms
can be proposed for this enzyme and are shown in figure 10 and supporting information , figure s12 .
although most of the p450-catalyzed c h bond
activation reaction has been shown to involve caged radical intermediates
rather than carbocations , several recently discovered p450-catalyzed reactions almost certainly
proceed through carbocationic mechanisms .
it
is possible that the various products produced by af510 result from a partitioning between radical pair and cationic
intermediates .
the first c o bond installation occurs
by c h bond
cleavage and hydroxylation at the c-5 of hydrocarbon 3 to form 5-hydroxybergamotene 8 .
next , the hydrogen
atom at c-9 is abstracted by the key ferryl - oxo intermediate ( fe = o , porphyrin cation radical ) to generate
the hydroxycyclobutylcarbinyl radical , a. this reactive
intermediate can either undergo an oxygen rebound to generate 14 or be converted to a hydroxycyclobutylcarbinyl cation ( b ) after one - electron oxidation by the oxidative ion ( iv)-hydroxo
intermediate . both reactions result in
a ferric heme center and resume the catalytic cycle of fma - p450 in
the presence of afcpr .
carbocation b can be subjected
to attack by the supernucleophile iron(iii)-peroxo
intermediate generated from the following p450 cycle .
the later reaction produces intermediate c and induces the subsequent ring - opening rearrangement through c5c8
bond cleavage to form the 8,9-epoxide 21 ( figure 10 ) , followed by a 1,2-epoxidation to generate 22 .
nucleophilic addition from water or ferric hydroxo ( fe oh ) complex to b also generates 14 .
this shunt pathway can readily take place , as indicated
by the high level of 14 observed in yeast ( figure 5a ) .
formation of the shunt -cis - bergamotene derivatives 1719 can
result from ring - opening / closure of the c-9 radical intermediate ( d ) to generate the isomeric radical ( or cation ) cis-5-hydroxybergamotene intermediate .
compounds 1719 can then readily be obtained through direct nucleophilic
attack of water or intervening 1,2-hydride shift .
lastly , if fma - p450
epoxidizes the 1,2-exomethylene double bond of 3 prior
to c-5 hydroxylation , the resulting epoxide intermediate could undergo
well precedented cationic rearrangement of its bicyclo[3.1.1 ] skeleton
to form the tetra - substituted sesquifenchol derivative 16 .
overall , the oxidative cleavage of 8 to 21 is particularly intriguing , and would
parallel the proposed conversion of loganin to secologanin , as well as that involved in the formation of
furanocoumarin . proposed mechanism of
fma - p450 .
radical mechanism from a to 21 was shown in supporting information ,
figure s12 .
the af490 ( fma - kr ) gene has previously been annotated
as a pks - like enzyme that harbors conserved motifs characteristic
of the pks - dh ( smart00826 ) superfamily and pks - kr ( smart00822 ) domains
as revealed by a ncbi conserved domain - search . the discovery that
the truncated pks gene af490 with only dh - kr domains
encodes a functional enzyme that catalyzes stereospecific 5-ketoreduction
of cyclohexanone 23 to 2 is intriguing given
that pks kr domain typically acts on -ketoacyl thioester substrates
attached on the acyl carrier proteins .
interestingly , a blast search
for similar fma - kr - like enzymes indicates that in a. fumigatus the hybrid pks / nrps ( psoa ) in pseurotin biosynthesis ( and the corresponding orthologues
in a. clavatus and m. anisopliae ) are the closest match with up to 56%
protein identity ( 65% nucleotide identity ) within the kr domain region .
hence , it appears that the fma - kr may be an evolutionary
product originating from classic gene duplication , divergence , and
differential loss , but with the mechanisms acting on the functional
domain level rather than on the entire gene .
although both a. clavatus and m. anisopliae have a close
orthologue of psoa , the fma - kr gene
appears to be absent , suggesting the duplication has only occurred
in a. fumigatus .
curiously , the pseurotin biosynthetic
gene cluster is intertwined with the fma gene cluster and was shown recently to be coregulated
by a single transcriptional factor ( fapr ) embedded in this fma - pso supercluster .
the possibility
that af490 originated from a duplicated partial psoa gene but neofunctionalized to participate in the fma pathway further adds to the complex evolutionary history
of this supercluster .
even more interesting , although we verified
the role of fma - kr in vitro and in s. cerevisiae , deletion of this gene in a. fumigatus did not lead
to complete abolition of fumagillin ( 1 ) biosynthesis .
it remains highly possible that the activity of the kr domain of psoa
can partially complement the deletion of af490 , therefore
maintaining the ketoreductase activity , albeit at lower efficiency
as indicated by the lowered titer of 1 in the af490 strain .
alternatively , other endogenous krs in a. fumigatus may also possess this activity and catalyze
the requisite reduction .
fma - abm encoded by af470 has been shown to be
the oxygenase responsible for the c10-c11 cleavage of the dodecapentaenoate of 4 and further
oxidation of the aldehyde intermediate to the decatetraenedioic ester 1 .
oxidative cleavage of olefins by a single oxygenase has
been implicated in the biosynthesis of carotenoids , as well as in the formation of microbial secondary metabolites
such as rifamycin .
the carotenoid oxygenases
are nonheme iron - dependent dioxygenases , while the cleavaging enzyme
in the rifamycin pathway is a p450 monooxygenase .
interestingly , fma - abm
belongs to a potentially new class of oxygenases that catalyze such
reactions .
it contains a duf4188 conserved domain of still unknown
function but homology modeling using phyre2 suggests that the main
portion of fma - abm is structurally related to aldoxime dehydratase
( supporting information , figure s7b ) which
is a heme - containing enzyme from bacillus sp .
, as well as the cofactor - free abm superfamily
of monooxygenases , which is typified by actva - orf6 in actinorhodin
biosynthesis and snoab in nogalonic acid biosynthesis . it is uncertain whether fma - abm requires a heme
as a prosthetic group as it is much smaller than aldoxime hydratase
( 275 versus 373 amino acids )
. further biochemical investigation of
fma - abm enzyme is warranted as a blast search reveals that fma - abm
homologues are ubiquitous in fungi .
we
have uncovered the roles of three oxygenases , fma - p450 , fma - c6h , and fma - abm , as well as an o - methyltransferase , fma - mt , involved in the biosynthesis
of fumagillin ( 1 ) . among these enzymes , fma - p450 , a p450 monooxygenase ,
catalyzes a multistep transformation
that includes a simple hydroxylation followed by ring - opening of the
bicyclic substrate coupled to generation of an epoxide , followed by
a second epoxidation that results in the conversion of -trans - bergamotene ( 3 ) to a highly oxygenated
structure , 5-keto - demethoxyfumagillol ( 22 ) . in the course of these studies we identified nine compounds as
off - pathway products that shed light on the mechanism of action and
catalytic potential of fma - p450 .
the catalytic versatility
of fma - p450 therefore significantly augments the already impressive
chemical virtuosity of this important class of enzymes .
we have
also characterized the function of fma - kr as the
ketoreductase that controls the configuration at c-5 of
hydroxylated intermediates that then undergo further acylation catalyzed
by fma - at .
this study has elucidated all the tailoring reactions in
the biosynthesis of 1 and will provide opportunities
for derivatization of 1 using enzymatic approaches .
a. fumigatus strain used in this study was a. fumigatus cea17 akub strain
( pyrg89 , akub ) , which is deficient
in nonhomologous end joining and was
maintained on czapek - dox agar or glucose minimal agar ( gmm ) .
e. coli top10 ( invitrogen ) and xl1-blue ( stratagene )
were used for dna manipulation , and bl21 ( de3 ) was used for protein
expression .
saccharomyces cerevisiae strain bj5464-npga
( mat ura352 his3- 200 leu2- 1
trp1 pep4::his3prb1 1.6r can1 gal ) was used as the
yeast expression host .
polymerase chain
reactions were performed using phusion dna polymerase ( new england
biolabs ) or platinum pfx dna polymerase ( invitrogen ) .
dna restriction
enzymes were used as recommended by the manufacturer ( new england
biolabs ) .
rna extraction was performed using a ribopure yeast kit
( ambion ) , and improm - ii reverse transcription system for rt - pcr ( invitrogen )
was used to synthesize complementary dna ( cdna ) from total rna .
pcr
products were subcloned to a pcr - blunt vector ( invitrogen ) and confirmed
by dna sequencing .
primers used to amplify the genes were synthesized
by integrated dna technologies and are listed in supporting information , table s2 . in vivo yeast recombination
cloning was performed by transforming the s. cerevisiae bj5464-npga with dna fragments with > 35 bp overlaps and includes
a 2 m plasmid backbone ( derived from yeplac195 or yeplac112 )
using an s.c .
easycomp transformation kit ( invitrogen ) . for production of 1 and
other metabolites , a. fumigatus and mutant strains
were cultured in cya medium (
total rna for rt - pcr was extracted from a.
fumigatus grown on czapek - dox liquid medium with 5 g / l yeast
extract ( cyb ) after 4 days of cultivation . for chemical complementation
of af520 , af510 , and
af480 mutants ,
purified compounds are supplemented
to the cya medium at 0.2 mg / ml individually .
lc ms analyses
of conversion of 7 to 8 by af480 and 7 to 2 by af480
and af390400 were performed on prevail 3
m , 2.1 100 mm c18 reversed - phase column
( alltech ) and separated on a 595% ( v / v ) ch3cn linear
gradient in h2o supplemented with 0.05% ( v / v ) formic acid
at a flow rate of 125 l / min .
all lc ms analyses except
the aforementioned were performed on a shimadzu 2010 ev lc
ms
( phenomenex luna , 5 m , 2.0 100 mm , c18 column )
using positive and negative mode electrospray ionization with a linear
gradient of 595% mecn - h2o in 30 min followed by
95% mecn for 15 min with a flow rate of 0.1 ml / min .
h , c , and 2d nmr spectra were obtained on bruker av500 spectrometer
with a 5 mm dual cryoprobe at the ucla molecular instrumentation center . af390 and
af400 were obtained as one transcript by rt - pcr verifying that af400
was misannotated ( for revised annotation , see supporting information ) .
the dna fragemnt of af390400 and af480 from cdna were inserted
into pet21 ( novagen ) digested with ndei and xhoi to yield pkw20174 and pkw20172 , respectively .
primers
used for the amplification and cloning are listed in supporting information , table s2 .
recombinant enzymes were
expressed with c - terminal his6-tagged in e. coli bl21 ( de3 ) and purified by nickel affinity chromatography .
the cells
were cultured at 37 c , 250 rpm in 500 ml of lb medium with 35
g / ml carbenicillin .
isopropylthio--d - galactoside
( iptg , 0.1 mm ) to induce protein expression was added at od600 between 0.4 to 0.6 and the cells were further cultured for 1216
h at 16 c .
the cells were then harvested by centrifugation ( 3500
rpm , 15 min , 4 c ) , resuspended in 25 ml of lysis buffer
( 100 mm tris - hcl , ph 7.4 , 0.1 m nacl , 20 mm imidazole ) , followed by
lysed by sonication on ice .
cell debris was removed by centrifugation
( 15 000 rpm , 30 min , 4 c ) .
the his6-tagged proteins were
purified by using ni - nta agarose ( qiagen ) according to manufacturer s
instructions .
purified enzyme was concentrated and exchanged into
buffer a ( 50 mm tris - hcl , ph 7.9 , 2 mm edta , 2 mm dtt ) + 10% glycerol
with the centriprep filters ( amicon ) and stored at 80 c
for enzyme assays .
for in vitro synthesis of 2 and 7 , 10 m
fma - c6h480 was incubated with 20 m substrate 6 , 1 mm sodium ascorbate , 1 mm -ketoglutarate , and 100 mm nacl
in 100 mm tri - hcl ( ph 7.4 ) . for in vitro synthesis of 2 , the assay was performed using the same condition as above with
additional 1 mm sam and 10 m fma - mt .
the organic phases
were dried and dissolved in 20 l of meoh and subjected for
analysis by lc
ms as described in chemical
analysis . for biotransformation
in s.c . of fma - p450 and afcpr , s. cerevisiae strain bj5464-npga harboring both fma - p450 and afcpr plasmid
were
inoculated to 4 ml of yeast synthetic drop - out medium without uracil
and leucine .
a 15 l aliquot of the seed culture was inoculated
with 2 ml of ypd ( 10 g yeast extract , 20 g peptone , and 950 ml of
milli - q water ) supplemented with 1% dextrose . 3 , 8 , and 9 ( 0.5 mg in 10 l of dmso )
was
added to the culture after 48 h at 28 c with shaking and the
cells were cultivated for another 24 h. the cultures were extracted
by hexanes - ethyl acetate ( 1:1 ) twice , the organic layers were concentrated in vacuo and redissolved in 100 l of meoh .
ms with the method
described in chemical analysis . for biotransformation
in s.c . of af470 ,
the culture of s. cerevisiae strain bj5464-npga harboring af470 was prepared by a similar method above and 4 ( 0.1 mg in 10 l of dmso ) was added to the culture after 48
h. details
in preparation of fma - p450 and afcpr - containing microsomes
for in vitro assay are shown in the supporting
information . for in vitro microsomes assay , 10 mg / ml ( wet weight )
microsomal fractions containing af510 and afcpr , 1 mm substrates , 2 mm nadph , and nadph regeneration
system ( bd ) solution a ( 5 l ) and b ( 1 l ) , and 100 mm
pbs , ph 7.4 were incubated in a 100 l reaction .
the reaction
was incubated at room temperature for overnight and extracted with
100 l of hexanes - ethyl acetate ( 1:1 ) twice .
the organic phase
was dried and redissolved in 20 l of meoh for analysis by lc ms .
the amount of protein in 10 mg / ml microsomes was calculated to be
180 g / ml based on a modified bradford assay against a bsa standard
curve ( protein samples were predenatured in 0.1 m naoh ) .
g / l
yeast extract , 20 g / l peptone ) supplemented with 1% dextrose and incubated
at 28 c with shaking for 72 h. the cells were harvested by centrifugation
( 3750 rpm at 4 c for 10 min ) , and the cell pellet was resuspended
in 20 ml of lysis buffer ( 50 mm nah2po4 , 150
mm nacl , 10 mm imidazole , ph 8.0 ) and lysed by sonication on ice in
one minute intervals until homogeneous . to remove cellular debris ,
the homogeneous mixture was centrifuged at 17000 rpm for 1 h at 4
c .
ni - nta agarose resin was added to the supernatant ( 2 ml )
and the solution was stirred at 4 c overnight .
soluble fma - kr
was purified by gravity - flow column chromatography with increasing
concentrations of imidazole in buffer a ( 50 mm tris - hcl , 500 mm nacl ,
20 mm250 mm imidazole , ph 7.9 ) .
purified protein was concentrated
and buffer was exchanged into buffer b ( 50 mm tris - hcl , 2 mm edta ,
100 mm nacl , ph 8.0 ) using an amicon ultra-15 centrifugal filter unit
and stored in 10% glycerol .
the purified fma - kr was analyzed by sds - page
( supporting information , figure s6 ) and
their concentration was calculated to be 8.7 mg / l , using the bradford
assay with bsa as a standard . for in vitro synthesis of 6 , 9 , and 2 , 10 m fma - kr
was incubated with 1 mm substrates 2123 , respectively , 2 mm nadph in 100 mm pbs , ph 7.4 in a total 100 l
reaction .
the reaction was incubated at room temperature overnight
and extracted with 100 l of hexanes ethyl acetate ( 1:1 )
twice .
the organic phase was dried and dissolved in 20 l of
meoh for analysis on lc ms .
the method of oxidation by pcc followed
that of asami et al . a suspension of
pcc ( 1 mg , 0.0046 mmol ) and
powdered molecular sieves 4a ( 3.0 mg ) was added to a solution of 2 ( 2.3 mg ) in 0.5 ml of ch2cl2 .
the
mixture was stirred in an ice water bath and for 2.5 h at room
temperature .
florisil and ch2cl2 were added
to the mixture , and the suspension was filtered through a combination
of celite and florisil .
the residue was purified by silica gel plate ( merck , tlc silica
gel 60 f254 , glass plates ) with 25% acetone - hexanes developed
by two times to give 23 ( 1.1 mg ) .
a similar method was
used as above to obtain 21 ( 0.8 mg ) and 22 ( 1.0 mg ) from 10 ( 2.2 mg ) and 11 ( 3.0
mg ) , respectively . | fumagillin ( 1 ) , a meroterpenoid
from aspergillus fumigatus , is known
for its antiangiogenic
activity due to binding to human methionine aminopeptidase 2 .
1 has a highly oxygenated structure containing a penta - substituted
cyclohexane that is generated by oxidative cleavage of the bicyclic
sesquiterpene -trans - bergamotene .
the chemical
nature , order , and biochemical mechanism of all the oxygenative tailoring
reactions has remained enigmatic despite the identification of the
biosynthetic gene cluster and the use of targeted - gene deletion experiments .
here , we report the identification and characterization of three oxygenases
from the fumagillin biosynthetic pathway , including a multifunctional
cytochrome p450 monooxygenase , a hydroxylating nonheme - iron - dependent
dioxygenase , and an abm family monooxygenase for oxidative cleavage
of the polyketide moiety .
most significantly , the p450 monooxygenase
is shown to catalyze successive hydroxylation , bicyclic ring - opening ,
and two epoxidations that generate the sesquiterpenoid core skeleton
of 1 .
we also characterized a truncated polyketide synthase
with a ketoreductase function that controls the configuration at c-5
of hydroxylated intermediates . |
unhealthy lifestyle behaviors including physical inactivity during childhood might track to adulthood , and in addition to their short - term health hazards , they might be associated with chronic diseases later in life .
sedentary lifestyle is a well - documented predisposing factor for obesity , metabolic syndrome , and non - alcoholic fatty liver disease in younger ages resulting in further chronic problems in adulthood , e.g. cardiovascular disease , diabetes mellitus , hyperlipidemia , hypertension , cancers and osteoporosis ( 13 ) .
the data clearly indicated that girls engaged in significantly less physical activity than their counterparts ( 4 ) in both childhood and adolescence ( 57 ) .
thus , there is a critical need to pay attention to girls physical activity patterns and influences .
this is a major concern globally and in iran because it has been reported that iranian girls are less physically active than boys ( 8) .
physical activity describes the actions or movements that one actually makes ( 9 ) and is influenced by a variety of factors ( 10 ) .
lindquist , reynolds and goran ( 1999 ) provided a hierarchal framework that included factors that affect children s physical activity behavior at four levels - physiological factors such as motor skill competence ( msc ) , psychological factors such as perceived motor competence ( pmc ) , socio - cultural factors including parents and family structure , and ecological factors such as the physical environment ( 11 ) .
motor skill competence is mastery of physical skills and movement patterns that enable enjoyable participation in physical activities ( 12 ) , and perceived motor competence is defined as an individual s awareness and belief of their capability to perform both gross and fine motor tasks ( 13 ) . in two separate models , both welk ( 14 ) and stodden et al .
although considering both perceived motor competence and motor skill competence in their conceptual models , they purported different competence more influential on children s physical activity behavior .
welk in his model proposed that although direct effects of motor skill competence on children s activity behavior are possible , indirect effects of perceived motor competence are perhaps more likely ( 14 ) . on the other hand , stodden et al .
, in their conceptual model proposed that the motor skill competence is the primary underlying mechanism that increases children s engagement in physical activity and perceived motor competence mediates relationbetween motor skill competence and physical activity ( 15 ) .
there is no consensus on the influence of perceived motor competence and motor skill competence on children s physical activity behavior .
previous researchers have studied the relation between physical activity and msc and pmc in children separately .
evidence revealed positive association between physical self perception ( 16 , 17 ) , perceived competence in physical education ( 18 ) , perceived athletic competence ( 19 ) , perceived sport competence ( 20 ) , and motor skill competence ( 2124 ) , and just one research was found which compared the influence of both pmc and msc on children s physical activity ( 25 ) . motor skill competence , but not perceived motor competence , influenced children s physical activity behavior significantly and explained 9% variance of 9 year - olds girl s physical activity level ( 25 ) .
this research was carried out to bulk out limited evidence of influence of perceived motor competence and motor skill competence on children s physical activity behavior .
therefore , this cross - sectional study aimed to investigate correlates of iranian girls physical activity and determine which variable best predicts third grade school girls physical activity : perceived motor competence or motor skill competence .
third grade girls from a state primary school located in the urban southwestern part of tehran province ( shahr - e - qods ) , iran in 2012 , were invited to voluntarily participate in the study . informed parental consent
was obtained for 352 girls ( mean age 8.7 , sd 0.3 yrs ) and the institutional review board of the corresponding author s university approved the present study .
the sample included children in the low - moderate range of socio - economic background , with no reported history of learning difficulties or behavioral , physical , neurological or orthopedic diagnoses .
accordingly , 352 girls constituted the final sample , who completed iranian version of test of gross motor development-2 ( tgmd-2 ; 26 ) , and physical activity questionnaire for older children ( paqoc ) ( 27 ) , physical ability sub - scale of self - description questionnaire ( 28 ) ( table 1 ) .
the physical activity questionnaire for older children ( paq - c ) was used to determine the participants physical activity level ( 29 ) .
paq - c requests participants to respond to the physical activity behaviours for the last 7 days by asking them to check a list of activities for frequency of participation on a scale from
no , 1 - 2 times per week , 3 - 4 , 5 - 6 , to 7 times or more .
questions are also asked about their physical activity behaviours during physical education ; recess , at lunch - time , right after school , and evenings , as well as
the last weekend. the paq - c demonstrates high test - retest reliability in a sample of 84 children in grades 4 8 and demonstrate interclass correlation coefficients of r = 0.82 for girls and significantly correlates with other measures of physical activity in elementary - age students ( 29 ) .
this questionnaire has been supported as a valid and reliable measure and cronbach s alpha of 0.894 was measured in iranian children ( 27 ) .
motor skill competence was assessed using the test of gross motor development-2 edition ( 30 ) .
the measure is made up of locomotor ( run , gallop , hop , horizontal jump , and slide ) and object control ( t - ball strike , stationary basketball dribble , catch , kick , over hand throw , and underhand roll ) subtests , each assessing six skills .
the tgmd-2 assesses the process of skill performance ( skill components ) rather than the outcome or product of performance ( 30 ) , and has proved to be a valid and reliable method for studying iranian children ( 26 ) . before testing each skill ,
participants were given a visual demonstration of skill by the researcher using the correct technique , but were not told what components of the skill were being assessed .
general encouragement but no verbal feedback on performance was given during or after the tests .
all skills were video - recorded and later assessed by one trained assessor who also administered the test .
after each performance the examiner recorded a score on her form corresponding to the evaluation of that particular skill component .
subtest scores were then summed to calculate each child s gross motor quotient ( gmq ) ( 30 ) .
the self - description questionnaire-1 ( sdq-1 ) was used to assess girls perceived motor competence .
the physical ability subscale of sdq-1 includes 8 items and uses a five - point response scale , with higher scores indicating more positive perception .
scores were summed to create a total perceived motor competence score ranging from 5 to 40 ( 31 ) .
previous researches support the internal consistency and construct validity of sdq-1 scores in iranian children ( 28 ) . in this sample ,
prior to gathering data , participants and parents were given written information about the nature of the study .
written permission was obtained from the participants , parents , and guardians prior to their child s involvement in the study .
no child had any reported history of learning difficulties or any behavioral , neurological or orthopedic problems that would qualify as exclusionary criteria for the history .
first , the tgmd-2 was executed in school yard within 4 weeks and inter - rater objectivity was measured 87% .
the physical subscale of sdq-1 was completed in the classroom , by all children at the same time .
latterly , physical activity questionnaire was completed by all children at the same time at the classroom .
the children were tested by the examiner and assistant who had been trained in the test protocols .
the initial analysis was descriptive in nature ( i.e. , means , standard deviations ) .
this was followed by multiple linear regression models to investigate the best predictor of girls physical activity .
third grade girls from a state primary school located in the urban southwestern part of tehran province ( shahr - e - qods ) , iran in 2012 , were invited to voluntarily participate in the study . informed parental consent
was obtained for 352 girls ( mean age 8.7 , sd 0.3 yrs ) and the institutional review board of the corresponding author s university approved the present study .
the sample included children in the low - moderate range of socio - economic background , with no reported history of learning difficulties or behavioral , physical , neurological or orthopedic diagnoses .
accordingly , 352 girls constituted the final sample , who completed iranian version of test of gross motor development-2 ( tgmd-2 ; 26 ) , and physical activity questionnaire for older children ( paqoc ) ( 27 ) , physical ability sub - scale of self - description questionnaire ( 28 ) ( table 1 ) .
the physical activity questionnaire for older children ( paq - c ) was used to determine the participants physical activity level ( 29 ) .
paq - c requests participants to respond to the physical activity behaviours for the last 7 days by asking them to check a list of activities for frequency of participation on a scale from
no , 1 - 2 times per week , 3 - 4 , 5 - 6 , to 7 times or more .
questions are also asked about their physical activity behaviours during physical education ; recess , at lunch - time , right after school , and evenings , as well as
the last weekend. the paq - c demonstrates high test - retest reliability in a sample of 84 children in grades 4 8 and demonstrate interclass correlation coefficients of r = 0.82 for girls and significantly correlates with other measures of physical activity in elementary - age students ( 29 ) .
this questionnaire has been supported as a valid and reliable measure and cronbach s alpha of 0.894 was measured in iranian children ( 27 ) .
motor skill competence was assessed using the test of gross motor development-2 edition ( 30 ) .
the measure is made up of locomotor ( run , gallop , hop , horizontal jump , and slide ) and object control ( t - ball strike , stationary basketball dribble , catch , kick , over hand throw , and underhand roll ) subtests , each assessing six skills .
the tgmd-2 assesses the process of skill performance ( skill components ) rather than the outcome or product of performance ( 30 ) , and has proved to be a valid and reliable method for studying iranian children ( 26 ) . before testing each skill ,
participants were given a visual demonstration of skill by the researcher using the correct technique , but were not told what components of the skill were being assessed .
general encouragement but no verbal feedback on performance was given during or after the tests .
all skills were video - recorded and later assessed by one trained assessor who also administered the test . after each performance the examiner recorded a score on her form corresponding to the evaluation of that particular skill component .
subtest scores were then summed to calculate each child s gross motor quotient ( gmq ) ( 30 ) .
the self - description questionnaire-1 ( sdq-1 ) was used to assess girls perceived motor competence .
the physical ability subscale of sdq-1 includes 8 items and uses a five - point response scale , with higher scores indicating more positive perception .
scores were summed to create a total perceived motor competence score ranging from 5 to 40 ( 31 ) .
previous researches support the internal consistency and construct validity of sdq-1 scores in iranian children ( 28 ) . in this sample ,
prior to gathering data , participants and parents were given written information about the nature of the study . written permission was obtained from the participants , parents , and guardians prior to their child s involvement in the study .
no child had any reported history of learning difficulties or any behavioral , neurological or orthopedic problems that would qualify as exclusionary criteria for the history .
first , the tgmd-2 was executed in school yard within 4 weeks and inter - rater objectivity was measured 87% .
the physical subscale of sdq-1 was completed in the classroom , by all children at the same time .
latterly , physical activity questionnaire was completed by all children at the same time at the classroom .
the children were tested by the examiner and assistant who had been trained in the test protocols .
the initial analysis was descriptive in nature ( i.e. , means , standard deviations ) .
this was followed by multiple linear regression models to investigate the best predictor of girls physical activity .
table 1 provides an overview of descriptive statistics . sample characteristics and descriptive statistics ( n=352 ) pearson correlations showed significant relationships among correlates ( table 2 ) .
a stepwise regression analysis indicates that gmq and pmc accounted for significant amounts of variance for moderate to vigorous physical activity ( r=0.21 , f=48.9 , p=0.001 ) . and ,
gmq ( r=0.15 , p= 0.001 ) resulted in more influence on pa in comparison with pmc ( r=0.06 , p=0.001 ( table 3 ) .
pearson s bivariate correlations among measures ( n=352 ) predictors of girls moderate to vigorous physical activity ( n=352 )
a stepwise regression analysis indicates that gmq and pmc accounted for significant amounts of variance for moderate to vigorous physical activity ( r=0.21 , f=48.9 , p=0.001 ) . and ,
gmq ( r=0.15 , p= 0.001 ) resulted in more influence on pa in comparison with pmc ( r=0.06 , p=0.001 ( table 3 ) .
pearson s bivariate correlations among measures ( n=352 ) predictors of girls moderate to vigorous physical activity ( n=352 )
this study describes the influential role of both pmc and msc on physical activity level of third grade school girls .
the multiple regression models showed that both pmc and msc are significant predictors of girls physical activity , and of particular interest was that msc had more influence in comparison with pmc on physical activity level .
our results suggest that having mastery of motor skills ( which includes locomotor and object control skills ) and high and positive perception of motor competence promote girls physical activity participation and as a result might reduce physical inactivity behavior .
this findings support stodden s conceptual model which shows that msc , not pmc , is a more influential factor on physical activity level in young children ( 15 ) .
the model showed that msc is underlying mechanism which influence children s physical activity participation ; and in middle and later childhood ( 6 - 10 year - olds ) , also higher levels of msc will offer a greater motor repertoire to engage in various physical activities , sports , and games ; in fact , msc drives physical activity levels ( 15 ) .
the theoretical basis explaining the mechanism of the more influential role of msc on physical activity , as we found among girls in the current study , suggests that proficiency in movement skills provides the behavioral competency required for participation in a variety of physical activity ( 32 ) , and may influence other determinants of behavior , such as perceptions of competence ( 20 ) . without the prerequisite movement competence
, children may opt out of opportunities for physical activity or may have preference for more sedentary pursuits ( 14 , 15 ) .
the results , also , support mcintyre s longitudinal study which msc was better predictor of children s physical activity than pmc(25 ) .
pmc was not a significant contributor to 9-year - olds girls physical activity levels ( 25 ) . however , our results along with previous studies revealed both msc ( 2023 ) and pmc ( 1620 ) are significant correlates of youth s physical activity .
mcintyre s study is the first longitudinal research which investigated influential roles of msc and pmc on physical activity level jointly ( 25 ) .
moreover , although pmc made significant contribution to our sample s physical activity , the variance just was 6% .
the video assessments of msc using a validated , process - oriented measure , was a method that reduced measurement bias in the current study .
however , physical activity assessment in this study may have been limited by the use of paqoc .
this questionnaire does not provide an estimate of caloric expenditure or specific frequency , time , and intensity information and does not discriminate between specific activity intensities ( 29 ) .
also , the findings of this research should be interpreted cautiously , since physical activity was assessed during autumn / winter and the data may also have been confounded by seasonal and weather - related variations .
high competent girls in motor skill have motor repertoire to engage in various physical activities , sports , and games .
this study highlights a need for intervention programs based on influential factors in order to promote children s physical activity behavior and healthy lifestyle in the future .
ethical issues including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . have been taken into consideration by authors . | abstractbackgroundthe main purpose of this study was to determine which correlate , perceived motor competence or motor skill competence , best predicts girls physical activity behavior.methodsa sample of 352 girls ( mean age=8.7 , sd=0.3 yr ) participated in this study . to assess motor skill competence and perceived motor competence , each child completed the test of gross motor development-2 and physical ability sub - scale of marsh s self - description questionnaire .
children s physical activity was assessed by the physical activity questionnaire for older children .
multiple linear regression model was used to determine whether perceived motor competence or motor skill competence best predicts moderate - to - vigorous self - report physical activity.resultsmultiple regression analysis indicated that motor skill competence and perceived motor competence predicted 21% variance in physical activity ( r2=0.21 , f=48.9 , p=0.001 ) , and motor skill competence ( r2=0.15 , =0.33 , p= 0.001 ) resulted in more variance than perceived motor competence ( r2=0.06 , =0.25 , p=0.001 ) in physical activity.conclusionresults revealed motor skill competence had more influence in comparison with perceived motor competence on physical activity level .
we suggest interventional programs based on motor skill competence and perceived motor competence should be administered or implemented to promote physical activity in young girls . |
middle aged men , even in a good state of health , frequently develop abdominal obesity , impaired glucose tolerance , metabolic syndrome , lack of energy and chronic fatigue , loss of libido , erectile dysfunction , change in body composition as well as coronary artery disease with its sequelae .
, these conditions are only in part attributed to ageing itself , and have been referred to a generalized decline of male hormones , including testosterone and dehydroepiandrosterone , which has been referred to the non - scientific term
andropause. of interest , most publications in this regard are review articles rather than data reports referring to clinical studies , since controversies exist regarding the clinical significance of the male climacterium or late - onset hypogonadism. the combination of androgen deficiency , sexual dysfunction and metabolic syndrome often presents a re - enforcing triad .
in addition , low levels of androgens have been associated with cardiovascular disease progression , especially coronary artery disease , and increased mortality. the metabolic syndrome itself is considered a risk factor for the development of generalized atherosclerosis , coronary artery disease , myocardial infarction and stroke . , the metabolic syndrome refers to the co - existence of diabetes mellitus or impaired glucose tolerance , hypertension , obesity , dyslipidemia and/or micro - albuminuria , based on definitions that slightly differ with regard to its predictive characteristics for the development of coronary artery disease between the american heart association / national heart / lung / blood institute criteria and others .
the contributing factor of a diet high in saturated fats and carbohydrates and lack of exercise further culminates in endothelial dysfunction as an early marker for an underlying vascular disease process .
several life - style changing programs and diets as well as exercise recommendations have been established to either prevent or reverse early metabolic syndrome with its consequences .
a recently published trial from germany reported a 50% reduction in the prevalence of the metabolic syndrome following a 12-week low - calorie diet . on the other hand ,
non - adherence to recommended life - style changes that are oftentimes associated with lack of adequate physician supervision , might reverse achievements in reduced body weight , fitness and improved cardiovascular profile over time and therefore , lack long - term success rates . using a more strict physician - supervised strategy , we tested a commercially available nutrition - exercise program with the addition of testosterone substitution in a group of partial androgen deficient obese middle - aged men and evaluated its effect on body weight and composition , glucose and lipid levels and potential side effects .
middle - aged men with overweight or obesity defined as a body mass index ( bmi ) above 26 kg / m were randomly selected from a commercially available nutritional and exercise program which was part of the cenegenics health evaluation .
the program has enrolled more than 20,000 individuals within usa with 2000 new patients per year .
most individuals were self - referrals to the program in order to initiate life - style changes , loose weight , or improve personal fitness .
patients from this pool were included in the study analysis if they fulfilled the following criteria : ( 1 ) overweight or obesity , i.e. , bmi > 26 kg / m ; ( 2 ) clinical signs of androgen deficiency such as abdominal obesity , lack of energy , fatigue , loss of libido or erectile dysfunction ; ( 3 ) total free testosterone serum levels below 100 pg / ml ; ( 4 ) willingness to undergo a life - style change including preparedness to adhere to a recommended , physician supervised and guided exercise and nutritional program ; and ( 5 ) consent to undergo testosterone replacement therapy by using weekly self - administered intramuscular injections as long as clinically indicated . in order to analyze a relative homogeneous group of individuals , only patients without a history of cardiovascular disease or any chronic debilitating diseases that might compromise participation in a scheduled exercise regimen were included .
additional exclusion criteria for the selection in the analysis was a history of diabetes mellitus , hypertension or hyperlipidemia , with or without treatment , elevated prostate specific antigen ( psa ) levels ( > 3 ng / ml ) and a history of prostate carcinoma . all individuals agreed that their data were analyzed blindly .
the study was approved by the review board of the cenegenics education and research foundation .
the following parameters were analyzed : ( 1 ) age , body weight , body composition with regard to body fat and muscle mass , baseline routine lab parameters ( lipid panel , fasting glucose , hemoglobin a1c ( hba1c ) , prostate specific antigen ( psa ) , free and total testosterone levels ) , and maximal exercise capacity ( mixed venous oxygen ( mvo2 ) ) at baseline ; ( 2 ) body weight and composition and lab parameters at follow up .
follow - up consisted of data collection , patient evaluation and laboratory tests between 6 months and 18 months after enrollment into the program .
the aim of the program , which is physician led and supervised , is risk factor management for aging men with the goal to assess risks and convert these into a proactive self management by use of a customized and individualized guide following a balanced low glycemic nutrition program to manage the effects of high glycemic foods on glucose metabolism without caloric restrictions and a balanced exercise program including aerobic activity and resistance training paired with high intensity exercise .
the exercise training program is designed on an individual basis based on personal questionnaires , individual goal , the fitness levels ( based on maximal oxygen consumption ) and exercise habits .
hormone optimization is based on clinical signs of androgen deficiency or partial androgen deficiency and on baseline free testosterone levels .
the entire program is directly guided by a physician with the assistance of trained nutritionists and personal trainers .
most importantly , the physician provides ongoing personal consultation , initially on a weekly base followed by monthly telephone check - ups as well as further follow - up interviews or tests as needed for the entire duration of the program .
all patients were instructed to report any side effects or adverse events immediately to the program physician as well as their primary care physicians , who were informed about the participation of their patients in the program .
in contrast to several other commercially available programs , the program described here was not intended to prevent aging and did not claim to increase longevity .
data of 56 males were analyzed ( age 52.3 7.8 years , median 52.5 , range 37 to 69 years , table 2 ) . of interest , 12 ( 21.4% )
as per the patients information , all participated in the recommended nutritional and exercise program that was slightly different among the participants since it was designed per the individual 's condition .
all men injected once or twice weekly testosterone cypionate between 80 mg and 200 mg per week , with an average dose of 113.0 27.7 mg ( 200 mg / ml ) , intramuscularly .
dose and frequency of testosterone injection ( once or twice weekly ) were chosen by the program physicians based on the individuals ' baseline testosterone levels .
baseline total testosterone measured 437.5 197.5 ng / dl , baseline free testosterone was 76.7 37.9 pg / ml ( 1.81 0.44 % free testosterone )
. baseline free testosterone of less than 100 pg / ml in combination with clinical signs of partial androgen deficiency was considered below acceptable levels .
psa was 0.91 0.67 ng / ml . at follow up , total testosterone was 890.0 488.9 ng / dl , free testosterone was 217.6 134.1 pg / ml ( 2.31 0.59% ) , p < 0.005 compared to baseline for all comparisons .
the average mvo2 at baseline was 28.6 6.0 ml / kg per minute , which , within the age group of 5059 year old males , is considered low , representing a significantly reduced physical capacity for the patients studied ( reference range : very low < 26.1 ; low 26.130.9 ; fair 31.035.7 , good 35.840.9 , excellent 41.045.3 , superior 45.3 ) .
follow up data on mvo2 after program enrollment was not available for the current analysis in this patient cohort .
tg : triglycerides ; chol : total cholesterol ; ldl : low density lipoprotein ; hdl : high density lipoprotein ; hba1c : hemoglobin a1c ; bmi : body mass index ; * p < 0.005 compared to baseline ( for details see text ) . upon self - referral ,
the weight was 233.9 30.0 lbs , average height was 178.6 6.7 cm , bmi was 33.2 3.3 kg / m , median 32.2 kg / m , range 27.5 to 45.0
forty seven patients lost weight , ranging from 1 lbs to 42.9 lbs , only 4 patients gained weight ranging from 4.8 lbs to 17.2 lbs . at follow up ,
weight was 221.3 25.1 lbs ( p < 0.001 vs. baseline , bmi was 31.3 2.8 kg / m , p < 0.0001 vs. baseline ) .
total body fat was 27.1% 5.2% at follow - up compared to 34.3 5.7% at baseline , p < 0.0001 .
total body fat was reduced in 50 patients , only two patients each had a slight increase in total body fat of 2.2% .
one of these patients also had an increase in total body weight , and one patient had no change .
upon examination of the patients ' medical records , it appeared that these two patients did not strictly adhere to the program recommendations for exercise and nutrition for more than one month after initiation of the program , supposedly because of lack of time and opportunity , as assessed in a personal interview at the end of the observation period .
for the entire group , no changes were found in total body bone density between baseline and follow up measurements . considering the normal range for fasting blood glucose between 70 mg / dl and 100 mg / dl , at baseline , 16 patients ( of 53 patients in whom data was available , 30.2% ) were considered above normal values even though no history of diabetes was known in any of the patients . at follow up
, only seven patients still had fasting glucose levels above 100 mg / dl ( 13% ) .
fasting glucose was reduced from 95.30 14.40 mg / dl at baseline to 87.51 12.58 mg / dl at follow up , p < 0.0001 .
hba1c measured 5.7% 0.6% at baseline vs. 5.5% 0.3% at follow up , p < 0.05 .
total fasting cholesterol was 195.4 33.0 mg / dl and reduced to 172.7 35.0 mg / dl at follow up , p < 0.005 .
triglyceride levels were reduced from 154.1 97.6 mg / dl to 100.2 38.5 mg / dl , and ldl was reduced from 122.4 29.8 mg / dl to 108.6 28.2 mg / dl at follow up ( p < 0.005 for both comparisons ) , whereas hdl was unchanged ( 46.7 11.5 mg / dl at baseline , 47.3 13.1 mg / dl at follow up , p = 0.33 ) .
no significant adverse events were reported from intramuscular testosterone injections except slight pain at the injection site in five cases , in one case development of a hematoma ( size not quantified ) , in another case the development of a hard nodule at the injection site that did not require any further intervention .
psa levels at follow up were slightly elevated 1.31 1.27 ng / ml vs. 0.91 0.67 at baseline , but within the normal range .
no follow up lab results resulted in cessation of testosterone supplementation , i.e. , there was no reported polycythemia , significant elevation of psa levels above normal , or significant side effects that prompted either the primary care physicians or the program physicians to stop testosterone injections or any portion of the program within the reported follow up period of 618 months after enrollment .
upon self - referral , the weight was 233.9 30.0 lbs , average height was 178.6 6.7 cm , bmi was 33.2 3.3 kg / m , median 32.2 kg / m , range 27.5 to 45.0 kg / m .
forty seven patients lost weight , ranging from 1 lbs to 42.9 lbs , only 4 patients gained weight ranging from 4.8 lbs to 17.2 lbs . at follow up ,
weight was 221.3 25.1 lbs ( p < 0.001 vs. baseline , bmi was 31.3 2.8 kg / m , p < 0.0001 vs. baseline ) .
total body fat was 27.1% 5.2% at follow - up compared to 34.3 5.7% at baseline , p < 0.0001 .
total body fat was reduced in 50 patients , only two patients each had a slight increase in total body fat of 2.2% .
one of these patients also had an increase in total body weight , and one patient had no change .
upon examination of the patients ' medical records , it appeared that these two patients did not strictly adhere to the program recommendations for exercise and nutrition for more than one month after initiation of the program , supposedly because of lack of time and opportunity , as assessed in a personal interview at the end of the observation period .
for the entire group , no changes were found in total body bone density between baseline and follow up measurements .
considering the normal range for fasting blood glucose between 70 mg / dl and 100 mg / dl , at baseline , 16 patients ( of 53 patients in whom data was available , 30.2% ) were considered above normal values even though no history of diabetes was known in any of the patients . at follow up , only seven patients still had fasting glucose levels above 100 mg / dl ( 13% ) .
fasting glucose was reduced from 95.30 14.40 mg / dl at baseline to 87.51 12.58 mg / dl at follow up , p < 0.0001 .
hba1c measured 5.7% 0.6% at baseline vs. 5.5% 0.3% at follow up , p < 0.05 .
total fasting cholesterol was 195.4 33.0 mg / dl and reduced to 172.7 35.0 mg / dl at follow up , p
triglyceride levels were reduced from 154.1 97.6 mg / dl to 100.2 38.5 mg / dl , and ldl was reduced from 122.4 29.8 mg / dl to 108.6 28.2 mg / dl at follow up ( p < 0.005 for both comparisons ) , whereas hdl was unchanged ( 46.7 11.5 mg / dl at baseline , 47.3 13.1 mg / dl at follow up , p = 0.33 ) .
no significant adverse events were reported from intramuscular testosterone injections except slight pain at the injection site in five cases , in one case development of a hematoma ( size not quantified ) , in another case the development of a hard nodule at the injection site that did not require any further intervention .
psa levels at follow up were slightly elevated 1.31 1.27 ng / ml vs. 0.91 0.67 at baseline , but within the normal range .
no follow up lab results resulted in cessation of testosterone supplementation , i.e. , there was no reported polycythemia , significant elevation of psa levels above normal , or significant side effects that prompted either the primary care physicians or the program physicians to stop testosterone injections or any portion of the program within the reported follow up period of 618 months after enrollment .
in the present analysis , we present data on 56 middle- aged obese men undergoing a physician - directed and supervised dietary and exercise program in order to improve general health , which is combined with an attempt to achieve hormonal balance with testosterone substitution in case of documented laboratory or clinical signs of androgen deficiency .
the motivation for participation was a desire to improve overall health , reduce weight , improve fitness and reduce cardiac ( coronary ) risk factors .
our data demonstrate that by conducting the recommended dietary and daily exercise changes in combination with testosterone substitution , obese males achieved significant weight loss and a significant reduction in common risk factors for the development of coronary artery disease . of importance
, there is a high prevalence of the metabolic syndrome among men who willingly undergo a life - style modification , as reported earlier .
moreover , the implementation of the prescribed program including the application of testosterone in men without contraindications did not result in any serious side effects and thus , is considered safe if adequately supervised and controlled by a physician appropriately trained in hormone therapy .
the fact that an exercise program can improve overall health and reduce risk factors is not innovative .
however , only few studies investigated a comprehensive nutritional and exercise program combined with hormone balancing in middle aged men . a recent study among sedentary women with metabolic syndrome showed that an incremental approach to 10,000 steps per day resulted in improvements of waist circumference and fasting glucose , bmi and resting heart rate .
similar to our data , a study from japan demonstrated that heart rate recovery also has been shown to be beneficially altered by exercise training among obese men with metabolic syndrome .
exercise by itself has been shown in several studies to promote weight loss , especially in women .
the combination of certain diets exercise ( resistance ) training appears to be more efficient in changing body composition . outside the frame of clinical trials , however , there is a lack of data on long - term success rates after cessation of the programs .
therefore , predictors have been stratified to identify individuals who can successfully undergo life - style modification programs including weight loss .
there is no head to head comparison between the exercise - dietary program described here with different strategies or programs .
the scientific analysis of additional androgen substitution , however , in this context is a novel approach that has not been described before in a similar manner .
in fact , a pubmed analysis with the search terms exercise training , weight loss , diet and testosterone does not reveal any studies performed in men .
in contrast , a vast number of publications on hormonal therapy in combination with life style changes in women were published .
its effects on ageing , the metabolic syndrome , diabetes mellitus , and atherosclerosis require further controlled studies . of interest ,
agents targeting the androgen receptor , called selective androgen receptor modulators , are under clinical trials to test for its anabolic and tissue preservation effects , as it has been reviewed recently .
controlled studies have revealed a positive effect of testosterone substitution on body composition , muscle strength , bone metabolism and erythropoesis .
moreover , a protective effect on the development of coronary artery disease can be expected by an improvement of the lipid profile , decrease of obesity and insulin resistance , i.e. , a reduction of risk factors for the development of progression of atherosclerosis .
contraindications for testosterone - supplementation such as carcinoma of the prostate and elevated psa - values as well as possible side effects of the treatment such as polyglobuly and sleep - apnea- syndrome require close attention before and observance during the replacement therapy . whether testosterone substitution is clinically indicated in cases as described here with clinical features of the partial androgen deficiency
testosterone therapy in our cases was initiated based on clinical findings of ( partial ) androgen deficiency in combination with a reduction in total free testosterone serum levels below 100 pg / ml .
this cut - off level might be debatable , however , in combination with the clinical findings and our past experience , raising free testosterone to levels above 120 - 150 pg / ml had been associated with improved well - being and reduction in coronary risk factors . despite our significant results with regard to weight loss and reduction in total body fat and coronary risk factors , our study has several limitations : ( 1 ) no control group has been studied .
however , the data represent the analysis of 56 patients following a systematic and physician supervised program that has not been reported in a similar way before .
( 2 ) since all patients were self - referred to a commercial life - style changing program , a selection bias might be considered since the participants were highly motivated to undergo and follow through the program . on the other hand
, this motivation may support compliance and usually provides a more strict adherence to the nutritional and exercise portions , which ensured analysis of a relative homogeneous group of patients .
also , hdl was unchanged but was considered normal even before starting the program , which most likely is caused by the fact that most of the individuals already did some degree of exercise training before but without appropriate guidance and supervision that was not adequate enough to loose weight .
the program described here did not consist of testosterone substitution alone but also of an exercise and nutritional program with the goal of weight loss and enhancing physical fitness .
no patients were studied without testosterone replacement therapy , but in our experience , the combination of weight loss nutrition , exercise and hormonal balancing had been more successful in achieving the targeted endpoints .
we present the first report of a combined commercially available exercise - nutrition program with testosterone replacement therapy in middle aged men with a partial androgen deficiency syndrome that resulted in a highly significant weight loss , reduction of total body fat and improvement in lipid and glucose levels among the participants .
testosterone substitution in this group appeared safe and might have supported the effects of weight reduction diet and exercising , as long as adequate physician supervision and follow - up was provided .
further studies on the long term effects of hormone replacement in partial androgen deficient men for the purposes of changes in body composition and reduction of risk factors for the development of chronic diseases such as atherosclerosis and coronary artery disease are required .
we present the first report of a combined commercially available exercise - nutrition program with testosterone replacement therapy in middle aged men with a partial androgen deficiency syndrome that resulted in a highly significant weight loss , reduction of total body fat and improvement in lipid and glucose levels among the participants .
testosterone substitution in this group appeared safe and might have supported the effects of weight reduction diet and exercising , as long as adequate physician supervision and follow - up was provided .
further studies on the long term effects of hormone replacement in partial androgen deficient men for the purposes of changes in body composition and reduction of risk factors for the development of chronic diseases such as atherosclerosis and coronary artery disease are required . | backgroundpartial androgen deficiency syndrome in the aging male is associated with signs of aging such as a development of abdominal obesity , sexual dysfunction , increase body fat , weight gain and the development of cardiac disease.objectivewe assessed the outcome of a commercially available physician supervised nutrition and exercise program with concomitant testosterone replacement therapy in middle age obese men with partial androgen deficiency in order to reduce cardiac risks factors.methodsfifty-six self referred men without diabetes mellitus , hypertension , or cardiovascular disease ( ages 52.3 7.8 years ) were randomly selected from a large cohort .
baseline weight , body fat composition , fasting glucose , hemoglobin a1c and fasting lipid levels , as well as free and total testosterone levels were assessed .
all patients were assessed and followed 618 months after initiation of the program . the program consisted of a low glycemic load balanced nutrition diet , a recommended structured daily exercise program of 3060 minutes , as well as once to twice weekly intramuscular testosterone injections ( 113.0 27.8 mg).resultsat follow up , weight was reduced from 233.9 30.0 pounds ( lbs ) to 221.3 25.1 lbs ( p < 0.001 ) , bmi was reduced from 33.2 3.3 kg / m2 to 31.3 2.8 kg / m2 ( p < 0.0001 ) .
total body fat was 27.1% 5.2% vs. 34.3% 5.7% at baseline ( p < 0.0001 ) .
fasting glucose was reduced from 95.3 14.4 mg / dl to 87.5 12.6 mg / dl ( p < 0.0001 ) .
total cholesterol was reduced from 195.4 33.0 mg / dl to 172.7 35.0 mg / dl ( p < 0.005 ) .
no clinically significant adverse events were recorded.conclusionstestosterone replacement therapy in middle aged obese men with partial androgen deficiency appeared safe and might have promoted the effects of a weight reduction diet and daily exercise program as long as an adequate physician supervision and follow up was granted .
the combination therapy significantly reduced coronary risk factors such as glucose intolerance and hyperlipidemia . |
since june 2002 , the finnish pediatric diabetes register and biobank has invited all pediatric patients diagnosed with diabetes in finland to participate in the register and the biobank . compared with the information provided by all the pediatric units in finland , the coverage of the register is 92% ( 25 ) . around 70% of the families participating in the register also provide blood samples for the biobank from the index case and/or the first - degree relatives as soon as possible after the diagnosis of the index case .
the samples are analyzed for diabetes - related autoantibodies ( ica , iaa , gada , and antibodies to the islet antigen 2 molecule [ ia-2a ] ) and hla - dr - dqa1-dqb1 haplotypes ( 26 ) .
families participating in the register receive a structured questionnaire ( supplementary data ) at the time of diagnosis of the index child .
a diabetes nurse or doctor answers the questions regarding clinical status and degree of metabolic decompensation at diagnosis and assists the family with questions on the family history of diabetes .
the total number of first - degree relatives ( parents and siblings ) is asked , but the number of second - degree relatives other than grandparents ( i.e. , siblings of parents ) is unknown . for parents , siblings , and grandparents , the diabetes status (
no , yes , or unknown ) and the diabetes type ( type 1 , type 2 , gestational , or other diabetes ) are asked separately , and families are asked to list any other relatives with type 1 diabetes . if the family is unsure of the diabetes type of a relative , the diabetes doctor or nurse classifies the disease according to the information provided by the family . for our analysis ,
however , parents with type 2 diabetes marked in the questionnaire were considered to have type 1 diabetes if autoantibodies other than iaa were detectable in their serum ( 8 of 2,916 , 0.3% ) .
the legal guardians of the children and their siblings 18 years of age or older give informed written consent .
the protocol has been approved by the ethics committee of the hospital district of helsinki and uusimaa . by october 2006 ,
the median age was 8.23 years ( range 016.98 ) , and 56.2% were male .
we excluded children with age at diagnosis > 15 years , no information on their relatives in the register , or incomplete diabetes - associated autoantibody analyses by april 2007 .
one child with an affected father and two affected brothers was excluded because a novel insulin gene mutation was recognized .
we planned to exclude any autoantibody - negative children with diabetes occurring in three successive generations as suspected mody ( maturity - onset diabetes of the young ) cases .
only the first child from any family to be diagnosed with diabetes and registered was included as an index case .
the demographic characteristics of the children included in or excluded from the study did not differ .
for most of the children ( 92.2% ) , the sample was drawn within 14 days .
those with the serum sample taken > 30 days after the diagnosis ( 103 of 1,488 , 6.9% ) were excluded from the autoantibody analysis . we have shown that insulin antibody levels 1 month after the diagnosis correlate more strongly with the iaa titers at diagnosis than with insulin antibody levels 3 months later ( unpublished data ) , indicating that even insulin antibodies detected 30 days after the diagnosis reflect an autoimmune response rather than a response to exogenous insulin .
1 displays the grouping of the case subjects . for the analyses , the following categories were used .
second , we used a classification into familial cases with affected first - degree relatives , familial with affected second - degree relatives , and familial with type 1 diabetes in both first- and second - degree relatives , as well as sporadic cases .
third , a closer analysis of familial cases was performed according to who in the immediate family ( parent or sibling ) or extended family ( paternal or maternal second - degree relative ) had diabetes .
grouping of the index case subjects according to who in the extended family was affected by type 1 diabetes .
the dashed lines refer to the case subjects with affected relatives from more than one category of relatives .
these case subjects are also included in the total number of each category of relatives .
icas were analyzed with indirect immunofluorescense on human group 0 donor pancreas with 2.5 jdfu as the detection limit ( 27 ) .
iaas , gadas , and ia-2as were quantified with specific radiobinding as - says ( 2830 ) .
the cutoff limits for antibody positivity were determined as the 99th percentiles in 354 finnish , nondiabetic children and adolescents , and were 2.80 relative units ( ru ) for iaa , 5.36 ru for gada , and 0.77 ru for ia-2a . according to the 2005 diabetes autoantibody standardization program ,
the disease sensitivities of these assays were 44 , 82 , and 72% and specificities were 98 , 97 , and 100% , respectively .
when calculating the median antibody titers , we included only samples at or above the cutoff for antibody positivity .
hla typing of major dr - dq haplotypes was performed with a pcr - based , lanthanide - labeled hybridization method using time - resolved fluorometry for detection ( 26 ) .
blood ph , plasma glucose , and -hydroxybutyrate levels of the index children were analyzed in local laboratories at the time of diagnosis .
hemoglobin a1c was not recorded because the methods used locally were not standardized and , accordingly , varied considerably between laboratories , hampering any nationwide comparison .
spss 17.0 and 19.0 statistical software packages ( spss , inc . , chicago , il ) were used for statistical analyses .
cross tabulation , statistics , and fisher exact test were applied for comparing frequencies of different variables .
differences in levels of variables were analyzed with student t test or one - way anova for parametric and mann - whitney u test / wilcoxon rank sum test or kruskal - wallis test with dunn post hoc test for nonparametric variables .
a two - tailed p value of 0.05 or less was considered statistically significant , and bonferroni correction for multiple comparisons was not applied due to its overly conservative nature .
since june 2002 , the finnish pediatric diabetes register and biobank has invited all pediatric patients diagnosed with diabetes in finland to participate in the register and the biobank . compared with the information provided by all the pediatric units in finland , the coverage of the register is 92% ( 25 ) . around 70% of the families participating in the register also provide blood samples for the biobank from the index case and/or the first - degree relatives as soon as possible after the diagnosis of the index case .
the samples are analyzed for diabetes - related autoantibodies ( ica , iaa , gada , and antibodies to the islet antigen 2 molecule [ ia-2a ] ) and hla - dr - dqa1-dqb1 haplotypes ( 26 ) .
families participating in the register receive a structured questionnaire ( supplementary data ) at the time of diagnosis of the index child .
a diabetes nurse or doctor answers the questions regarding clinical status and degree of metabolic decompensation at diagnosis and assists the family with questions on the family history of diabetes .
the total number of first - degree relatives ( parents and siblings ) is asked , but the number of second - degree relatives other than grandparents ( i.e. , siblings of parents ) is unknown . for parents , siblings , and grandparents , the diabetes status (
no , yes , or unknown ) and the diabetes type ( type 1 , type 2 , gestational , or other diabetes ) are asked separately , and families are asked to list any other relatives with type 1 diabetes . if the family is unsure of the diabetes type of a relative , the diabetes doctor or nurse classifies the disease according to the information provided by the family . for our analysis ,
however , parents with type 2 diabetes marked in the questionnaire were considered to have type 1 diabetes if autoantibodies other than iaa were detectable in their serum ( 8 of 2,916 , 0.3% ) .
the legal guardians of the children and their siblings 18 years of age or older give informed written consent .
the protocol has been approved by the ethics committee of the hospital district of helsinki and uusimaa . by october 2006 ,
the median age was 8.23 years ( range 016.98 ) , and 56.2% were male .
we excluded children with age at diagnosis > 15 years , no information on their relatives in the register , or incomplete diabetes - associated autoantibody analyses by april 2007 .
one child with an affected father and two affected brothers was excluded because a novel insulin gene mutation was recognized .
we planned to exclude any autoantibody - negative children with diabetes occurring in three successive generations as suspected mody ( maturity - onset diabetes of the young ) cases .
only the first child from any family to be diagnosed with diabetes and registered was included as an index case .
the demographic characteristics of the children included in or excluded from the study did not differ .
for most of the children ( 92.2% ) , the sample was drawn within 14 days .
those with the serum sample taken > 30 days after the diagnosis ( 103 of 1,488 , 6.9% ) were excluded from the autoantibody analysis . we have shown that insulin antibody levels 1 month after the diagnosis correlate more strongly with the iaa titers at diagnosis than with insulin antibody levels 3 months later ( unpublished data ) , indicating that even insulin antibodies detected 30 days after the diagnosis reflect an autoimmune response rather than a response to exogenous insulin .
1 displays the grouping of the case subjects . for the analyses , the following categories were used .
second , we used a classification into familial cases with affected first - degree relatives , familial with affected second - degree relatives , and familial with type 1 diabetes in both first- and second - degree relatives , as well as sporadic cases .
third , a closer analysis of familial cases was performed according to who in the immediate family ( parent or sibling ) or extended family ( paternal or maternal second - degree relative ) had diabetes .
grouping of the index case subjects according to who in the extended family was affected by type 1 diabetes .
the dashed lines refer to the case subjects with affected relatives from more than one category of relatives .
these case subjects are also included in the total number of each category of relatives .
icas were analyzed with indirect immunofluorescense on human group 0 donor pancreas with 2.5 jdfu as the detection limit ( 27 ) .
iaas , gadas , and ia-2as were quantified with specific radiobinding as - says ( 2830 ) .
the cutoff limits for antibody positivity were determined as the 99th percentiles in 354 finnish , nondiabetic children and adolescents , and were 2.80 relative units ( ru ) for iaa , 5.36 ru for gada , and 0.77 ru for ia-2a . according to the 2005 diabetes autoantibody standardization program ,
the disease sensitivities of these assays were 44 , 82 , and 72% and specificities were 98 , 97 , and 100% , respectively .
when calculating the median antibody titers , we included only samples at or above the cutoff for antibody positivity .
hla typing of major dr - dq haplotypes was performed with a pcr - based , lanthanide - labeled hybridization method using time - resolved fluorometry for detection ( 26 ) .
blood ph , plasma glucose , and -hydroxybutyrate levels of the index children were analyzed in local laboratories at the time of diagnosis .
hemoglobin a1c was not recorded because the methods used locally were not standardized and , accordingly , varied considerably between laboratories , hampering any nationwide comparison .
statistics , and fisher exact test were applied for comparing frequencies of different variables .
differences in levels of variables were analyzed with student t test or one - way anova for parametric and mann - whitney u test / wilcoxon rank sum test or kruskal - wallis test with dunn post hoc test for nonparametric variables .
a two - tailed p value of 0.05 or less was considered statistically significant , and bonferroni correction for multiple comparisons was not applied due to its overly conservative nature .
at diagnosis , the index cases were between 0.28 and 14.99 years of age ( median 8.23 ) .
a majority of the subjects were male ( 846 of 1,488 , 56.9% ) , and 80.6% ( 1,200 of 1488 ) had at least one biological sibling .
the proportion of children with a first - degree relative affected by type 1 diabetes was 12.2% ( fig .
twelve children had two first - degree relatives with type 1 diabetes ( 0.8% ) and one had four ( 0.1% ) .
ninety children ( 6.2% ) had a father with type 1 diabetes , and 47 ( 3.2% ) had an affected mother .
fifty seven subjects ( 3.9% out of a total of 1,488 children or 4.8% out of 1,200 children with at least one sibling ) had a sibling with type 1 diabetes .
of the 1,488 children , 177 ( 11.9% ) had a second - degree relative affected by type 1 diabetes ( fig .
1 ) . the number of these relatives per child varied between one and five .
thirty eight ( 2.8% ) had a paternal grandparent and 40 ( 2.8% ) a maternal grandparent affected by type 1 diabetes , whereas 54 ( 3.7% ) had an affected paternal sibling and 63 ( 4.2% ) an affected maternal sibling .
taken together , 88 ( 6.0% ) index children had an affected second - degree relative from the paternal side and 97 ( 6.5% ) from the maternal side of the family .
the proportions of children with affected grandfathers and grandmothers were similar , with 41 ( 2.9% ) children having an affected grandfather and 37 ( 2.6% ) an affected grandmother . among the 5,580 grandparents with information on their diabetes status , 79 ( 1.4% ) had type 1 diabetes . in summary ,
324 children ( 21.8% ) had a first- and/or second - degree relative with type 1 diabetes , and 35 ( 2.4% ) had both an affected first- and second - degree relative .
the age of the index cases at diagnosis did not differ between any categories of comparisons .
both sexes equally often had a first - degree ( 12.2% of the boys and 12.3% of the girls , p = 1.00 ) or second - degree relative ( 12.4% of boys and 11.2% of girls , p = 0.53 ) with type 1 diabetes .
similarly , both sexes equally often had a father with type 1 diabetes ( 6.4% of the boys and 6.0% of the girls , p = 0.82 ) , a mother with type 1 diabetes ( 3.3% of boys and 3.0% of the girls , p = 0.83 ) , or a sibling with type 1 diabetes ( 3.7% of boys and 4.1% of girls , p = 0.79 )
. however , the proportion of children with an affected paternal second - degree relative was 4.9% among boys and 7.5% among girls ( p = 0.06 ) .
the respective proportion for affected maternal second - degree relatives was 7.8% for boys and 4.8% for girls
( p = 0.03 ) . a conspicuous majority of children with an affected maternal second - degree relative were boys ( 71.9% ) , when compared with those with an affected paternal relative ( 46.6% ) or sporadic cases ( 56.6% , p = 0.002 ) .
at the time of diagnosis , the clinical condition was significantly poorer in sporadic than familial cases ( table 1 ) .
accordingly , the sporadic cases had ketoacidosis ( p < 0.001 ) and impaired level of consciousness ( p = 0.02 ) more often than the familial cases . moreover , their weight loss was greater ( p < 0.001 ) , -hydroxybutyrate ( p < 0.001 ) and plasma glucose concentrations ( p <
0.001 ) were higher , and blood ph was lower ( p < 0.001 ) ( table 1 ) .
when comparing four groups ( table 2 ) , it became evident that the less severe clinical condition at diagnosis was also true for children with an affected second - degree relative .
demographic , metabolic , immunological , and genetic markers in index children with familial type 1 diabetes and sporadic cases demographic , metabolic , immunological , and genetic markers in index children with affected first - degree ( group i ) , second - degree ( group ii ) , or both first- and second - degree relatives ( group iii ) and sporadic cases ( group iv ) no significant differences in the frequencies or titers of various autoantibodies were observed when familial children were compared with sporadic cases ( table 1 ) , or when comparing four groups ( table 2 ) .
however , the children with both affected first- and second - degree relatives tended to have a lower number of positive antibody responses ( table 2 ) .
there were no major differences in the levels or frequencies of autoantibodies between the index children with affected parents or siblings .
the children with familial type 1 diabetes carried the drb1 * 0401/2/4/5-dqa1 * 0301-dqb1 * 0302 ( dr4-dq8 ) haplotype more often than sporadic children ( 74.0 vs. 67.0% , p = 0.02 ) ( table 1 ) . comparison between the four groups ( presented in table 2 ) revealed that the only significant difference was in the frequency of the dr4-dq8 haplotype between sporadic cases and those with an affected second - degree relative ( 67.0 vs. 78.4% , p < 0.04 ) . when comparing the sporadic cases with those with an affected maternal or paternal second - degree relative , the index cases with affected maternal relatives more often carried the dr4-dq8 haplotype than the sporadic cases ( 80.7 vs. 67.0% , p = 0.01 ) .
this finding differed significantly between the sexes , in that boys with affected maternal relatives more often carried the dr4-dq8 haplotype compared with boys with affected paternal relatives ( 81.0 vs. 58.5% , p = 0.03 ) or sporadic cases ( 81.0 vs. 66.5% , p = 0.03 ) ( supplementary table 1 ) .
in contrast , girls with affected paternal relatives had dr4-dq8 ( 84.1 vs.67.7% , p = 0.04 ) and girls with affected maternal relatives had the dr4/non - dr3 genotype ( 68.0 vs. 45.1% , p = 0.04 ) more frequently when compared with the sporadic group ( supplementary table 1 ) .
there were no differences in the hla genetics of the index children with affected parents or siblings .
at the time of diagnosis , the clinical condition was significantly poorer in sporadic than familial cases ( table 1 ) .
accordingly , the sporadic cases had ketoacidosis ( p < 0.001 ) and impaired level of consciousness ( p = 0.02 ) more often than the familial cases . moreover , their weight loss was greater ( p < 0.001 ) , -hydroxybutyrate ( p < 0.001 ) and plasma glucose concentrations ( p <
0.001 ) were higher , and blood ph was lower ( p < 0.001 ) ( table 1 ) . when comparing four groups ( table 2 ) , it became evident that the less severe clinical condition at diagnosis was also true for children with an affected second - degree relative .
demographic , metabolic , immunological , and genetic markers in index children with familial type 1 diabetes and sporadic cases demographic , metabolic , immunological , and genetic markers in index children with affected first - degree ( group i ) , second - degree ( group ii ) , or both first- and second - degree relatives ( group iii ) and sporadic cases ( group iv )
no significant differences in the frequencies or titers of various autoantibodies were observed when familial children were compared with sporadic cases ( table 1 ) , or when comparing four groups ( table 2 ) .
however , the children with both affected first- and second - degree relatives tended to have a lower number of positive antibody responses ( table 2 ) .
there were no major differences in the levels or frequencies of autoantibodies between the index children with affected parents or siblings .
the children with familial type 1 diabetes carried the drb1 * 0401/2/4/5-dqa1 * 0301-dqb1 * 0302 ( dr4-dq8 ) haplotype more often than sporadic children ( 74.0 vs. 67.0% , p = 0.02 ) ( table 1 ) . comparison between the four groups ( presented in table 2 ) revealed that the only significant difference was in the frequency of the dr4-dq8 haplotype between sporadic cases and those with an affected second - degree relative ( 67.0 vs. 78.4% , p < 0.04 ) . when comparing the sporadic cases with those with an affected maternal or paternal second - degree relative , the index cases with affected maternal relatives more often carried the dr4-dq8 haplotype than the sporadic cases ( 80.7 vs. 67.0% , p = 0.01 ) .
this finding differed significantly between the sexes , in that boys with affected maternal relatives more often carried the dr4-dq8 haplotype compared with boys with affected paternal relatives ( 81.0 vs. 58.5% , p = 0.03 ) or sporadic cases ( 81.0 vs. 66.5% , p = 0.03 ) ( supplementary table 1 ) .
in contrast , girls with affected paternal relatives had dr4-dq8 ( 84.1 vs.67.7% , p = 0.04 ) and girls with affected maternal relatives had the dr4/non - dr3 genotype ( 68.0 vs. 45.1% , p = 0.04 ) more frequently when compared with the sporadic group ( supplementary table 1 ) .
there were no differences in the hla genetics of the index children with affected parents or siblings .
this study had an optimal setting to characterize familial and sporadic type 1 diabetes in that the study subjects were derived from a nationwide register with a high level of ascertainment in the country with the highest incidence of type 1 diabetes globally . in keeping with earlier findings
, we observed that 12.2% of the children with newly diagnosed type 1 diabetes had at least one affected first - degree relative ( 811 ) . in previous studies ,
the proportion of type 1 diabetes patients with affected second - degree relatives has varied from 4.8 to 16.7% ( 1,5,11,1719 ) , with a proportion of 16% reported for newly diagnosed pediatric patients ( 5,11 ) . in some studies , third - degree relatives (
5,17,19 ) or only grandparents ( 18 ) have been included in the analyses , which hampers any subsequent comparison .
in addition , misclassification of type 1 diabetes as type 2 diabetes in older individuals might affect the results , as type 1 diabetes in such individuals might be milder at diagnosis and not requiring immediate insulin treatment ( 18,31 ) . in our study ,
11.9% of the children had type 1 diabetes in their second - degree relatives , a proportion similar to that of children with affected first - degree relatives .
taken together , 21.8% had an affected relative among first- and/or second - degree relatives , making the true proportion of children with newly diagnosed sporadic type 1 diabetes < 80% . in accordance with earlier reports ( 812,15,17 ) , a higher proportion of fathers with type 1 diabetes than affected mothers
reports on reduced risk of type 1 diabetes in the same - sex offspring of an affected parent , i.e. , fathers transmitting preferentially to daughters and mothers to sons , are controversial ( 3,4,9,12,16 ) . based on the data at hand
our results suggest , however , a similar phenomenon at the level of second - degree relatives ; affected boys more often had maternal than paternal second - degree relatives with type 1 diabetes . although the statistical power of the analysis is limited , hla genetics support this finding ; boys with affected maternal relatives and girls with affected paternal relatives had higher frequencies of the high - risk hla haplotype dr4-dq8 . in general , the case subjects having affected grandfathers ( 2.9% ) and grandmothers ( 2.6% ) were equally distributed , and the proportions of type 1 diabetes from maternal ( 6.5% ) and paternal ( 6.0% ) second - degree relatives were equal .
as our case subjects were ascertained through offspring rather than affected parents , differential transmission from fathers and mothers as well as reduced risk in the same - sex offspring could theoretically result from the male preponderance among type 1 diabetes cases ( 32 ) .
sex differences in fecundity of affected fathers and mothers might cause a similar bias , but this could be excluded because affected fathers had an average of 2.18 children , compared with 2.02 among diabetic mothers ( p = 0.53 ) .
as expected , those with prior experiences of type 1 diabetes in the immediate family had a milder metabolic decompensation at diagnosis than sporadic cases ( 8,13,21 ) .
this can be readily explained by the better awareness of the parents in terms of early symptoms of type 1 diabetes facilitating a swift diagnosis and initiation of treatment .
recognition of hyperglycemia with blood glucose self - measurement equipment readily available in these families could be part of the explanation .
importantly , our results indicate that the metabolic status at diagnosis is ameliorated also if the relative with type 1 diabetes is a grandparent or parental sibling . in these families ,
the small group of children with type 1 diabetes in both first- and second - degree relatives demonstrated a tendency to have even less metabolic decompensation at diagnosis than those with only one affected patient in the family , emphasizing the accumulated knowledge of the disease in such families .
as reported previously from finland ( 8) , the frequencies or levels of disease - associated autoantibodies in sporadic and familial cases of type 1 diabetes were alike .
we were unable to reproduce the recent findings from israel where higher frequencies of iaa as well as a higher number of positive antibody responses were observed among familial cases ( 13 ) .
as hypothesized , the index cases with affected relatives in the extended family , especially among second - degree relatives , carried the dr4-dq8 haplotype more often than the sporadic cases .
( 8) have reported in familial type 1 diabetes a higher frequency of dr3/dr4 heterozygosity and lower frequency of protective alleles ( dqb1 * 0602 , 0603 , or 0301 ) .
similar differences have been observed in other studies ( 20,22,23 ) , but not in all ( 24 ) .
our observations suggest that the diabetes status of second - degree relatives affects the frequency of the dr4-dq8 haplotype in the index case more than that of the first - degree relatives .
this finding is difficult to explain , although affected grandparents have been shown to transmit their hla haplotypes to affected grandchildren more often than expected ( 18 ) .
our findings of genetic differences between familial and sporadic cases implicate a weak contributing effect of differential hla dr - dq haplotype frequencies to the familial clustering of type 1 diabetes .
the data should be interpreted with caution , however , since these haplotypes are relatively frequent in the general population , and the differences are of borderline statistical significance and only seen in terms of one haplotype .
a limitation of our study is the lack of knowledge of the total number of second - degree relatives except grandparents , which prevents us from reporting the actual prevalence of diabetes in parental siblings . in addition
, a questionnaire - based study design is prone to recall bias either by over- or underreporting family members with type 1 diabetes .
in this survey , information on second - degree relatives was insufficient for completion of all the fields in the questionnaire in 122 families ( 8.2% ) .
given that these cases were counted as nonfamilial cases in the analysis , our results are likely to be conservative and somewhat underestimate the prevalence of familial diabetes .
our study setting lacked a control group of nondiabetic finnish children , and we are thus not able to directly compare the prevalence of type 1 diabetes in families of diabetic and nondiabetic children .
however , the prevalence of 2.9% of type 1 diabetes in siblings observed here can be compared with 0.5% in the general population < 20 years of age in finland in 2006 . according to these estimates , the siblings of affected children have , compared with the general population , a sixfold risk of type 1 diabetes .
our register - based data on the time of diagnosis is a point estimation , and thus the life - time risk of type 1 diabetes in relatives , especially among siblings , is underestimated .
our study shows that , considering the extended family history , the proportion of true sporadic cases of type 1 diabetes at diagnosis may be < 80% .
the proportion of case subjects with type 1 diabetes in first- and second - degree relatives was similar .
the case subjects had equally affected paternal and maternal relatives , but an interesting difference in the distribution of sexes was observed between these groups .
a novel observation is that the metabolic status at diagnosis is ameliorated in familial type 1 diabetes also when the affected person is a second - degree relative .
children with familial type 1 diabetes had an autoantibody profile very similar to that of sporadic cases , implying similar pathogenetic disease mechanisms .
familial cases carried the dr4-dq8 haplotype more often than sporadic cases , implying that the enrichment of hla class ii
| objectiveto determine the frequency of newly diagnosed diabetic children with first- and second - degree relatives affected by type 1 diabetes and to characterize the effects of this positive family history on clinical markers , signs of -cell autoimmunity , and hla genotype in the index case.research design and methodschildren ( n = 1,488 ) with type 1 diabetes diagnosed under 15 years of age were included in a cross - sectional study from the finnish pediatric diabetes register .
data on family history of diabetes and metabolic decompensation at diagnosis were collected using a questionnaire .
antibodies to -cell autoantigens ( islet cell antibodies , insulin autoantibodies , gad antibodies , and antibodies to the islet antigen 2 molecule ) and hla genotypes were analyzed.resultsa total of 12.2% of the subjects had a first - degree relative with type 1 diabetes ( father 6.2% , mother 3.2% , and sibling 4.8% ) and 11.9% had an affected second - degree relative .
children without affected relatives had lower ph ( p < 0.001 ) , higher plasma glucose ( p < 0.001 ) and -hydroxybutyrate concentrations ( p < 0.001 ) , a higher rate of impaired consciousness ( p = 0.02 ) , and greater weight loss ( p < 0.001 ) .
there were no differences in signs of -cell autoimmunity .
the familial cases carried the hla dr4-dq8 haplotype more frequently than sporadic cases ( 74.0 vs. 67.0% , p = 0.02).conclusionswhen the extended family history of type 1 diabetes is considered , the proportion of sporadic diabetes cases may be reduced to < 80% . a positive family history for type 1 diabetes associates with a less severe metabolic decompensation at diagnosis , even when only second - degree relatives are affected .
autoantibody profiles are similar in familial and sporadic type 1 diabetes , suggesting similar pathogenetic mechanisms . |
the word forensic , derived from the latin word , forensis ; means the art or study of public .
forensic science refers to the area of endeavor that can be used in a judicial setting and accepted by the court and the general scientific community to separate truth from untruth .
there are many well - known implanted methods of human identification , one of the most interesting emerging method of human identification which originates from the criminal and forensic practice , is human lips recognition .
cheiloscopy is a forensic investigation procedure which deals with human identification based on lip traces . in 1902 , fischer was the first to describe the biological phenomenon of systems of furrows on the red part of human lips .
use of lip prints in personal identification and criminalization was first recommended in france by edmond locard . since 1950 ,
the japanese have carried out extensive research in the matter . based on the research done by two japanese scientists tsuchihashi and suzuki ,
it was established that the arrangement of lines on the human lips is individual and unique for each human being .
mc donell in 1972 conducted a study on lip prints between two identical twins and reported that two identical twins seemed to be indistinguishable by every other mean , but their lip prints were different .
cheiloscopy is one of the special techniques used for personal identification was reported by cottone , in 1981 , in his book outline of forensic dentistry . in 1990 , kasprzak conducted research to elaborate the practical use of cheiloscopy .
vahanwala in 2000 conducted a study of lip patterns to promote the importance of cheiloscopy in forensic science identification .
sivapathasundharam et al . in 2001 studied the incidence of particular lip print patterns in the indo - dravidian population .
the objective of this study was to assess the distribution of lip outline pattern among males and females and to evaluate the lip outlines patterns for their uniqueness .
in 1902 , fischer was the first to describe the biological phenomenon of systems of furrows on the red part of human lips .
use of lip prints in personal identification and criminalization was first recommended in france by edmond locard . since 1950 ,
based on the research done by two japanese scientists tsuchihashi and suzuki , it was established that the arrangement of lines on the human lips is individual and unique for each human being .
mc donell in 1972 conducted a study on lip prints between two identical twins and reported that two identical twins seemed to be indistinguishable by every other mean , but their lip prints were different .
cheiloscopy is one of the special techniques used for personal identification was reported by cottone , in 1981 , in his book outline of forensic dentistry . in 1990 , kasprzak conducted research to elaborate the practical use of cheiloscopy .
vahanwala in 2000 conducted a study of lip patterns to promote the importance of cheiloscopy in forensic science identification .
sivapathasundharam et al . in 2001 studied the incidence of particular lip print patterns in the indo - dravidian population .
the objective of this study was to assess the distribution of lip outline pattern among males and females and to evaluate the lip outlines patterns for their uniqueness .
a sample of 200 individuals comprising 100 females and100 males were included in the study .
the subjects were selected whose lips were free from any pathology such as inflammation , mucocele , cicatrization , and deformities such as cut marks or lesions .
informed consent was taken from each of the subjects . to classify the lip outline pattern in this study , the classification scheme proposed by chandrashekaran
were : upper lip outline pattern lower lip outline pattern
lip liner / lipstick of a bright red color and non glossytransparent cellophane tape , glued on one sidescissorswhite bond paperadhesive tapecottonscalemagnifying lenspen / pencil for labeling the individual details .
lip liner / lipstick of a bright red color and non glossy transparent cellophane tape , glued on one side pen / pencil for labeling the individual details .
the lips of individuals were cleaned , and a dark red colored lip liner was used to mark upper and lower vermilion border .
a lip outline impression was made on strip of cellophane tape on glued portion , by dabbing it in the center first and then pressing it uniformly toward the corners of the lips [ figure 1 ] .
the cellophane strip was then stuck to the white chart paper for permanent record purpose and then visualized by magnifying lens [ figure 2 ] .
the recording was done by noting outline pattern in both upper and lower lips , in both males and females . taking lip outline pattern on a cellophane tape cellophane tape with lip outline pattern stuck on white chart paper for permanent record
a sample of 200 individuals comprising 100 females and100 males were included in the study .
the subjects were selected whose lips were free from any pathology such as inflammation , mucocele , cicatrization , and deformities such as cut marks or lesions .
to classify the lip outline pattern in this study , the classification scheme proposed by chandrashekaran was followed [ tables 1 and 2 ] .
materials used were : upper lip outline pattern lower lip outline pattern
lip liner / lipstick of a bright red color and non glossytransparent cellophane tape , glued on one sidescissorswhite bond paperadhesive tapecottonscalemagnifying lenspen / pencil for labeling the individual details .
lip liner / lipstick of a bright red color and non glossy transparent cellophane tape , glued on one side pen / pencil for labeling the individual details .
the lips of individuals were cleaned , and a dark red colored lip liner was used to mark upper and lower vermilion border . a lip outline impression was made on strip of cellophane tape on glued portion , by dabbing it in the center first and then pressing it uniformly toward the corners of the lips [ figure 1 ] .
the cellophane strip was then stuck to the white chart paper for permanent record purpose and then visualized by magnifying lens [ figure 2 ] .
the recording was done by noting outline pattern in both upper and lower lips , in both males and females . taking lip outline pattern on a cellophane tape cellophane tape with lip outline pattern stuck on white chart paper for permanent record
a total of 200 individuals were included in the study , comprising of 100 males and 100 females each , in the age group of 1725 years .
the lip outline pattern obtained from each individual was carefully examined with an illuminated magnifying lens and checked for the pattern .
it was observed in our study that ten different upper lip outline patterns in females and nine different patterns in males [ figures 3 and 4 ] .
most common upper lip outline pattern in females was uneven peak ( 28.5% ) [ figure 5 ] , followed by twin peak , double convex , dome , flat , butterfly , flying bird , cantilever , butte , suspension bridge , and flat .
most common upper lip outline pattern in males was twin peak ( 32.9% ) [ figure 6 ] followed by an uneven peak , double convex , flat , dome , butterfly , suspension bridge , butte , and cantilever .
upper lip out line patterns showing ( a ) twin peak ( b ) uneven peak ( c ) flying bird and ( d ) double convex lower lip out line pattern showing ( a ) companulate ( b ) boat ( c ) basin and ( d ) apple bottom lip outline patterns in females showing ( a ) twin peak ( b ) uneven peak ( c ) companulate and ( d ) basin patterns lip outline patterns in males showing ( a ) twin peak ( b ) uneven peak ( c ) boat and ( d ) companulate in males , the most common lower lip outline pattern observed in our study was companulate ( 28.6% ) followed by basin , boat , flat , apple bottom , and concave arc .
we observed most common lower lip outline pattern in females was companulate ( 34.4% ) followed by boat , basin , flat , apple bottom , and concave arc .
personal identification is required for an unknown deceased person in homicide , suicide , accident , mass disaster , etc . , and for living individual who are missing or culprits hiding their identity .
identification can be made by comparing the antimortem record with that of the postmortem record .
if a definite description of the different parts of the upper lip and lower lip are established for an individual by detailed study , this antimortem record can be scanned and preserved for matching the details of lip outlines patterns in postmortem records for personal identification .
lips are two fleshy folds surrounding the oral orifice , lined externally by the skin and internally by mucosa .
the skin is continuous with the mucosa at transitional or vermilion border , a reddish zone covered by thin keratinized epithelium .
the junction between the vermilion border and the skin forms the lip outline , produced by characteristic shape in the labial mucosal surface , unique for the individuals like finger prints .
these lip outline patterns can be employed in forensic investigations as an adjuvant tool along with the lip prints .
the presence of lip outline on materials left in the crime scene is considered as an evidence of a relationship between the suspect and the crime scene .
research studies and information regarding the use of lip outline patterns as evidence in personal identification and criminal investigation in forensic dentistry is very much scanty . in spite of few studies available ,
the study of chandrashekaran gives a standard classification of his own for different types of lip outline patterns . keeping this classification as the basis
, the current study was conducted to study the lip outline pattern of different individuals , to establish facts so as to aid in giving further details of lip outline patterns .
analysis of lip outline pattern in females showed that ten different upper lip outline patterns .
most common upper lip outline pattern in females was uneven peak ( 28.5% ) , [ figure 7 ] followed by twin peak , double convex , dome , flat , butterfly , flying bird , cantilever , butte , suspension bridge , and flat .
most common upper lip outline pattern in males was twin peak [ 32.9% ] , followed by uneven peak , double convex , flat , dome , butterfly , suspension bridge , butte , cantilever .
common lip outline patterns in females showing ( a ) uneven peak ( b ) twin peak in males , the most common lower lip outline pattern observed was companulate ( 28.6% ) [ figure 8 ] followed by basin , boat , flat , apple bottom , and concave arc .
we observed most common lower lip outline pattern in females was companulate ( 34.4% ) followed by boat , basin , flat , apple bottom , and concave arc .
common lip outline patterns in males showing ( a ) twin peak ( b ) uneven peak
cheiloscopy is a relatively new field among the large number of identification tools available to the forensic expert .
lip outlines are unique to an individual and does not change with time so it can also be used as a method for identification
further work on the subject can help to make lip outline pattern a practical reality at the ground level of the forensic identification process .
| introduction : personal identification is becoming increasingly important not only in legal medicine but also in crime / criminal investigation and identification . sometimes establishing a person 's identity can be a very difficult process .
dental , fingerprint , and dna comparisons are probably the most common technique used .
however , there are many well - known implanted methods of human identification , one of the most interesting emerging methods of human identification which originates from the criminal and forensic practice , is human lips recognition .
cheiloscopy is a forensic investigation technique that deals with the identification based on lip traces .
the lip outline of every person is unique and can be used to fix the personal identity.aim:the aim of this study was to assess the distribution of lip outline patterns among males and females , and to evaluate the uniqueness of lip outline pattern.materials and methods : the study group comprised of 200 individuals from kamineni institute of dental sciences .
lip outline patterns were obtained and were transferred to the proforma sheet for analysis.results:the results of the study revealed that the lip outline patterns for each individual were unique.conclusion:this study showed that lip outline patterns are unique to each individual and can be used for personal identification . |
renal cell carcinoma ( rcc ) has a potential to metastasize to almost any site . in descending order of frequency , the most common sites of metastasis are the lung , lymph nodes , liver , bones , adrenal glands , kidneys , brain , heart , spleen , intestine , and skin .
it can involve any part of the bowel and accounts for 7.1% of all metastatic tumors to the small intestine .
duodenal metastasis from rcc is very uncommon and only few cases have been described in the literature ( table 1 ) .
also duodenal metastasis generally occurs when there is widespread nodal and visceral involvement and evidence of metastatic disease elsewhere in the body .
commonly , renal cell metastases present many years after initial treatment , with recurrences reported up to 17.5 years after initial surgery .
most cases of duodenal metastasis from rcc present with upper gastrointestinal bleeding or obstructive symptoms , and sequelae may include anemia , melena , fatigue , and early satiety .
these include a variety of surgical and interventional therapy options that have been shown to provide effective survival benefits . here
we report on a patient with solitary duodenal metastasis who presented with gastrointestinal bleeding 13 years after nephrectomy .
a 66-year - old male presented with progressively worsening shortness of breath , fatigue and generalized weakness for the last 3 weeks .
he also complained of loss of appetite and 15 pounds unintentional weight loss over the last few weeks .
his past medical history was significant for hypertension , bilateral rcc status post right nephrectomy and partial left nephrectomy done 13 years earlier , ileocolectomy for perforated cecal diverticulitis , and prostate cancer treated with radiation .
he had a 50 pack year history of smoking , but denied any alcohol use . on physical exam
laboratory investigations on admission were significant for microcytic hypochromic anemia with hemoglobin 5 g / dl , hematocrit 16.8% , mcv 72 fl and mch 21.7
esophagogastroduodenoscopy showed an actively bleeding , 4 cm irregular , polypoid , ulcerative mass in the second portion of the duodenum , adjacent to but not involving the papilla ( fig .
biopsies were obtained from this mass , following which the patient started having severe bleeding from the lesion .
visceral angiography was done which demonstrated no extravasation or pseudoaneurysm in the duodenal vascular distribution or pancreaticoduodenal arcade .
however , empiric angiographic embolization of the gastroduodenal artery was performed . despite these endoscopic and radiologic embolization attempts ,
bleeding continued , requiring another endoscopic procedure when the lesion was treated with argon plasma coagulation with successful control of the bleeding .
the patient received a total transfusion of 11 units of packed red blood cells during his hospital course .
further investigations included a computed tomography scan of the abdomen which showed an ill - defined mass measuring 5.4 3.3 cm at the junction of the second and third portion of the duodenum , adjacent to but not involving the head of the pancreas ( fig .
2 ) . there was no evidence of liver or visceral metastasis , and the visceral vessels and lymph nodes appeared normal .
ultrasound of the abdomen showed normal pancreaticobiliary system with no common bile duct dilatation suggestive of obstruction .
subsequently , the patient underwent exploratory laparotomy which revealed a 7 4 cm mass in the second portion of the duodenum
. there was no evidence of malignant ascites , carcinomatosis , omental implants , or involvement of the pancreas or liver .
he underwent pylorus - sparing pancreaticoduodenectomy , choledochojejunostomy , handsewn pancreaticojejunostomy and gastrojejunostomy with removal of the mass .
postoperatively , the patient 's hospital course was complicated by leakage of the pancreaticojejunostomy fistula and sepsis , and he died two weeks later .
gross examination of the surgical specimen revealed a 2.0 1.5 0.8 cm tan - red polypoid , pedunculated tumor protruding into the duodenum approximately 0.5 cm away from the ampulla of vater .
the tumor appeared confined grossly to the mucosa and submucosa of the duodenum and did not appear to extend into or through the muscularis .
eight lymph nodes were identified in the resection specimen , and all were negative for metastatic tumor with successful excision of all involved structures with clear margins and with no lymph node involvement .
microscopically , the tumor was composed of clear cells arranged in a trabecular and alveolar pattern ( fig .
immunohistochemically the tumor was positive for vimentin , cd10 , ae1/ae3 and epithelial membrane antigen and negative for ck7 , ck20 and pnra ( fig .
4 ) . histopathological features and immunostaining findings were compatible with a diagnosis of metastatic rcc of clear cell type .
small bowel involvement by metastatic tumors is rare and has been reported in only 12% of autopsy cases [ 2 , 4 ] .
common metastatic malignancies known to involve the small bowel are melanomas , lung cancer , cervical carcinomas , rccs , thyroid carcinomas , hepatomas and merkel cell carcinomas .
solitary duodenal metastasis from rcc is exceedingly rare and most frequently involves the periampullary region or the duodenal bulb [ 6 , 7 ] .
we have included patients with the bulk of the gross tumor in the duodenal or ampullary regions , distinct from pancreatic involvement . the majority of patients were found to have metastasis within 1 year after nephrectomy though it could be seen even after several years .
the mean duration post nephrectomy to diagnosis of these solitary duodenal metastases was 7.9 4.7 years ( median 8 years ) .
the range was 0 to 13 years , with one patient presenting with a synchronous metastasis .
males were more commonly affected ( male : female ratio 3:1 ) and the incidence of metastasis increased with age .
mean age at diagnosis of duodenal metastasis was 60.7 14 years ( median age 63 years , range 1686 years ) .
disease eradication and cure are possible after nephrectomy ; however , there is also the possibility of a long period of disease latency , followed by recurrence of metastatic disease at unsuspected anatomic locations .
rcc can metastasize via the lymphatic or hematogenous route , as well as by peritoneal dissemination or direct invasion into adjacent anatomic structures .
the duodenum is an exceptionally rare site of metastasis in rcc , which is perhaps counterintuitive given its retroperitoneal proximity to the right kidney , though a majority ( around 70% ) occur from the right kidney .
the most common clinical presentation of gastrointestinal metastases of rcc is gastrointestinal bleeding , resulting from the invasion of intestinal vessels by the neoplastic disease and/or intestinal obstruction [ 7 , 9 ] .
most of the patients with solitary duodenal rcc metastasis present with gastrointestinal bleeding ( 69% ) , anemia and fatigue , whereas others present with early satiety , bowel obstruction , abdominal pain or jaundice from biliary obstruction [ 4 , 7 , 10 , 11 ] . patients post nephrectomy for rcc presenting with gastrointestinal symptoms should undergo complete diagnostic work - up with both endoscopic and radiologic evaluation , for detection and evaluation of the extent of metastatic disease . on endoscopy
the lesion can be seen as a submucosal mass with ulceration of the tip , multiple nodules of varying sizes or raised plaques .
endoscopic biopsy of suspicious lesions provides tissue for histologic diagnosis of metastasis and helps to distinguish primary gastrointestinal malignancy from metastatic disease .
the treatment options in a case of solitary duodenal rcc metastasis depend upon the extent and location of the lesion and therapy must be individually tailored .
procedures ranging from classic pancreaticoduodenectomy ( whipple procedure ) to interventional embolization have been reported ( table 1 ) .
any patient with solitary metastatic rcc to the duodenum should be considered a candidate for complete surgical excision if medically and technically feasible , both for palliation of symptoms and because it provides the opportunity for meaningful disease - free survival .
a curative role for pancreaticoduodenectomy in patients with solitary duodenal metastasis has been reported and has been shown to improve survival [ 11 , 13 , 14 , 15 , 16 ] .
hemostasis of gastrointestinal bleeding occurring due to metastasis or invasion of malignant tumor is hard to manage endoscopically , and data on endoscopic therapy of bleeding from these duodenal lesions are limited . in selected cases , intractable gastrointestinal bleeding can be treated with arterial embolization of tumor - supplying arteries that has been reported to control gastrointestinal bleeding effectively , but there are no long - term follow - up data [ 17 , 18 , 19 ] .
however , embolotherapy is only palliative while the tumor develops other collateral vessels and has potential for re - bleeding .
also , in these cases , the physician should keep in mind that embolization for control of hemorrhage in the small bowel carries a significant risk of bowel infarction . for disseminated malignancy
, treatment is mainly supportive and palliative , in the form of palliative surgery , radiotherapy , chemotherapy or immune - stimulating agents ( interleukin-2 ) [ 10 , 21 , 22 ] . patients with metastatic disease have poor survival despite the above treatment .
the average survival is about 4 months and only 10% of them survive for 1 year .
this case report highlights the importance of vigilance and high index of suspicion in post nephrectomy patients upon presentation of new clinical symptoms .
appropriate awareness , recognition and aggressive work - up of gastrointestinal symptoms in patients post nephrectomy for rcc are of paramount importance .
all authors declare that there are no potential conflicts ( financial , professional , or personal ) relevant to this paper . | massive upper gastrointestinal bleeding due to malignancy is relatively uncommon and the duodenum is the least frequently involved site .
duodenal metastasis is rare in renal cell carcinoma ( rcc ) and early detection , especially in case of a solitary mass , helps in planning further therapy .
we report a case of intractable upper gastrointestinal bleeding from metastatic rcc to the duodenum . the patient presented with melena and anemia , 13 years after nephrectomy for rcc .
on esophagogastroduodenoscopy , a submucosal mass was noted in the duodenum , biopsies of which revealed metastatic rcc . in conclusion ,
metastasis from rcc should be considered in nephrectomized patients presenting with gastrointestinal symptoms and a complete evaluation , especially endoscopic examination followed by biopsy , is suggested . |
zabardasti claims the molecule is aromatic , whereas korkin suggests the hcno monomer spontaneously trimerizes in order to reach a more stable configuration , a feature which if proved correct would indicate that aromaticity is an indication of stabilization energy .
korkin discovered that it manifests strong stabilization energy and likewise tarko revealed it has descriptors of aromaticity and one of their targets is this same hexahydrotriazine derivative . molecular structure and electronic behavior ( charge distribution ) of cyanuric acid
has also been the focus of x - ray and neutron difraction studies [ 57 ] . in this context , the important point is that the planarity of the molecule and its electronic delocalization has been achieved , both of which are important characteristics for evaluating aromaticity .
theoretical calculations of these molecules have been carried out for the purpose of studying an array of topics .
the marked capability of cyanuric acid for generating hydrogen bonds has been analyzed in terms of nanostructures and intrinsic capability .
furthermore analogs containing sulfur atoms instead of oxygen atoms in the carbonyl groups have also been theoretically studied and compared with cyanuric acid [ 10 , 11 ] .
hydrogen bonded supramolecular clusters of cyanuric acid and melamine in particular have been the focus of several studies because these clusters display interesting electrical and material behavior . besides , some adducts to a metallic complex have been prepared , showing promising characteristics in terms of their supramolecular chemistry for the development of new materials .
another particularity of cyanuric acid , also derived from this characteristic of facile hydrogen bond formation is the formation of hamilton complexes [ 1618 ] .
these kinds of complexes result from the interaction of a substrate containing electron donor ends with terminal hydrogen atoms , which have the capacity to easily interact with negative charge densities .
this feature offers several possibilities for preparing novel compounds with applications in different fields , for example biochemistry , synthesis or in the pharmaceutical industry , as in the case of barbituric acid [ 1618 ] .
the goal of the present manuscript is to evaluate from different points of view , the intrinsic electronic characteristics that we foresee helping to explain the possible aromatic character of these compounds .
the evaluation was carried out by applying the homa method [ 19 , 20 ] and the ase approximation , employing isodesmic reactions [ 2224 ] .
all structures were optimized in the gas phase using the jaguar 4.0 package . a pure dft method which included
becke s gradient correction for exchange and perdew - wang s for correlation was used for the energy evaluations .
frequency calculations were carried out at the same level of theory in order to confirm that the optimized structures were at the minimum of the potential surface .
these frequencies were then used to evaluate the zero - point vibrational energy , the thermal vibrational corrections to the enthalpy and the total enthalpy values , calculated at 273k .
the bond lengths of the optimized structures were incorporated into the harmonic oscillator model of aromaticity ( homa ) method [ 19 , 20 ] used to study aromaticity .
in almost all cases , the molecules in this study resemble the general structure depicted in fig .
1general structure of the compounds in this study general structure of the compounds in this study the calculation of the tautomer of compound ( 5 ) that corresponds to the structure shown in fig . 2
2tautomer of compound ( 5 ) tautomer of compound ( 5 ) it is a known fact that in the majority of cases , the keto form of any species manifesting keto - enolic tautomerism is more stable that its corresponding enol form .
this phenomenon is due to the fact that a stronger bond exists between carbon and oxygen than the bond between carbon - carbon ( considering bonds ) .
however , the presence of non bonding electrons in heteroatoms close to carbonyl groups , in this case causes a cyclic delocalization , resulting in an aromatic system ( 4n + 2 ) , n = 1 .
surprisingly , the calculation of both tautomers gives a total energy difference of 28.87 kj mol , with the keto form taking the lead .
this result contradicts the previous premise concerning aromatics because the enol form shows the typical delocalization of an aromatic compound , in contrast to the keto form which does not ; thus we return to the initial question
the ase results come from the evaluation of the isodesmic reactions based on cyanic acid ( ncoh ) and its interaction with several simple molecules ; the set of reactions is as follows :
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\begin{document}$$ { \hbox{3ncoh } } \to { { \hbox{c}}_3}{{\hbox{n}}_3}{{\hbox{o}}_3}{{\hbox{h}}_3 } $ $ \end{document}2\documentclass[12pt]{minimal }
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\begin{document}$$ { \hbox{3ncoh } } + { \hbox { c}}{{\hbox{h}}_3}{\hbox{n}}{{\hbox{h}}_2 } + { \hbox { c}}{{\hbox{h}}_3 } - { \hbox{c}}{{\hbox{h}}_3 } \to { { \hbox{c}}_6}{{\hbox{n}}_3}{{\hbox{o}}_3}{{\hbox{h}}_9 } + { \hbox { n}}{{\hbox{h}}_3 } $ $ \end{document}3\documentclass[12pt]{minimal }
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\begin{document}$$ { \hbox{3ncoh } } + { } { { \hbox{h}}_2}{\hbox{c}}{{\hbox{o}}_2 } + { } { { \hbox{o}}_2 } \to { { \hbox{c}}_3}{{\hbox{n}}_3}{{\hbox{o}}_6}{{\hbox{h}}_3 } + { } { { \hbox{h}}_2}{\hbox{co } } $ $ \end{document}4\documentclass[12pt]{minimal }
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\begin{document}$$ { \hbox{3ncoh } } + { \hbox { 3p}}{{\hbox{h}}_3 } + { \hbox { 2c}}{{\hbox{h}}_4 } \to { { \hbox{c}}_3}{{\hbox{n}}_3}{{\hbox{p}}_3}{{\hbox{o}}_3}{{\hbox{h}}_6 } + { \hbox { c}}{{\hbox{h}}_3 } - { \hbox{c}}{{\hbox{h}}_3 } + { 4}{{\hbox{h}}_2 } $ $ \end{document}5\documentclass[12pt]{minimal }
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\begin{document}$$ { \hbox{3ncoh } } + { \hbox { 3n}}{{\hbox{h}}_3 } + { \hbox { 2c}}{{\hbox{h}}_4 } \to { { \hbox{c}}_3}{{\hbox{n}}_6}{{\hbox{o}}_3}{{\hbox{h}}_6 } + { \hbox { c}}{{\hbox{h}}_3 } - { \hbox{c}}{{\hbox{h}}_3 } + { 4}{{\hbox{h}}_2}. $ $ \end{document } the isodesmic reaction ( 1 ) yields unsubstituted cyanuric acid , the ch3- derivative is obtained by a means reaction ( 2 ) , whereas oh- , ph2 and nh2 derivatives are the result of reactions ( 3 ) , ( 4 ) , and ( 5 ) respectively .
the ase values relating to these isodesmic reactions are shown in table 1 .
table 1ase values from the isodesmic reactionsmoleculeasehydoh92.87 kj / molhydch3119.23 kj / molhydh135.56 kj / molhydph82.20 kj / molhydnh253.97 kj / mol ase values from the isodesmic reactions the greatest stabilization energy corresponds to unsubstituted cyanuric acid ( hydh ) ; therefore the inductive effect has little importance in this kind of molecule .
first , the most aromatic species in this set is the enol form of cyanuric acid ( 5b ) , with the next strongest value corresponding to the keto form of the same derivative .
likewise , this result suggests that the enol form will turn out to be the most stable isomer .
however , the energy results indicate the contrary , and as outlined above the explanation for this phenomenon arises from the existence of a zwitterionic species which converts from 5 to become 5b , based on the proton mobility of both species . on the other hand ,
the same zwitterionic species is also described as having a canonical structure as in 5 , presented in scheme 1
benzene homa is 0.999moleculehomahydch3 ( 1)0.710hydoh ( 2)0.691hydn2 ( 3)0.6hydph2 ( 4)0.612hydh ( 5)0.7373hydrol ( 5b)0.998scheme 1contributing structures in cyanuric acid direct homa for the compounds being considered . for comparison purposes
benzene homa is 0.999 contributing structures in cyanuric acid these results can be compared to those for a different molecule that has little resemblance to cyanuric acid , in order to evaluate the suitability of the homa method for the present study .
3 ) . with respect to this species , it has the same three oh substituents , but in this case it is expected to be very difficult to detect the phenomenon of tautomerism because the hydrogen atoms which leave the hydroxyl groups converting to the keto form should unite with the free carbon atoms on the ring , resulting in a severe distortion .
the homa results for the enol and keto forms of this species are 0.9995 and 0.357 respectively .
the difference between these two cases is larger than those in the case of cyanuric acid because this last is constituted from nitrogen atoms with dangling bonds which favor the keto - enolic equilibrium .
thus our comparison is useful in this context to validate the use of the homa method .
( a ) enol form ( b ) keto form the cyanuric acid molecule presents two zones of electronic flow ; the first one is the typical delocalization zone in the ring , generated by the movement of the three electronic pairs inherent in the c = n bond .
the second zone is that generated by the electrons associated with the three oxygen atoms in the carbonyl groups .
thus , two contributions to the stabilization of the molecule exist , relating to these two sets of electrons that will be analyzed with reference to the molecular orbitals analysis . a diagram of molecular orbital ( mo ) interaction is shown in fig
first , cyanuric acid seems to represent a very strange case of aromaticity because of its particular mo arrangement .
several aromatic compounds manifest the set of six delocalized electrons in one single mo ( homo ) and a two - fold degenerate set ( homo-1 ) which causes the resonant interaction , generating the aromatic cloud .
the cyanuric acid molecule belongs to the d3h symmetry group , therefore the sets that contain the six electrons of the resonant group belong to the e and a2 irreducible representations .
however , the energy of the three orbitals is practically the same , so that we have a strange case of an accidental triple degenerated set .
this set keeps the electrons that participate in the aromatic flow at the center of the molecule , indeed this set is very curious because it is not common to find that all the mo s manifest the same energy and neither is it common to find this set representing the homo-1 group .
besides , although it might be expected that the homo would participate in aromaticity , at first sight this is not the case , because it seems that all central aromaticity is found in the homo-1 group .
irreducible representation that occupies almost the same region as the homo , therefore , it seems to be a classical set of three orbitals , separated by 1.115 ev depicting an aromatic set .
however the set of the homo-1 orbitals is localized at the middle of the scheme .
5 .
fig . 4interaction mo diagram showing both sets of electronic interaction for compound 5fig .
( b ) classic aromaticity set formed by homo and homo-2 interaction mo diagram showing both sets of electronic interaction for compound 5 ( a ) accidental triple degenerated set corresponding to homo-1 .
( b ) classic aromaticity set formed by homo and homo-2 the conclusion to all this is that we have two aromatic sets ( double aromaticity [ 33 , 34 ] ) ; homo-1 represents one set , and homo , homo-2 the other one , and there is interaction between them .
these two sets do not occupy the same space , although there is some overlap .
this means that there must be competition between them and that they should thus shield each other . in other words ,
thus , the molecule should have a very weak aromatic character concurring with the result obtained when aromaticity indexes are used . applying this framework we can reveal the inductive effect due to the different substituents , but a drastic change
is not expected because the strong interaction of molecular orbitals causes the molecule to imitate the same electronic behavior . in scheme 1
the scheme is relatively simple in the case where the substituents are hydrogen atoms ; however it can become very complicated in cases where compounds of this kind contain other types of substituents .
firstly ; other substituents may not have the same mobility as hydrogen , therefore , it is to be expected that they will remain in their position on the nitrogen atom , secondly ; it is a known fact that certain substituents have inductive effects , strongly affecting the electronic behavior of the aromatic ring .
the zwitterionic form continues to be aromatic and display interesting electronic properties . in order to evaluate the aromaticity of this family ,
the homa method is once again applied , however in this case we are using the theory designed by krygowski in order to develop an equation with specific characteristics , permitting a comparison to be made between the different aromaticity values for these substances .
the keto form of the cyanuric acid ( molecule 5 in the last table ) has the most geometrically regular structure of any of the molecules in this study , therefore , we consider this species to represent the ideal model and establish that this is the most aromatic example out of the entire family .
thus the parameters of this molecule are applied to the krygowski scheme , in the following way .
krygowski s general equation is presented as :
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\begin{document}$$ { \hbox{homa } } = { 1 } --{\hbox{a}}/{\hbox{n}}{({{\hbox{r}}_{\rm{opt } } } - { { \hbox{r}}_{\rm{ij}}})^2 } , $ $ \end{document}where corresponds to an empirical constant that helps take into account the length and angles of a particular ring and is evaluated in terms of
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\begin{document}$$ { \hbox{a } } = { 2}{\left ( { { { \left [ { { \hbox{r}}\left ( { \hbox{s } } \right ) { } --{{\hbox{r}}_{\rm{opt } } } } \right]}^2 } + { } { { \left [ { { \hbox{r}}\left ( { \hbox{d } } \right ) - { { \hbox{r}}_{\rm{opt } } } } \right]}^2 } } \right)^ { - { 1}}}. $ $ \end{document } by taking ( 6 ) and ( 7 ) , an equation was developed for the purpose of evaluating the changes in cyanuric acid and its derivatives .
considering the calculated measurements of cyanuric acid itself as a pattern for evaluating , substituting r(s ) = 1.465 ( the length of a single bond n - c as is described in the same work by krygowski ) , r(d ) = 1.269 ( the length of a double bond between n and c as described by krygowski ) and ropt = 1.387 in eq .
7 yields a value of 99.96 for . all these parameters were included in eq .
6 and the relative homa aromaticity ( named internal homa ) for all species are presented in table 3 .
homa for the compounds included in this study the order is the same as that in table 2 , but this time the molecules with substituents , which extract electronic charge from the ring ( -cooh , -cn and no2 ) have also been included .
it should be noted that in the case of hydno2 , the value of aromaticity is increased , almost to the level of the reference molecule ( hydh ) .
this phenomenon is due to the presence of high electronic density in the substituent , which strongly reinforces the electronic flow on the periphery of the ring , in strong contrast to classic aromatic molecules .
cyanuric acid and several derivatives were calculated in order to study their possible aromatic character .
all species are aromatic in the enol form , but the keto form is more stable than the enol one . therefore the aromaticity of all species in keto form was analyzed using ase and homa methods .
the explanation is thus found in the frontier molecular orbitals analysis , where it is possible to identify two aromatic systems for cyanuric acid in keto form but it seems these systems are opposed , thus tending to decrease the overall aromatic character . as a result | this study analyzes the aromatic nature of cyanuric acid ( hexahydrotriazine ) and some of its derivatives , in terms of aromatic stabilization energy ( ase ) and electronic behavior . the simplest molecule ( c3n3o3h3 )
is the most aromatic item out of the entire set , but some of the others also display aromatic character .
the structure of all the rings is analyzed considering their molecular orbitals as well as studying the inductive effect . |
gami - jakyak gamcho buja decoction ( mecasin ) was developed for treating amyotrophic lateral sclerosis patients with pain , joint contracture and muscular weakness .
mecasin , which is the major component of mecasin , has been used in traditional medicine to relieve pain , muscle spasms and cold syndrome due to blood deficiency .
a recent study showed that jakyak gamcho decoction ( jgt ) and its constituents have a protective effect against tert - butyl hydroperoxide ( t - bhp)-induced cytotoxicity in the hippocampal ht22 cell line .
in addition , mecasin , which consists of jgt and radix aconiti lateralis preparata , is believed to be useful for suppressing the progress of osteoarthritis because of its anti - inflammatory effects and its ability to reduce pain with histopathological efficacy .
because of these facts , mecasin has been studied for treating patients with joint pain , muscle spasms , and arthralgia due to cold .
the constituents of mecasin are radix paeoniae alba , radix glycyrrhizae , radix aconiti lateralis preparata , radix salviae miltiorrhizae , rhizoma gastrodiae , radix polygalae , curcuma root , fructus chaenomelis , rhizoma atractylodis japonicae .
several studies on the relation between the toxicity and the composition of mecasin have been conducted .
studied radix glycyrrhizae consumption as a causing of severe hypokalemic paralysis elinav et al . conducted a study on the single - dose toxicity of aconitum kusnezoffii reichb .
studied the cytotoxic effect of radix salviae miltiorrhizae against l1210 cells sun et al . conducted a study on the acute oral toxicity of radix polygalae extract and suggested that water soluble extract of radix polygalae had no acute oral toxicity and that the oral ld50 value was over 4,000 mg / kg in sprague - dawley ( sd ) rats roh et al
conducted a philological study on the poisoning and the side effects of rhizoma gastrodiae kim et al . conducted a study on the cytotoxicity and the antimicrobial effects of extracts from the atractylodes japonica koidzumi choi et al
nevertheless , objective oral toxicity testing of mecasin , which is complex combination of herbs , has not been conducted yet .
the current research trend for oral toxicity testing of extracts is to study acute and subacute toxicity through good laboratory practice ( glp ) regulations .
all the experiments for this research were conducted at medvill , an institution authorized to perform non - clinical studies , under the glp regulations .
this study was performed to analyze the oral toxicity and the lethal dose of mecasin in rats .
the mecasin was prepared in a sterile room at the hanpoong pharm & food co. , ltd .
, the ph was controlled to between 7.0 and 7.5 nacl was added to make a 0.9% isotonic solution .
the reason sd rats were chosen is that they have been widely used in safety test in the field of medicine , so the results can be easily compared with many other databases .
the mean weights of the rats were 229.1 246.6 g and 169.7 199.0 g , respectively , for the male and the female rats at the time of mecasin administration .
for all animals , a visual inspection was conducted ; all animals were weighed at the beginning . during 6 days of acclimatization
the temperature of the lab was 19.8 24.3c , and the humidity was 47.7% 68.1% .
animals were selected if their weights were close to the mean weight . in total , 20 male rats and 20 female rats were selected .
the animals were randomly distributed into 4 groups ( 5 male and 5 female rats per group ) as shown in ( table 1 ) . in this study
2,000 mg / kg was set as a high - dose , and 1,000 mg / kg and 500 mg / kg were set as mid and low doses , respectively . in the control group ,
mecasin and normal saline were administered into the mouths of the rats in all groups by using disposable syringes .
this study was conducted under the approval of the institutional animal ethics committee of medvill co. , ltd . from the 1 day to the 14 day after treatment , the general symptoms were examined once a day . on the day of dosing ( day 0 ) , the general symptoms ( side effects , revealing time , recovery time , etc .
, ) as well as the mortality , were examined at 30 minutes and at 1 , 2 , 4 , and 6 hours after injection .
the weights were measured immediately before treatment and at 1 , 3 , 7 and 14 days after treatment .
after observations had been terminated , we conducted a necropsy of the rats after cutting the abdominal aorta and vein under co2 anesthesia , and the organs of all surviving animals were visually inspected and microscopically examined .
the weight data from the experiments were analyzed by using spss program ( spss 16.0 ) .
a levene test was conducted to evaluate the homogeneity of the variance and the significance .
the one - way analysis of variance ( anova ) test was conducted when the homogeneity of the variance was recognized .
in this study , no deaths or abnormalities occurred in any of the groups . in weight examinations , slight decreases in the weights of four female rats ( one female rat in the control group , two female rats in the 500-mg / kg group , and one female rat in the 1,000-mg / kg group )
however , no significant changes in weight were observed in the other rats ( table 2 ) finally , in both the control and the experimental groups , no meaningful changes in the necropsy were noted .
radix paeoniae alba is a herbal medicine that has been used for treating gastrointestinal disorders for hundreds of years .
conducted a study providing in - vitro and in - vivo evaluations of the use of radix paeoniae alba for peripheral nerve regeneration .
they suggested that radix paeoniae alba extract could be a potential nerve growth - promoting factor , being salutary in helping the growth of injured peripheral nerves huang et al .
radix glycyrrhizae has been commonly used in the orient for treating a variety of diseases .
reported that administration of radix glycyrrhizae improved spatial learning and memory and reduced stress - induced anxiety .
they concluded that radix glycyrrhizae had the potential to attenuate the behavioral and neurochemical impairments caused by stress park et al .
radix aconiti lateralis preparata has been widely used for the improvement of symptoms such as heart failure , inflammation , pain , and diarrhea for thousands of years in the orient .
however , the toxicity of radix aconiti lateralis preparata has been the subject of controversy .
some kinds of diester diterpenoid - type aconitum alkaloids , such as aconitine and mesaconitine , are major toxic ingredients .
some processing techniques such as pressure - steaming are known to be able to reduce the toxicity of aconitum alkaloids .
these methods use the facts that alkaloids can hydrolyze highly toxic diester - diterpene aconitum alkaloids to compounds of much lower toxicity such as aconine , benzoylaconine and benzoylmesaconine . recently
, radix aconiti lateralis preparata was found to have the ability to activate the proliferation of mouse bone - marrow mesenchymal stem cells and to induce osteogenic lineage differentiation .
radix salviae miltiorrhizae has been used for treating heart disease for several thousand years in the orient .
it has been found to exhibit an anti - atherosclerosis effect , which improves the patient s heart function recovery . reported that treatment with radix salviae miltiorrhizae protected the myocardium against ischemia and reperfusion injury .
they also suggested that radix salviae miltiorrhizae decoction could act as an anti - apoptotic and anti - ion stunning agent to protect hearts against ischemia and reperfusion injury hu et al . conducted a study of the effects of radix salviae miltiorrhizae on neuronal apoptosis following intracerebral hemorrhage in rats .
rhizoma gastrodiae has been used for treating diseases such as headache , dizziness , paralysis , and convulsion and for enhancing health .
it also has been used for balancing the liver and extinguishing wind for the efficacies of moistening viscera , for calming and protecting the human body , and for calming the liver and suppressing liver - yang .
radix polygalae is used as an anti - inflammatory , sedative that is thought to exert a variety of neuropsychiatric effects .
reported that radix polygalae extract could protect against n - methyl d - aspartate ( nmda ) neurotoxicity and induced brain - derived neurotrophic factor expression , suggesting modulatory roles at glutamatergic synapses and possible antidepressant action shin et al .
curcuma root is a spice that not only is used in preparing asian curries dishes but also is a component of some ancient herbal remedies for various diseases such as cardiovascular disease .
nowadays , curcumin , a component of curcuma root , has been demonstrated to have a variety of beneficial health effects , including anti - atherosclerosis , anti - oxidative , anti - inflammatory and anticancer effects ; thus , it has been recently used as a herbal medicine medicine qin et al . performed a study to investigate the effects of fructus chaenomelis water
extract on the production of inflammatory mediators in raw 264.7 cell mouse macrophages stimulated with lipopolysaccharide ( lps ) ryu et al .
they showed that fructus chaenomelis significantly inhibited increases in il-2 , il-10 , il-12p70 , tnf- , gm - csf , m - csf , lif , vegf , no and ca in lps - induced raw 264.7 cells without causing toxicity .
these results suggested that fructus chaenomelis had an anti - inflammatory effect on controlling the over - inflammatory reaction by the raw 264.7 cells ryu et al .
in addition , fructus chaenomelis pharmacopuncture at the joksamni is known to play an important role in controlling immune reactions and suppressing the inflammatory response to collagen - induced rheumatoid arthritis . although mecasin has been used in clinics , safety studies on mecasin are insufficient .
toxicity tests provide important data and are essential for evaluating the safety of test substances in medications .
doses of 500 mg / kg , 1,000 mg / kg , and 2,000 mg / kg of mecasin were administered to the experimental groups , and a dose of 10 ml / kg of normal saline solution was administered to the control group . in all four groups ,
no significant differences in the clinical signs or weights were noted between the control group and the experimental groups . in necropsy for checking for abnormalities in organs , no significant findings were noted . to assess the oral toxicity of mecasin , we need to study its acute and chronic side effects more .
the korea food & drug administration has testing protocol guidelines for the study of toxicity , and all experiments should be conducted following glp regulations .
the results of our study showed that administration of 2,000 mg / kg of mecasin did not cause any changes in the weights of sd rats or in the results of necropsy examinations .
it also did not result in any mortality , which indicates that mecasin administration can be used as a safe treatment .
the results showed that administration of 500 2,000 mg/ kg of mecasin did not cause any changes in weight or in the results of necropsy examinations .
it also did not result in any mortalities , which indicated that the lethal dose of mecasin was higher than 2,000 mg / kg .
the results obtained in this study suggest that mecasin administration can be used as a safe treatment . | objectives : in this study , we investigated the oral toxicity of gami - jakyak gamcho buja decoction ( mecasin ) to develop safe treatmentsmethods : all experiments were conducted at the medvill , an institution authorized to perform non - clinical studies , under the good laboratory practice ( glp ) regulations . in order to investigate the oral toxicity of mecasin , we administered mecasin orally to rats .
sprague - dawley rats were divided into four groups of five male and five female animals per group : group 1 being the control group and groups 2 , 3 , and 4 being the experimental groups .
doses of mecasin , 500 mg / kg , 1,000 mg / kg and 2,000 mg / kg , were administered to the experimental groups , and a dose of normal saline solution , 10 ml / kg , was administered to the control group .
we examined the survival rate , weight , clinical signs , and gross findings .
this study was conducted under the approval of the institutional animal ethics committee.results:no deaths or abnormalities occurred in any of the four groups .
although slight decreases in the weights of some female rats were noted on the third day , no significant changes in weights or gross findings between the control group and the experimental groups were observed . to check for abnormalities in organs ,
we used microscopy to examine representative histological sections of each specified organ ; the results showed no significant differences in any of the organs.conclusion:the results showed that administration of 500 2,000 mg / kg of mecasin did not cause any changes in weight or in the results of necropsy examinations .
it also did not result in any mortalities .
the above findings suggest that treatment with mecasin is relatively safe .
further studies on this subject are needed to yield more concrete evidence . |
the success of root canal treatment depends on the eradication of microorganisms from the root canal system and prevention of re - infection . due to its complex anatomy , with multiple fins , isthmuses , ramifications and accessory canals , it is virtually impossible for mechanical instrumentation to shape and clean the entire root canal system . according to a study conducted by bystrm and sundqvist ,
the number of intracanal microorganisms reduced 1001000 fold following hand instrumentation with stainless steel files and using sterile saline as a root canal irrigant .
thus , using an appropriate irrigation solution is essential to remove residual tissue and to kill microorganisms .
the antimicrobial effectiveness of naocl is based on its high ph ( hydroxyl ions action ) .
the high ph of naocl disturbes the completeness of the cytoplasmic membrane via inhibiting enzymes irreversibly , biosynthetic changes in cellular metabolism and depriving phospholipids observed in peroxidation of lipids . furthermore , naocl possesses excellent tissue dissolving ability .
in other words , tetracyclines are absorbed to dentin immediately and are deliver thereafter without missing their antibacterial effectiveness . in the field of endodontics ,
tetracyclines have been used to irrigate root - end cavities , to remove the smear layer from the instrumented canals and as an intra - canal medicament . currently , there are two antibiotic - based root canal irrigation solutions in the field of endodontics .
in addition , its antibacterial substantivity has been proved in some studies [ 10 , 11 ] .
the other antibiotic - based irrigant , tetraclean , ( ogna laboratori farmaceutici , muggi , italy ) , similar to mtad , is a mixture of an antibiotic , an acid and two detergents ( propylene glycol and cetrimide ) .
however , the concentration of the antibiotic , doxycycline ( 50 mg ml ) and the type of detergent ( polypropylene glycol and cetrimide ) differ from those of mtad . recently ,
mohammadi et al . showed that the substantivity of tetraclean was significantly greater than mtad and 5.25% naocl .
mtad as well as tetraclean do not dissolve organic tissues . therefore , naocl should be used to dissolve organic tissues .
. showed that the antimicrobial substantivity of mtad was reduced when mtad was applied to 1.3% naocl - irrigated dentin .
recently , mohammadi et al . showed that substantivity of tetraclean was reduced in naocl - irrigated dentin .
a study demonstrated that pretreatment of naocl - irrigated dentin with ascorbic acid prevented the decrease in mtad substantivity . considering the closed similarity between the composition of mtad and tetraclean , the aim of this study was to assess the effect of pretreatment of naocl - irrigated dentin with ascorbic acid on the substantivity of tetraclean .
the method of the present study was modified from that described by haapasalo and rstavik .
intact bovine central incisor teeth were used . in order to clean and disinfect the surface ,
the teeth were kept in 0.5% naocl solution ( merck , germany ) for up to 7 days .
the clinical crown and apical third were removed from each tooth using a rotary diamond saw at 1000 rpm ( isomet plus precision saw , buehler , il , usa ) under water - cooling .
the cementum was removed using polish paper ( ecomet 3 , variable - speed grinder - polisher , buehler , il , usa ) , which resulted in a center - holed piece of root dentin with a 6-mm outer diameter ( fig 1 ) . using a diamond saw ,
the remained piece of each tooth was cut into 4-mm - thick slices . using an iso 023 slow speed round bur ,
the canals of the 4-mm blocks were enlarged ( standardized ) . during the procedures , all teeth and dentin slices
were preserved in vials containing tap water to prevent dehydration . in order to remove the smear layer ,
each dentin block was placed inside 3 ml of 17% edta with a ph of 7.2 placed in an ultrasonic bath for 10 min and irrigated under tap water for 1 hour .
thereafter , the mentioned process was conducted with 5.25% naocl ( merck , darmstadt , germany ) instead of edta .
the specimens were then placed in bhi broth ( oxoid , basingstoke , uk ) and autoclaved at 121 c with 1 atm pressure for 15 minutes .
the specimens were kept in an incubator at 37c for 24 h to monitor the efficacy of the sterilization .
a total of 80 specimens were randomly divided into five groups as follows :
group 1 ( 20 specimens ) : tetraclean ( ogna laboratori farmaceutici , muggi , italy ) irrigationgroup 2 ( 20 specimens ) : 5.25% naocl and tetraclean irrigationgroup 3 ( 20 specimens ) : 5.25% naocl , ascorbic acid and tetraclean irrigation group 1 ( 20 specimens ) : tetraclean ( ogna laboratori farmaceutici , muggi , italy ) irrigation group 2 ( 20 specimens ) : 5.25% naocl and tetraclean irrigation group 3 ( 20 specimens ) : 5.25% naocl , ascorbic acid and tetraclean irrigation furthermore , 10 sterile and 10 infected dentin tubes were used as negative and positive control groups , respectively . in order to contaminate the dentin tubes , isolated 24-h colonies of pure cultures of e. faecalis ( atcc 29212 ) suspended in 5 ml of bhi
the bottles containing each specimen in groups 1 , 2 , 3 and 4 were opened under laminar flow .
two milliliters of sterile bhi was removed with sterile pipettes and replaced with 2 ml of bacterial inoculum .
the bottles were closed and kept at 37c for 14 days . to prevent medium saturation ,
1 ml of freshly prepared bhi was replaced by 1 ml of contaminated bhi every 2 days .
each specimen was removed from its bottle under aseptic conditions after 14 days and the canal was irrigated with 5 ml of sterile saline and dried with sterile paper points .
the outer surface of the specimens was covered with two layers of nail varnish to prevent contact of the medicament with the external surface .
afterwards , specimens were fixed at the bottom of wells of 24-well cell culture plates using decontaminated sticky wax , which also obliterated the apical surface of the root canal .
finally , the irrigating solutions were inserted into the canal lumen with sterile 3-ml plastic syringes and 27-gauge needles until the dentin tubes were totally filled .
solutions were removed using sterile paper points ten minutes after placement into the lumen . in group 2
the naocl solution was then removed with sterile paper points and dentin tubes were filled with tetraclean for 10 minutes . however , in group 3 samples , dentin tubes were filled with 10% ascorbic acid solution for 10 minutes before using tetraclean and after removing 5.25% naocl solution .
the specimens were then incubated at 37c for 28 days to maintain humidity . at experimental times of 0 , 7 , 14 , 21 and 28 days
, dentin chips were removed from the canals with sequential sterile low - speed round burs with increasing diameters of 025 , 027 , 029 , 031 and 033 iso sizes , respectively .
the powder dentin samples obtained with each bur were immediately collected in separate test tubes containing 3 ml of freshly prepared bhi .
the positive control group showed viable bacteria at all experimental times , which indicated the efficiency of the method .
in contrast , the negative control group showed no viable bacteria at all experimental times .
the number of cfu was minimum in the first cultures ( day 0 ) in all experimental groups and there were statistically significant differences between the obtained results at any time period ( p = 0.03 ) .
the tetraclean group showed the most effective antibacterial action at all five experimental periods ( p < 0.001 ) .
the naocl / ascorbic acid / tetraclean group showed similar antibacterial substantivity to the tetraclean group at all time periods ( p= 0.002 ) .
enterococcus faecalis was chosen for the following reasons : ( i ) its prevalence in root - filled teeth with periradicular lesions using culturing and polymerase chain reaction ( pcr ) methods is 2470% and 6777% , respectively .
( ii ) its capacity to endure prolonged periods of starvation until an adequate nutritional supply becomes available has been demonstrated .
the method of the present study was a modification of that proposed by haapasalo and orstavik .
using the mentioned method , each consecutive bur size removes only 0.1 mm of the dentin thickness around the root canal . according to the microcomputed tomography ( ct ) studies , comparatively huge areas of the root canal surfaces leave untouched by files .
therefore , using an irrigation solution is necessary to improve the debridement of the canals . for improvement of the efficacy of root canal irrigants
the closeness of this approximation is related to the wettability of the irrigant on dentin surface and wettability of irrigants has a strict correlation with their surface tension .
the force between molecules that produces a tendency for the surface area of a liquid to decrease .
surface tension is the force between molecules that decreases the surface area of the irrigant .
this force reduces the penetration depth of the liquid into a capillary tube . enhancing the wettability of an antimicrobial irrigant
increase its protein solubility and improves its efficacy in the uninstrumented parts of the root canal . despite the strong antimicrobial activity of naocl against e. faecalis , even in the form of biofilm ,
its high surface tension does not allow it to reach bacterial stocks in the depth of dentinal tubules and canal irregularities . to overcome this problem , some antibiotic - based irrigation solutions ( i.e. mtad and tetraclean ) with low surface tension have been introduced .
new generation of endodontic irrigants combine a powerful detergent effect with a strong antimicrobial efficacy .
in fact , in the present study , the effect of ascorbic acid ( vitamin c ) was assessed on the reversing the oxidative potential of naocl as a reducing agent .
findings demonstrated that treatment of naocl - irrigated dentin with 10% ascorbic acid for 10 minutes prevents reduction of the substantivity of tetraclean by prohibiting oxidation of doxycycline by naocl . reducing the activity of ascorbic acid
ascorbic acid also prevents photodegradation of tetracycline isomers ( doxycycline and minocycline ) [ 27 , 28 ] . using a human dentin tube model , mohammadi and shahriari demonstrated that residual antibacterial activity of mtad was significantly greater than chx . in another study using bovine dentin tube model , mohammadi et al
. demonstrated that the residual antibacterial activity of tetraclean was significantly greater than mtad and 5.25% naocl .
another study demonstrated that the residual antibacterial activity of tetraclean was significantly greater than hypoclean ( a naocl - based irrigation solution with reduced surface tension ) and 5.25% naocl .
. demonstrated that when mtad was used as initial rinse followed by the use of various concentrations of naocl as final rinse , a chemical reaction happened between naocl and the residual mtad , which caused formation of a brown solution in the root canal .
they proposed that such a phenomenon might be caused by the dentinal absorption and release of doxycycline present in mtad and the final rinse of naocl .
the reaction of naocl with either doxycycline or citric acid in mtad is not an acid - base reaction .
rather , it is a redox reaction that involves the oxidation of doxycycline by naocl .
examined the effect of naocl - mtad interaction on the antimicrobial substantivity of mtad in dentin .
findings demonstrated that the use of 1.3% naocl as an initial rinse resulted in a 30% reduction in the substantivity of mtad .
this study also showed that treating the naocl - irrigated dentin with ascorbic acid prevented the interaction between naocl and mtad . in another study ,
mohammadi et al . found that substantivity of tetraclean in naocl - treated dentin reduced significantly .
the reduction of the substantivity of tetraclean and mtad can be attributed to the partial loss in the substantivity from the doxycycline component .
within the limitations of the present study , in naocl - irrigated dentin , ascorbic acid prevented the decrease in the substantivity of tetraclean . | objective : the purpose of this study was to assess the in vitro effect of ascorbic acid on the antibacterial substantivity of tetraclean in bovine root dentin pretreated with sodium hypochlorite ( naocl).materials and methods : eighty dentin tubes prepared from bovine incisor teeth were infected with enterococcus faecalis for 14 days .
the specimens were divided into five groups as follows : tetraclean ; 5.25% naocl / tetraclean ; 5.25% naocl / ascorbic acid / tetraclean ; infected dentin tubes ( positive control ) ; and sterile dentin tubes ( negative control ) . at experimental times of 0 , 7 , 14 , 21 and 28 days , dentin chips were removed from the canals by sequential sterile low - speed round burs with increasing diameters of 025 , 027 , 029 , 031 and 033 iso sizes , respectively . after culturing ,
the number of colony - forming units ( cfu ) was counted.results:in all experimental groups , the number of cfu was minimum in the first cultures and the results obtained were significantly different at any time period ( p < 0.05 ) .
the tetraclean group showed the most effective antibacterial action at all five experimental periods ( p < 0.05 ) .
naocl / tetraclean group showed the least antibacterial activity at all time periods .
the naocl / ascorbic acid / tetraclean group showed similar antibacterial substantivity to the tetraclean group at all time periods ( p > 0.05).conclusion : ascorbic acid prevents the decrease of residual antibacterial activity of tetraclean in dentin samples pretreated with naocl . |
polymethyl methacrylate ( pmma ) is primarily used for removable partial and complete denture fabrication as a base material .
reinforcements such as fibers , glass1 and polyethylene or activation and processing techniques such as injection - molding and microwave2 activation provided new benefits in polymer knowledge . however , this material still has some limitations about mechanical properties such as dimensional stability , color and flexural strength
. eventually fracture of acrylic resin denture base is known to be the common clinical problem in prosthodontic practice .
causes of such fractures are known to be related to porosity , residual monomer , poor fit of denture base , occlusal imbalance , design and fabrication failures , material choices , stress after long clinical usage and accidents.3,4 besides , in certain circumstances before complete fracture , a crack formation may be propagated bound to different types of stresses that denture base materials are subjected to .
it is known that rough surfaces present suitable conditions for microorganism colonisation and/or biofilm formation,5,6 denture base cracks may become to be one of the best sites for microorganism propagation .
biofilm is a microbial community that has dense and complex structure and may represent multiple organisms.7 they are often encapsulated within a matrix of exopolymeric material that consists of intricate networks of cells attached to biotic surfaces .
they resist antimicrobials and immune cell challenge8 and are deeply embedded into cracks and porosities of dental materials as mentioned before .
metallic and non - metallic medical devices like catheters , implants , dental materials are suitable sites for colonization of various types of microorganisms.9 corrosion at the interfacial surfaces of non - metallic materials usually starts with swelling after infiltration of little molecules or microorganisms .
interfacial electrochemical process may be activated with the formation of biofilm on metallic and non - metallic surfaces resulting in an increased corrosion of colonized substratum .
this development can detoriorate the materials with the presence of biofilm and is termed as biocorrosion.9 three - dimensional structure of biofilm is known to provide a highly complex arrangement of microorganisms.10 several studies regarding the developments and structures of biofilms on different dental materials including denture bases and their effects over oral health have been constituted.8 - 13 however the relationship between the biofilm related biocorrosion and crack and/or fracture formation still remains complicated even undefined .
the aim of this study was to investigate the destructive effects of biofilm formation and/or biocorrosive activity of 6 different oral microorganisms by evaluating the diffusion potential of the microorganisms , fracture propagation and scanning electron microscope ( sem ) images ( calculating biofilm covered surfaces ) on three different denture base materials .
three different heat polymerized acrylic resins ( table 1 ) were used to prepare 50 15 4 mm ( type a ; for three point bending ( tpb ) test , n=210 ) , 8 8 1 mm ( type b ; for sem analysis , n=54 ) and 2 2 2 mm ( type c ; for spectrophotometer analysis , n=180 ) samples ( table 2 ) .
next , each specimen were deflasked and finished with 320 , 400 and 600-grit silicone carbid papers . to simulate a crack line on the denture base , " v " type notch was carved in the middle of each specimen of impact test groups ( a type ) along with the 15 mm surface by using a milling machine and a milling tool as shown in the fig .
all type of specimens were ultrasonically cleaned for 20 minutes and immersed in distilled water for 48 hours at 37 before tests .
s. aureus , e. faecalis , p. aeruginosa , e. coli , s. mutans and c. albicans strains were inoculated in trypticase soy broth media and grown to stationary phase overnight .
the samples were diluted 1:100 and each diluted bacterial culture ( 200 l ) was inoculated into each well in a fresh 96-well flat - bottom microtiter polystyrene plates , which also contain " c " type acrylic resin samples .
plates were incubated for 48 hours at 37 and visualized by staining with 0.5% crystal violet for five minutes after washing with water .
the biofilm was quantified in duplicate , after adding 100 l of 95% ethanol and the contents were transferred to new wells of microtiter plate
. optical density ( od ) of stained adherent bacteria was determined with a micro elisa auto reader at wavelength of 620 nm spectrophotometrically.14,15 these od values were calculated as : " od - control " and considered as an index of bacteria adhering to acrylic surfaces and forming biofilms .
ten specimens of type " a " , 3 specimens of type " b " and 10 specimens of type " c " denture base materials from 3 different brands ( table 1 ) were randomly inoculated into one of ; staphylococcus aureus ( atcc 6538 ) , streptococcus mutans ( atcc 35688 ) , enterococcus faecalis ( atcc 10541 ) , escherichia coli ( atcc 25922 ) , pseudomonas aeruginosa ( atcc 2327 ) or candida albicans ( atcc 18804 ) culture as indicated in table 2 at 0.5 mcfarland scale which corresponds to 10 cfu / ml for 168 hours at 37. a previously described method was modified and performed for biofilm formation.11 in brief , 1 ml aliquot of the bacterial and yeast cultures were introduced into 500 ml of brain heart infusion broth media in conical flasks and prepared denture base materials were inoculated into media . for the maintenance of bacterial and yeast density near the steady - state growth phase ,
50% of the media were drained and replaced with the equal amount of a fresh sterile medium every two days and on day seven , the denture base materials were retrieved from the inoculated media for sem examination .
the control group was composed of 10 non - contaminated " a " type samples of each denture base material ( 30 total ) and was kept in distilled water at 37 until the tpb test .
the contaminated samples of type " a " , were rinsed with pbs and kept in running water for 15 minutes .
the impact values of the specimens were measured under 2500 n maximum load ( 1 n preload ) with 1 mm / min cross - head speed with tpb test by using an universal testing machine ( lloyd lrx , west sussex , uk ) .
the specimens were then supported on the jigs with a diameter of 3.2 mm with span length 50 mm .
the " v notch " was placed face down on the jigs and the load was applied to the centre of the specimens .
the data of the measurements were transferred to a personal computer , and the results were recorded .
after incubation , each specimen of type " b " was removed and kept in an ultrasonic cleaner for 1 minute and rinsed with pbs to remove non or weak adhered microorganisms .
the samples were fixed in 2.5% glutaraldehyde solution for 1h at room temperature , then rinsed with pbs .
ethanol solutions with concentrations graded from 75% to 95% were used in 5 steps to dehydrate the specimens .
specimens were then dried and placed on stubs to coat with 20 a gold / palladium for sem ( jeol 6400 , jeol corp .
digital photographs as tiff files at 5,000 magnification were obtained from three different regions of each sample surface ( fig .
the images were transferred to a personal computer to calculate total area and area fractions of biofilms using image j software.16 since cleaning and dehydrating processes were performed accurately , the area aspects apart from denture surface were all accepted as biofilm surface of that microorganism . area fraction of each image was recorded as the data of that sample .
descriptive data were expressed as median , maximum , minimum and mean standard deviation .
statistical analysis was performed using " pasw 18.0 statistics " and " statistica-7 " statistical software .
when normality assumptions were satisfied analysis of variance ( anova ) otherwise the equivalent non - parametric test : kruskal - wallis was used for group comparisons . when significant differences found between groups , we used tukey and dunnett 's test ( after anova ) and dunn 's test ( after kruskall wallis ) for multiple group comparisons .
table 3 shows the mean quantitative biofilm values and statistical significances according to microorganism and denture base material . there were significant differences among the adhesion potential of 6 different microorganisms and there were significant differences among their adhesion onto 3 different denture base materials .
the highest median value within all the denture base materials was obtained with p. aeruginosa ( 0.095 0.018 ) followed by s. aureus ( 0.085 0.014 ) , c. albicans ( 0.081 0.017 ) , s. mutans ( 0.079 0.010 ) , e. faecalis ( 0.070 0.013 ) and e. coli ( 0.070 0.012 ) .
the difference of adhesion between p. aeruginosa and s. aureus was not significant however there were significant differences between p. aeruginosa and other 4 microorganisms .
the adhesion potential of the microorganisms over lucitone denture base material was higher than the other materials .
the difference was not significant compared with qc-20 but it was significant when compared with ivocap plus .
p. aeruginosa exhibited the highest median value on lucitone 550 denture base material surface ( 0.105 0.020 ) while c. albicans exhibited the lowest value on ivocap plus ( 0.067 0.021 ) .
sem images revealed the regions of typical biofilm formation of each selected microorganism on denture base materials ( fig .
2 ) . table 4 shows the mean biofilm covered regions ( % ) on denture base materials and statistical significances according to microorganism and denture base material .
the type of denture base material did not alter the diffusion potential of the microorganisms significantly .
the percentages of biofilm covered areas of denture base materials ranged from 52.57% to 70.96% .
s. aureus and p. aeruginosa had significantly higher ( 68.66% ) diffusion potential than the other tested microorganisms , but the difference among them was not significant .
e. coli had the least diffusion potential and was significantly different from all the tested groups .
table 5 shows the mean and standard deviation values of tpb test and statistical significances according to microorganism and denture base material .
biofilm formation of the tested microorganisms decreased the tpb test values compared to the control group and this was statistically significant .
lucitone 550 and ivocup plus denture base materials were significantly more resistant than qc-20 when mean tpb test values were consulted .
e. coli produced the minimum destructive effect to the base materials when mean tpb test values of materials were evaluated , however this was not significantly different from e. faecalis ( p=.699>.05 ) , s. mutans and c. albicans .
this study investigated the effects of biofilm formation and/or biocorrosive activity of 6 different oral microorganisms on three different denture base materials .
all tested microorganisms significantly decreased the tpb test values of the tested denture base materials .
after contamination with microorganisms , the tpb test values of denture base materials were decreased significantly compared with the control groups . it may be speculated that the structure of all the denture base materials were decomposed by the biofilm formation and/or biocorrosive activity of microorganisms .
several studies have investigated the fracture resistance of denture materials.17 - 20 in this study ivocap plus appears to be the most resistant base material when the peak data of control group and mean values at tpb tests were evaluated .
this was compatible with the results from the study of hedzelek and gajdus.21 however , when the percentages of tpb test value reduction were examined , lucitone 550 was the most resistant material .
the mean tpb test value of samples exhibited 18% resistance reduction at lucitone 550 samples , 19% at ivocap plus and 24% at qc-20 denture base materials compared with control group after contamination processes of 6 different microorganisms . evaluating the subgroups data of denture base materials and microorganisms of this study
, the highest tpb test value reduction was observed in p. aeruginosa - qc group with 36% .
even e coli , known to have the least degenerative bioactivity,12 decreased the tpb test values of lucitone 550 by 8% . eventually considering all the mean tpb test values of three denture bases of our study it can be affirmed that microorganisms had biocorrosive activity and deteriorated at least 15% of the initial physical composition of tested denture base materials .
however , the difference of mean biofilm covered regions on denture base materials at sem display were not statistically significant the mean quantitative biofilm values of microorganisms on ivocap plus was significantly different from lucitone 550 .
this may be due to the dissimilar accumulation of microorganisms on the material surface ( fig .
2 ) that varies according to the production of extracellular polysaccharides which can only be demonstrated using advanced techniques like three - dimensional confocal scanning laser microscopy.13 consequently , the two - dimensional sem display may have inhibited quantitative estimation of this phenomenon .
serrano - granger et al.22 claimed that there was no relationship between the microorganism adhesion and acrylic resin type and/or composition .
this was particularly compatible with our study ; when biofilm covered regions were evaluated when we observed that microorganisms showed minimal adhesion to ivocap plus .
this result was not statistically significant when compared with qc but significant compared with lucitone 550 .
this may be attributed to the special fabrication technique of this material ( injection molding ) which may form samples with smoother surface and lesser microporosities .
the other reason for minimal adhesion may be the higher residual monomer release after using injection molding technique.23 it is known that the pmma monomer is toxic for living cells.23,24 during the experimental process , with the mentioned destructive effect , pmma monomer may have inhibited the survival and/or adhesion of microorganisms tested on denture base materials . on the contrary , the higher adhesion to lucitone 550 may be attributed to the reinforcing materials that poorly adhere to the polymer matrix forming microporosities that become suitable for microorganism accumulation.25 c. albicans was shown to have higher adhesion potential to denture base materials in most of the studies.10 conversely , in our study c. albicans - ivocap plus group exhibited the least adhesion potential when all the subgroups of quantitative biofilm values were examined .
this result was compatible with the literature that p. aeruginosa and s. aureus have higher survival success over the plastic and/or metallic devices used in medicine.26 additionally the mean biofilm covered regions on denture base materials at sem display indicated the highest percentages for p. aeruginosa and s. aureus confirming the results of mean quantitative biofilm values of our study . within the limitations of this study
it was shown that microorganisms diffused at least 52% of the denture base surface that can not be neglected in dental practice .
however it was a remarkable result that this rate did not exceed 71% ( table 4 ) which may be attributed to the environmental or local conditions of this study or the floral equilibrium of microorganisms in certain circumstance .
it can be reported that all the tested microorganisms had destructive effect over the structure and composition of the denture base materials . | purposethe aim of this study was to investigate the destructive effects of biofilm formation and/or biocorrosive activity of 6 different oral microorganisms.materials and methodsthree different heat polymerized acrylic resins ( ivocap plus , lucitone 550 , qc 20 ) were used to prepare three different types of samples .
type " a " samples with " v " type notch was used to measure the fracture strength , " b " type to evaluate the surfaces with scanning electron microscopy and " c " type for quantitative biofilm assay .
development and calculation of biofilm covered surfaces on denture base materials were accomplished by sem and quantitative biofilm assay . according to normality assumptions
anova or kruskal - wallis was selected for statistical analysis ( =0.05).resultssignificant differences were obtained among the adhesion potential of 6 different microorganisms and there were significant differences among their adhesion onto 3 different denture base materials . compared to the control groups after contamination with the microorganisms , the three point bending test values of denture base materials decreased significantly ( p<.05 ) ; microorganisms diffused at least 52% of the denture base surface .
the highest median quantitative biofilm value within all the denture base materials was obtained with p. aeruginosa on lucitone 550 .
the type of denture base material did not alter the diffusion potential of the microorganisms significantly ( p>.05).conclusionall the tested microorganisms had destructive effect over the structure and composition of the denture base materials . |
cannabis sativa , better known as marijuana , has been used in traditional medicine for millennia to treat various ailments [ 14 ] .
development of cannabinoids from the cannabis plant as therapeutic agents has been hindered by their recreational abuse and addictive properties [ 5 , 6 ] .
medical marijuana is primarily used to treat glaucoma and to stimulate appetite and prevent weight loss in aids and cancer patients [ 14 ] . at present ,
the food and drug administration has approved two cannabinoid medications , marinol containing a psychoactive phytocannabinoid and cesamet consisting of a synthetic cannabinoid , for the treatment of nausea , emesis , and cachexia [ 68 ] . in several other countries , sativex has been approved to treat spasticity and neuropathic pain in multiple sclerosis patients and pain in patients with advanced cancer [ 1 , 9 ] .
it contains an equimolar combination of psychoactive -tetrahydrocannabinol and nonpsychoactive cannabidiol , which are phytocannabinoids [ 1 , 9 ] .
nevertheless , the beneficial health effects of cannabinoids , for the most part , remain empirical and anecdotal .
however , discovery of two major cannabinoid receptors and endogenous cannabinoids in humans provides the opportunity to understand the mechanisms of action and to develop approaches for manipulating the cannabinoid system as effective treatment for particular human diseases .
familiar effects of marijuana result from phytocannabinoids acting as neurotransmitters or modulating neurotransmitter release that , in turn , causes euphoria , diminished pain , altered sensory perception , impaired memory , and enhanced appetite [ 6 , 7 ] .
numerous studies report that cannabinoids suppress in vitro functions of human and animal immune cells , and animals exposed to cannabinoids have decreased host resistance to various pathogens and tumors ( reviewed in [ 1012 ] ) .
chronic cannabis use is associated with increased incidence of rhinitis , pharyngitis , asthma , bronchitis , and sexually transmitted diseases [ 5 , 9 , 13 , 14 ] .
besides social behavior contributing to an increased rate of sexually transmitted diseases , depressed immune functions could enhance susceptibility of marijuana users to infections .
because all immune cells examined so far express cannabinoid receptors regardless of their cell lineage , all types of immunity are sensitive to cannabinoid modulation [ 1012 ] .
the importance of the cannabinoid system in regulating immune competency is revealed by altered immune status in mice genetically deficient in cannabinoid receptors . in terms of adaptive immunity
, cannabinoids usually suppress primary antibody responses to t cell - dependent antigens , induction of cytotoxic cd8 t cells , and cytokine production by helper cd4 t cells , whereas other adaptive immune responses are unaffected or enhanced [ 1012 ] .
the current view is that cannabinoid exposure skews t cell responses leading to suppression of cell - mediated immunity and inflammatory reactions [ 1012 ] .
furthermore , cannabinoids impact innate immunity that mediates inflammatory responses and promotes initiation of adaptive immune responses .
for example , alveolar macrophages isolated from chronic marijuana users have compromised phagocytosis of microorganisms , ability to kill bacteria , and production of proinflammatory cytokines [ 16 , 17 ] .
these consequences of drug use parallel in vitro cannabinoid suppression of immune functions by monocytes , macrophages , and macrophage cell lines of human and rodent origins [ 1012 ] .
my laboratory reported that cannabinoids impair the ability of murine macrophages to function as antigen - presenting cells resulting in depressed helper cd4 t cell responses [ 1820 ] .
furthermore , macrophages from mice lacking cannabinoid receptor expression are refractory to cannabinoid suppression of antigen - presenting cell function [ 21 , 22 ] .
therefore , cannabinoids can exert their influence on an immune response before helper cd4 t cell activation .
the endocannabinoid system consists not only of cannabinoid receptors , but also of endogenous cannabinoids and their biosynthetic and metabolizing enzymes .
both exogenous and endogenous cannabinoids inhibit proinflammatory cytokine production by macrophages stimulated through toll - like receptors ( tlrs ) .
conversely , activation of macrophages via tlrs alters their expression of cannabinoid receptors and levels of endogenous cannabinoids .
this review discusses the endocannabinoid system and tlr family and evaluates the interaction between them with emphasis on the innate immune system .
central cannabinoid receptor type 1 ( cb1 ) and peripheral cannabinoid receptor type 2 ( cb2 ) are the predominant receptors and share approximately 44% homology [ 2730 ] .
endogenous cannabinoids also bind transient receptor potential vanilloid 1 receptor , a capsaicin receptor , which is structurally different from cb1 and cb2 receptors [ 2426 ]
. the orphan receptor gpr55 may be another receptor subtype , although it has low homology to the other cannabinoid receptors [ 2426 ] .
cb1 receptor , originally identified in rat cerebral cortex , is primarily expressed in the central nervous system [ 27 , 28 ] .
this receptor subtype is also expressed in various peripheral tissues , such as testis , vascular endothelium , and small intestine [ 27 , 28 ] .
its expression is heterogeneous within the nervous system and is mainly responsible for cannabinoid psychoactive properties .
in contrast , cb2 receptor was originally identified in the promyelocytic leukemic cell line hl60 and is prevalent within the immune system [ 29 , 30 ] .
all lineages of immune cells express the cb2 receptor , although its expression level varies among the cell types . in rank order ,
b cells express the highest level followed by natural killer cells , macrophages , monocytes , polymorphonuclear cells , and t cells .
cb2 receptor expression in healthy brains is limited to a few neurons in the brain stem [ 32 , 33 ] .
however , during neurological diseases , such as multiple sclerosis and alzheimer 's disease , microglial cells , which are brain macrophages , express a high level of cb2 receptor [ 33 , 34 ] .
some immune cells , including monocytes , also express the cb1 receptor [ 3537 ] .
when both receptor subtypes are present in immune cells , the cb2 receptor is usually expressed at a significantly higher level than the cb1 receptor [ 3537 ] .
unlike cb1 receptor - mediated cell activation , signal transduction through the cb2 receptor lacks psychotropic effects [ 38 , 39 ] making it an attractive target for immunotherapy .
cannabis is a complex mixture of over 100 cannabinoids along with other classes of compounds that have pharmacological and biological activities .
numerous synthetic analogues have been produced based on structure - activity relationship studies [ 24 , 25 ] .
many synthetic cannabinoid analogues have biological effects similar to their natural counterparts [ 40 , 41 ] .
cannabimimetic compounds are lipophilic molecules and are classified into four main groups based on their chemical structure [ 24 , 25 ] .
the classical group consists of dibenzopyran derivatives , and the phytocannabinoids fall into this group [ 24 , 25 ] .
it is a partial agonist binding both cb1 and cb2 receptors and is an ingredient of marinol and sativex .
cannabidiol does not activate cannabinoid receptors and yet has biological activity , including immune suppression . instead , cannabidiol appears to behave as a potent antagonist or inverse agonist [ 24 , 25 ] .
major homologues of -tetrahydrocannabinol and cannabidiol are -tetrahydrocannabivarin and cannabidivarin , respectively , with propyl side chains rather than pentyl side chains .
other members are synthetic analogues of phytocannabinoids , and some synthetic compounds are selective receptor agonists with higher affinity for one or other cannabinoid receptor subtype .
the best - studied member is ( )-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl ) cyclohexanol referred to as cp55,940 , which is a full nonselective agonist with higher potency than -tetrahydrocannabinol .
cannabinoids belonging to the aminoalkylindole group are very structurally different from classical and nonclassical compounds [ 24 , 25 ] .
the most widely studied member is ( r)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo [ 1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone called win 55,212 - 2 with slightly higher cb2 than cb1 receptor affinity .
eicosanoids are the endogenous compounds and are oxidized derivatives of 20-carbon fatty acids [ 24 , 25 ] .
arachidonoyl - ethanolamide also called anandamide was the first one isolated from porcine brain and was found to have cannabimimetic activity .
it behaves as a partial agonist and has lower intrinsic activity for cb2 than for cb1 receptor .
another prominent member is 2-arachionoyl glycerol , a monoglyceride , which is more potent than anandamide .
initially , 2-arachionoyl glycerol was isolated from intestine and brain [ 44 , 45 ] , and its concentration in the brain is approximately 170-fold higher than that of anandamide . because 2-arachionoyl glycerol favors the cb2 receptor , it is viewed as the main endocannabinoid to modulate immune functions .
classical and nonclassical cannabinoids and aminoalkylindoles are far more active than their corresponding enantiomer or stereoisomer [ 24 , 25 ] .
cannabinoid receptors are seven - transmembrane - spanning g protein - coupled receptors [ 2730 ] . the first evidence that cannabinoid receptors are gi / o protein - coupled receptors was cannabinoid - induced inhibition of adenylate cyclase leading to decreased intracellular camp level
. pertussis toxin inhibition of a cannabinoid effect confirms gi / o protein - mediated signal transduction .
g proteins are heterotrimers , and upon activation the subunit dissociates from the dimer ( figure 1 ) . the gi / o subunit inhibits adenylate cyclase decreasing intracellular camp levels [ 40 , 41 ] . in turn
, camp - dependent protein kinase a activity diminishes leading to less active transcription factor camp response element - binding protein affecting gene expression [ 40 , 41 ] .
the g protein dimer eventually leads to activation of the mitogen - activated protein kinase ( mapk ) pathways and phosphatidylinositol-3 kinase ( pi-3k ) [ 49 , 50 ] .
mapk and akt regulation by cannabinoid receptor signaling pathways is not well understood ( figure 1 ) .
pi-3k inhibitors attenuate cannabinoid - induced activation of mapk in chinese hamster ovary cells transfected with human cb1 receptor cdna .
this finding indicates that pi-3k leads to akt activation eventually causing p42/44 and p38 mapk activation .
similarly , -tetrahydrocannabinol activates the pi-3k / akt pathway in epithelial cells leading to raf-1-mediated activation of p42/p44 mapk .
in contrast , stimulation of rat microglial cells with 2-arachionoyl glycerol causes mapk activation that is dependent on protein kinase c , not pi-3k .
conversely , win 55,212 - 2 has the opposite effect and inhibits p42/p44 mapk activation in murine splenic immune cells .
akt may activate mammalian target of rapamycin ( mtor ) present in complex 1 ( mtorc1 ) , and , in turn , mtorc1 regulates protein synthesis , glucose metabolism , and autophagy .
rapamycin , a mtor inhibitor , blocks cannabinoid - induced neural progenitor cell proliferation and cannabinoid - enhanced oligodendrocyte differentiation [ 53 , 54 ] . on the other hand
furthermore , stimulation of promyelocytic hl60 cells with 2-arachionoyl glycerol or other cannabinoid agonists does not activate akt or mtor .
thus , cannabinoids may or may not activate akt , mapks , and mtor depending on the cannabinoid group type , cannabinoid concentration , and/or cell type with differential cannabinoid receptor expression . while cb1 and cb2 receptors share many signaling steps ,
cb1 receptor signaling can cause increased intracellular ca level , which may result from phospholipase c activation .
stimulation through the cb1 receptor also activates a - type and inwardly rectifying potassium channels and inhibits n- and p / q - type calcium currents in neural cells [ 40 , 41 , 49 ] .
most notably , cb1 receptor signaling may increase intracellular camp levels and camp - dependent protein kinase a activity due to the receptor associating with gs proteins .
furthermore , the cb1 receptor may associate with gq proteins leading to phospholipase d activation . while the cb1 receptor may couple to gs or gq proteins , the cb2 receptor does not . within the central nervous system ,
anandamide and 2-arachionoyl glycerol are not preformed molecules stored in vesicles but rather are synthetized when cells are stimulated , such as depolarization of neurons [ 56 , 57 ] .
their synthesis depends on increased intracellular ca concentration mainly due to ca dependency of the biosynthetic enzymes [ 5861 ] .
anandamide is predominantly produced in a two - step enzymatic process starting with phosphatidylethanolamine that is catalyzed by ca - dependent n - acyltransferase followed by n - acylphosphatidylethanolamine - hydrolyzing phospholipase d ( reviewed in [ 6265 ] ) .
the critical precursors of 2-arachionoyl glycerol are sn-1-acyl-2-arachidonoylgylerols , and multiple enzymatic pathways generate 2-arachionoyl precursors .
the main pathway involves phosphoinositide - selective phospholipase c or similar phospholipases followed by two sn-1-selective diacylglycerol lipase isoenzymes ( reviewed in [ 6265 ] ) .
in contrast to anandamide , various stimuli in addition to ca - mobilization trigger 2-arachionoyl glycerol synthesis in neural cells , epithelial cells , and macrophages [ 56 , 64 ] . unlike anandamide ,
2-arachionoyl glycerol is an important precursor of other molecules , which may explain their differential resting levels . in most cases , enhanced anandamide and 2-arachionoyl glycerol biosynthesis
is limited in time and location , and , thus , their release affects only cells in the nearby vicinity . after release
, endocannabinoids are rapidly internalized into cells by an undefined mechanism , and a proposed transporter has not been definitely identified .
intracellular anandamide is principally hydrolyzed by fatty acid amide hydrolase , an integral plasma membrane protein .
similarly , internalized 2-arachionoyl glycerol is primarily hydrolyzed by monoacylglycerol lipase associated with the plasma membrane .
reaction products do not activate cannabinoid receptors and may recycle back into their respective biosynthetic pathways .
one hydrolysis product , arachidonic acid , may be metabolized by various enzymes to produce prostaglandins , thromboxanes , leukotrienes , and other biologically active compounds , which are potent inflammatory mediators or immune suppressors .
anandamide and 2-arachionoyl glycerol themselves may be oxidized by cytochrome p450 , cyclooxygenase-2 , and 5- and 12-lipoxygenases [ 63 , 65 ] .
some lipoxygenase products bind both cb1 and cb2 receptors [ 25 , 56 , 57 ] .
hence , results from metabolic enzyme inhibitors or enzyme deficient mice should not presume to be caused by only increased endocannabinoid levels .
a fundamental characteristic of the immune system is the ability to distinguish between self and non - self - molecules or antigens .
immune cells are particularly adept at detecting microbial antigens , and the subsequent immune response can clear an infection and provide protection against a future infection .
innate immunity represents the first line of defense against infectious diseases , and cells , such as neutrophils and macrophages , are the first responders .
inflammation is the initial immune response against infectious agents , and the inflammatory response promotes initiation of an adaptive immune response by antigen - specific t and b cells .
however , cells of innate immunity do not express antigen - specific receptors , unlike t and b cells of adaptive immunity .
decades ago , janeway proposed a hypothesis that innate immune cells utilize germline - encoded receptors that are antigen - selective , and such receptors were eventually identified many years later .
innate immune cells express pattern - recognition receptors that bind conserved molecular patterns , and engagement of these receptors transduces a signal allowing cells to sense danger in the form of a pathogen or host cellular damage [ 69 , 70 ] .
these receptors are present at the cell surface , in endocytic organelles , or in the cytoplasm permitting perception of both extracellular and intracellular dangers .
the major receptor gene families based on protein domain homology include tlrs , retinoid acid - inducible gene-1-like receptors , absent in melanoma-2 receptors , c - type lectin receptors , intracellular dna sensors , and nucleotide - binding domain , leucine - rich repeat - containing receptors ( or nucleotide - binding , oligomerization domain - like receptors ) that are discussed in several comprehensive reviews [ 7076 ] .
this review focuses on the tlr family , which has been extensively investigated and was the first one discovered .
toll gene was first identified in drosophila as essential in regulating embryonic development of dorsal - ventral polarity [ 77 , 78 ] .
in addition , toll has a critical role in resistance to fungal infections in adult flies .
this latter discovery was key to understanding innate inflammatory triggers and led to finding the mammalian homologues [ 80 , 81 ] .
although the number of family members varies among mammalian species , tlrs are evolutionarily conserved type i transmembrane proteins [ 7076 ] .
tlrs are predominantly expressed by innate immune cells , especially dendritic cells and macrophages , while adaptive immune b and t cells , and nonimmune cells , including fibroblasts and epithelial cells , have limited tlr expression [ 7076 ] .
family members have a highly conserved cytoplasmic toll / interleukin-1 receptor ( tir ) domain that initiates signal transduction via recruitment of adaptor proteins [ 7076 ] . at present , 10 tlr chains have been identified in humans , while tlr10 is a pseudogene in mice , but three additional chains ( tlr11 to tlr13 ) are expressed in mice [ 7076 ] .
promiscuous ligand recognition , which is determined by the leucine - rich repeat extracellular domain , is indispensable for the handful of tlrs to mediate effective immune defenses against an array of diverse pathogens .
regarding mammalian host resistance , tlrs bind pathogen - associated molecular patterns ( pamps ) present within microbial molecules , but absent from mammalian molecules [ 7075 ] .
tlrs are divided into two subfamilies based on their cellular location , and the following discussion will focus on the tlr chains expressed in humans .
tlr1 and tlr2 , tlr4 through tlr6 , and tlr10 are plasma membrane proteins , whereas tlr3 and tlr7 through tlr9 reside in membranes of endocytic organelles .
cell surface tlrs sense fungal cell wall components ( tlr2 ) , bacterial lipopolysaccharides ( tlr4 ) , lipoproteins ( tlr1 , tlr2 , and tlr6 ) , flagella ( tlr5 ) , or peptidoglycans ( tlr2 ) .
on the other hand , intracellular tlrs are specific for viral double - stranded rna ( tlr3 ) , single - stranded rna ( tlr7 and tlr8 ) , dna ( tlr9 ) , or unmethylated cpg dna ( tlr9 ) .
for example , endosomal tlrs engage when virulent intracellular pathogens infect cells , but do not signal when the pathogen remains outside the cell .
although all tlrs possess a similar tir domain , cell surface and intracellular receptors , in general , utilize different adaptor molecules to transduce a signal [ 7075 ] .
cell surface tlr signaling mainly relies on tir - containing adaptor protein ( tirap ) and protein myeloid differentiation primary response 88 ( myd88 ) .
furthermore , tir domain - containing adaptor - inducing interferon- ( trif ) and trif - related adaptor molecule ( tram ) , also , transmit tlr signals .
the different pairs of adaptor molecules activate distinct transcription factors , and , hence , influence the nature and outcome of the inflammatory response . for many years
, one puzzling aspect of innate immunity has been inflammatory responses in the absence of an infection that contribute to tissue damage during autoimmune diseases and chronic inflammatory diseases .
matzinger proposed that the immune system does not distinguish between self and non - self - antigens per se but rather is designed to detect danger .
the apparent paradox of sterile inflammation is resolved by realization that pattern - recognition receptors , including tlrs , recognize endogenous ligands .
the endogenous molecules called damage - associated molecular patterns ( damps ) are created or released upon tissue injury or cell death [ 69 , 76 ] .
heat shock protein 60 was the first candidate damp reported to induce tlr4 activation . within months
, necrotic cells were shown to induce proinflammatory gene expression and dendritic cell maturation via tlr2 [ 84 , 85 ] . since then , several damps have been identified , and some are intracellular molecules to which innate immune cells are not normally exposed .
analogous to pamps , cell surface and intracellular tlrs recognize different types of damps , although endogenous ligands have not been identified for all tlrs [ 69 , 76 ] .
tlr2 and tlr4 ligands include heat shock proteins , serum amyloid a , and oxidized low - density lipoprotein .
recent evidence indicates that danger signals provided by pamps and damps synergistically activate inflammatory responses .
functional tlrs form dimers and particular receptors associate with a coreceptor and/or an accessory molecule [ 6976 , 86 ] .
most tlrs form homodimers with tlr2 as the notable exception pairing with tlr1 or tlr6 .
when a ligand binds , the tir domains oligomerize recruiting adaptor proteins that initiate downstream signaling events .
tlr4 , the prototype and best - studied receptor , is unique among tlrs using four adaptor molecules .
the classic tlr4 ligand is lipopolysaccharide ( lps ) from gram - negative bacteria , which causes septic shock .
the lps response requires a molecular complex ( figure 2(a ) ) consisting of tlr4 homodimer , lps - binding protein , cd14 , and myeloid differentiation-2 protein ( md-2 ) [ 6976 ] .
complex concentrates within cholesterol - rich lipid rafts containing cd14 , which is anchored to the plasma membrane by glycosylphosphatidylinositol .
the lps - binding protein , a soluble serum protein that binds lipid a moiety , initiates sequential transfer of lps monomers to coreceptor cd14 and then to md-2 . gioannini and weiss estimate that lps monomers extracted from one bacterium are sufficient to activate 1,000 macrophages .
hence , sequential lps transfer is thought to heighten sensitivity of innate immune cells , in particular macrophages .
adaptor tirap binds the tir domain of tlr4 but lacks a signaling domain [ 7075 , 86 ] .
tirap itself contains a tir domain that subsequently recruits adaptor myd88 to the tlr4 complex ( figure 2(a ) ) .
the myd88-dependent signaling pathway triggers il-1r - associated kinases and mapks culminating in activation of transcription factors , nuclear factor- ( nf- ) b , and activator protein-1 .
all tlrs except tlr3 utilize myd88 , and the other cell surface receptors , also , interact with tirap similar to tlr4 and lead to nf-b activation [ 6976 , 86 ] .
similar to tlr4 , tlr2 heterodimers localize to lipid rafts and require myd88 via tirap for signal transduction .
however , tlr2/tlr1 activation involves coreceptor cd14 , whereas cd36 serves as the coreceptor for tlr2/6 [ 70 , 88 ] .
cd36 functions as a scavenger receptor in monocytes and macrophages and participates in phagocytosis and endocytosis .
unlike other tlrs , tlr4 signals involve a second pair of adaptor proteins resulting in type i interferon production ( figure 2(a ) ) and a second wave of nf-b activation [ 6976 , 86 ] . in this pathway , adaptor tram and trif molecules drive cell activation .
analogous to tirap , tram lacks a signaling domain and recruits a second adaptor molecule , in this case trif , to the plasma membrane .
trif recruits signaling molecules that , in turn , recruit inducible ib kinase - i and tank - binding kinase-1 to the complex .
ultimately , transcription factor interferon regulatory factor- ( irf- ) 3 becomes activated leading to type i interferon synthesis , especially interferon-. receptor - interacting protein kinase-1 activity eventually leads to trif - dependent mapks and nf-b activation . both tirap and tram
localize to the plasma membrane ; however their binding to tlr4 appears to be mutually exclusive .
colocalization , time kinetics , and endocytosis inhibition studies indicate that tram engages after tlr4 internalization [ 70 , 7275 , 86 ] .
a novel model of tlr4 signaling proposes that cd14 mediates trafficking of tlr4 to the endosomes , whereupon the trif - dependent pathway is induced [ 70 , 7275 , 86 ] .
hence , the myd88-dependent and trif - dependent signaling pathways are sequestered from each other , and commence at distinct cellular locations .
intracellular tlrs stimulate cells by divergent pathways compared with tlr4 [ 70 , 7275 , 86 ] .
instead , engagement of these receptors induces type i interferon production via irf7 and nf-b activation in a myd88-dependent manner .
the alternative myd88 pathway triggers il-1r - associated kinases to activate transcription factors irf7 and nf-b . on the other hand
, tlr3 employs the adaptor trif protein ( figure 2(b ) ) but does not need either tram or myd88 .
the high affinity tir domain of tlr3 directly binds trif in the absence of tram .
notably , a point mutation within the tir domain of tlr3 switches its specificity from trif to myd88 .
similar to tlr4 , this trif - dependent pathway activates transcription factor irf3 via tank - binding kinase-1 inducing robust interferon- secretion .
critical regulatory mechanisms range from tlr trafficking and cleavage to protein modification of signaling molecules . furthermore , negative regulators are important in preventing autoimmune and inflammatory diseases .
tlr recognition of pamps is crucial for host resistance to infectious diseases , and individuals with defective tlr responses are immune compromised . on the flip side , abnormal tlr activation by pamps , mutations in tlr signaling molecules , and tlr activation by damps are associated with the development and pathogenesis of numerous diseases , including autoimmune diseases , hypersensitivities , chronic inflammatory diseases , cancer , and cardiovascular diseases . hence , cannabinoid modulation of tlr signal transduction pathways may have beneficial or detrimental consequences on human health .
important innate immune cells expressing tlrs are monocytes , macrophages , microglial cells , and dendritic cells .
monocytes circulate in the blood and mature into macrophages or dendritic cells depending on the stimulus .
all these cells express cannabinoid receptors , and cannabinoids influence their immune functions . of note ,
alveolar macrophages isolated from chronic marijuana users are compromised in secreting proinflammatory cytokines , such as tumor necrosis factor- and interleukin-6 , in response to lps [ 16 , 17 ] .
this review discusses cannabinoid modulation of tlr ligand responses by innate immune cells , and vice versa .
cannabinoid studies regarding tlrs have concentrated on bacterial lps responses via tlr4 as a classic model for inflammation ( table 1 ) . for the most part ,
exogenous and endogenous cannabinoids interfere with proinflammatory cytokine and nitric oxide production by lps- or lps / interferon--stimulated monocytes , macrophages , microglia , and macrophage cell lines in culture [ 1012 , 92 , 93 ] . however , one study reported increased interleukin-1 secretion by lps - activated resident peritoneal cells caused by -tetrahydrocannabinol in opposition to other investigations [ 95 , 96 ] .
cannabinoids display biphasic dose - response curves for cytokine secretion in some culture systems [ 96 , 97 ] , which may account for this apparent discrepancy .
interestingly , -tetrahydrocannabinol decreases lps - induced cyclooxygenase-2 expression in mouse macrophage j774 cell line , which would diminish endocannabinoid 2-arachidonoyl - glycerol metabolism , thereby augmenting immune suppression .
in addition , chronic marijuana use increases cb1 and cb2 receptor expression on peripheral blood monocytes , which may enhance cannabinoid sensitivity .
thus , exogenous cannabinoids may alter the endocannabinoid system leading to greater suppression of the lps response .
-tetrahydrocannabinol perturbs posttranslational processing of tumor necrosis factor- protein in lps - activated mouse macrophages [ 99 , 100 ] .
furthermore , cannabinoids diminish proinflammatory cytokine production in lps - stimulated mouse microglial bv-2 cell line that is accompanied by decreased transcription factor nf-b activity and corresponding cytokine mrna levels .
lps stimulates nitric oxide production and release through induction of nitric oxide synthase-2 expression under nf-b regulation .
similar to cytokine suppression , cannabinoids attenuate nitric oxide release , nitric oxide synthase-2 gene expression and enzymatic activity , and transcription factor nf-b activation in lps- or lps / interferon--activated myeloid cells [ 92 , 102104 ] .
analogously , cannabinoids suppress peptidoglycan - stimulated cell growth of a glioma cell line via tlr2 with concomitant decreased nf-b activation .
these results indicate that cannabinoids directly interfere with tlr signal transduction . on the other hand
for example , a cb2 receptor - selective agonist prevents lps - upregulated tlr4 expression on mouse bone marrow dendritic cells rendering the cells less lps responsive , although a similar cannabinoid effect upon tlr4 expression has not been reported for other innate immune cells .
moreover , cannabinoids induce apoptosis that has been proposed as an immunosuppressive mechanism [ 107 , 108 ] . however , the majority of findings regarding cannabinoid inhibition of tlr - mediated responses can not be attributed to apoptosis or cell toxicity .
anandamide and a cb2 receptor - selective agonist induce production of interleukin-10 , an inhibitory cytokine , in lps / interferon--activated myeloid cells [ 109 , 110 ] .
cp55,940 induces interleukin-1 receptor antagonist expression in lps - stimulated microglial cells , which interferes with interleukin-1 signals .
thus , multiple modes of action may mediate cannabinoid suppression of in vitro lps responses .
lps administration in animals is frequently used as in vivo models of inflammation and bacterial sepsis ( table 2 ) .
a synthetic cannabinoid diminishes lps - stimulated proinflammatory cytokine levels in the brain and blood of rats . in the lps - induced pulmonary inflammation model , exogenous and endogenous cannabinoids dose - dependently
decrease tumor necrosis factor- level in bronchoalveolar fluid and reduce neutrophil infiltration into the lungs in mice .
synthetic cannabinoids rescue c. parvum - primed mice from lps lethality and diminish serum proinflammatory cytokine levels .
likewise , a nonpsychoactive synthetic cannabinoid abolishes lps - induced hypotensive response in rats and rescues mice from the lethal effects of lps and d - galactosamine coadministration .
a cb2 receptor - selective agonist also protects against mortality and acute liver failure , decreases proinflammatory cytokines levels , and increases inhibitory interleukin-10 level in mice given lps and d - galactosamine . upon in vitro lps stimulation , cytokine and nitric oxide production by macrophages from mice previously given -tetrahydrocannabinol remain suppressed without additional drug in the cultures .
therefore , the effects of cannabinoids on lps activation in several animal models , in general , parallel the in vitro findings .
one set of criteria is cannabinoid receptor - selective agonists causing inhibition and cannabinoid receptor - selective antagonists reversing inhibition .
cannabinoid suppression of proinflammatory cytokine and nitric oxide production is cb2 receptor - mediated in lps - stimulated cultured cells , lps - induced pulmonary inflammation , and lps / galactosamine - induced acute liver failure [ 113 , 116 ] ( table 2 ) . on the other hand , protection of c. parvum - primed mice from lps lethality and diminished nf - kb activity in tlr2 ligand - stimulated glioma cells are mediated through the cb1 receptor [ 105 , 114 ] , whereas both cannabinoid receptors participate in decreasing cytokine levels after in vivo lps administration .
cb2 receptor - deficient mice are highly susceptible to induced inflammatory diseases , including contact dermatitis , experimental autoimmune encephalomyelitis , atherosclerosis , and carbon tetrachloride - induced liver damage [ 15 , 117 ] . incidence and severity of the diseases are exacerbated in the cb2 receptor - deficient mice compared to wild - type mice [ 15 , 117 ] .
importantly , the cb2 receptor - deficient mice have lower survival , more pronounced tissue damage , and increased serum interleukin-6 levels in a sepsis model . mice lacking both cannabinoid receptors have markedly heightened allergic inflammation leading to exacerbated contact dermatitis , delayed - type hypersensitivity , and inflammatory responses to influenza virus [ 119121 ] .
alveolar macrophages and bone marrow dendritic cells from mice deficient in both cannabinoid receptors have a more mature phenotype , have increased expression of major histocompatibility complex class i and class ii molecules , and are more efficient in activating t cells [ 120 , 121 ] , suggesting that the absence of endocannabinoid signals may alter differentiation and maturation of innate immune cells towards hyperresponsiveness .
perhaps , the role of the cb1 receptor is more readily detected in in vivo models due to interactions among various cell types .
the absence of both cannabinoid receptors has a more dramatic impact on the immune system , indicating the possible interplay of cannabinoid receptors during disease processes , which has important implications in developing cannabinoids as therapeutic agents for inflammatory diseases .
microglial cells play an important role in neuroinflammation and appear to be a special case in terms of cannabinoid receptor - mediated immune suppression .
general consensus is that resting microglial cells express a low level of the cb1 receptor and lack cb2 receptor expression [ 12 , 56 , 57 ] .
however , microglial cells from diseased tissues or microglial cells activated in culture gain cb2 receptor expression [ 12 , 56 , 57 ] .
criteria used for cannabinoid receptor involvement in diminished proinflammatory cytokine secretion and nitric oxide release from lps - activated microglial cells in culture reveal all possible outcomes encompassing both cannabinoid receptors , or the cb1 , cb2 , or no cannabinoid receptor [ 103 , 109 , 111 , 123125 ] . a receptor - independent mechanism despite cannabinoid receptor expression on the cells implies that cannabinoid receptor expression is too low to exert a biological effect , or the receptors are inactive .
a receptor - independent mechanism may involve disruption of lipid rafts due to the hydrophobicity of cannabinoids .
as discussed above , lipid rafts are critical for proper assembly of the tlr4 and tlr2 complexes , and their disruption would contribute to a decreased tlr response .
differences in agonist or antagonist concentrations , and cell activation state among the studies , may contribute to disparate findings concerning cannabinoid receptor participation .
cannabinoids exert immune suppression when innate immune cells are activated , but not when the cells are resting or quiescent .
when direct suppression is cannabinoid receptor - mediated , the two signal transduction pathways ( figures 1 and 2(a ) ) would cross - talk .
transcription factor nf-b is activated through the myd88-dependent signal transduction pathway via both tlr4 and tlr2 .
hence , cannabinoids must interfere with the myd88-dependent signal transduction pathway to decrease nf-b activity along with diminishing cytokine production and cell growth through tlr4 and tlr2 . in some cases , cell permeable camp to counteract the active gi / o subunit
reverses cannabinoid inhibition of cytokine secretion and nitric oxide release [ 1012 , 102 , 124 ] suggesting the involvement of decreased protein kinase a activity in mediating suppression .
however , protein kinase a activity is not necessary for cytokine gene expression in tlr - activated cells ( figure 2 ) .
other studies suggest cannabinoid regulation of p42/p44 mapk activation participates in immune suppression [ 41 , 52 ] .
activation of the mapk pathways is required for cytokine gene transcription . in this scenario ,
although a remaining question is what is the link between the cannabinoid and tlrs signaling pathways , mapks are attractive candidates .
very few studies have examined the impact of cannabinoids on tlr3 signal transduction and interferon- production ( figure 2(b ) ) , and , thus , studies with nonimmune cells are discussed below .
interferon- is a type i interferon with antiviral and anti - inflammatory activities and is a treatment for multiple sclerosis patients .
win 55,212 - 2 does not suppress but rather enhances interferon- mrna expression in polyinosinic : polycytidylic acid - activated hek293 cell line transfected with tlr3 cdna .
interferon- transcript upregulation is accompanied by increased mapk and transcription factor irf3 activities ; however tumor necrosis factor- secretion and transcription factor nf-b activity decrease in the tlr3-transfected cells .
analogously , win 55,212 - 2 augments interferon- mrna expression but decreases tumor necrosis factor- secretion in polyinosinic : polycytidylic acid - activated primary astrocytes and peripheral blood mononuclear cells from multiple sclerosis patients [ 126 , 127 ] .
perhaps , excessive mapk activation leads to decreased nf-b activity as discussed for the lps response.the opposing effects on irf3 and nf-b activities indicate that the cannabinoid alters a signaling step downstream of trif binding tlr3 . in striking contrast ,
win 55,212 - 2 has the opposite effect on interferon- mrna and transcription factor irf3 in tlr4-transfected hek293 cells stimulated with lps ( table 1 ) .
likewise , -tetrahydrocannabinol and nonpsychoactive cannabidiol inhibits interferon- mrna expression and protein secretion in lps - activated mouse microglial bv-2 cell line .
an antagonist of peroxisome proliferator - activated receptor- blocks the enhanced interferon- level without affecting tumor necrosis factor- level in tlr3-transfected hek293 cells , indicating sensitivity of transcription factor irf3 , but not nf-b , activity to peroxisome proliferator - activated receptor-. however , sensitivity of interferon- inhibition to the peroxisome proliferator - activated receptor- antagonist was not examined in tlr4-transfected hek293 cells . the opposing cannabinoid effects on interferon-
induced via tlr3 versus tlr4 signaling pathways ( figure 2 ) raise the question of why peroxisome proliferator - activated receptor- does not enhance interferon- production in lps - stimulated cells exposed to cannabinoids , unless the receptor is not activated during tlr4 signaling . the disparate cannabinoid effect on interferon-
production implies two different molecular targets in the tlr3 and tlr4 pathways . perhaps , the tram : trif and trif adaptors , or traf3 and traf6 used by tlr4 and tlr3 , respectively , have differential sensitivity to cannabinoid immune modulation .
cannabinoid receptor expression by immune cells varies depending on the cell type , maturational stage , and activation state .
innate immune cells have a high degree of plasticity , and their cannabinoid receptor expression can be manipulated intentionally ( table 3 ) .
human peripheral blood monocytes , human dendritic cells , and some monocyte / macrophage cell lines constitutively express both cb1 and cb2 receptors [ 129 , 138 , 143 ] .
maturation of human peripheral blood monocytes and monocytic thp-1 cells and differentiation of human promyelocytic hl60 cell line into macrophages by phorbol esters upregulate cannabinoid receptor expression [ 143145 ] .
on the other hand , resident mouse macrophages lack detectable cb1 and cb2 receptor mrna , whereas inflammatory thioglycollate - elicited macrophages express a high level of the cb2 receptor , and interferon- stimulation may further increase cb2 receptor expression .
microglial cells express no / low level of cb1 receptor mrna , and cb2 receptor mrna is undetectable [ 128 , 129 , 146 ] .
stimulation of rodent microglial cells with interferon- induces cb2 receptor mrna expression [ 129 , 130 ] .
the level of cannabinoid receptor expression on immune cells affects cannabinoid immune modulation . for example
, a nonpsychoactive cannabinoid inhibits lps - induced interleukin-6 expression only after human monocytes mature into macrophages .
hence , particular stimuli induce or upregulate cannabinoid receptor expression during myeloid cell differentiation , maturation , and activation affecting their sensitivity to cannabinoids .
the influence of only tlr4 signal transduction has been investigated on the endocannabinoid system in myeloid cells ( table 3 ) .
lps modulation of cannabinoid receptor expression in myeloid cells depends on the experimental system ( figure 3 ) .
for example , cb2 receptor mrna level drops in thioglycollate - elicited macrophages in response to lps [ 128 , 131 ] .
in contrast , lps activation of mouse macrophage raw 264.7 cell line induces cb1 receptor mrna and upregulates cb2 receptor mrna expression [ 129 , 132 , 139 ] .
protein kinases a and c inhibitors partially block the lps effect on cb1 and cb2 receptor expression , although involvement of the camp - protein kinase a pathway in lps signaling is controversial .
likewise , in vivo lps administration to rodents upregulates cb2 receptor expression in microglial cells [ 132 , 133 ] .
importantly , various pathological conditions are associated with altered cannabinoid receptor expression , usually increased cb2 receptor expression [ 10 , 57 ] . hence
, the differential lps impact on cannabinoid receptor expression may reflect plasticity of innate immune cells to cues from their microenvironment .
evidence is growing that endocannabinoid levels change during various disease processes due to altered catalytic activities of the biosynthetic or metabolizing enzymes . in several animal disease models ,
modified endocannabinoid levels exert pro- or anti - inflammatory effects on innate immune cells based on enzyme inhibitors , transporter inhibitors , and mice genetically deficient in the enzymes .
this research area is discussed in detail elsewhere [ 56 , 57 , 92 , 133 , 149 ] .
lps administration in vivo decreases metabolizing fatty acid amide hydrolase activity in mouse peripheral blood mononuclear cells , which would increase anandamide levels ( table 3 ) .
similarly , lps administration also diminishes 2-arachidonyl glycerol hydrolytic activity within the spleen and liver .
anandamide is barely detected in rat monocytes and macrophage j774 cell line , whereas the cells contain substantial anandamide levels upon lps stimulation [ 136 , 140 ] .
in addition , lps activation of rat macrophages and j774 cells also increases 2-arachidonyl glycerol levels due to decreased 2-arachidonyl glycerol hydrolytic activity in the cells .
human immature dendritic cells contain enhanced 2-arachidonyl glycerol , but not anandamide , levels upon lps activation .
conversely , low dose - lps activation of macrophage raw 264.7 cell line increases anandamide , but not 2-arachidonyl glycerol , levels along with enhanced biosynthetic n - acyltransferase and phospholipase d activities [ 23 , 137 ] .
the rapid time kinetics of augmented anandamide level indicates a direct effect of the tlr4 signal transduction pathway as opposed to autocrine stimulation by secreted cytokines . in support of this possibility ,
increased anandamide level in raw 264.7 cells is prevented by mapk and nf-b inhibitors , indicating involvement of the myd88-dependent signal transduction pathway ( figure 2(a ) ) .
although what endocannabinoids increase during lps stimulation appears to depend on the cell type and experimental conditions , higher endocannabinoid levels are accompanied by increased biosynthetic enzyme activity and/or decreased metabolizing enzyme activity .
when the immune system encounters a pathogen , innate immune cells recognize the pathogen via tlrs and other pattern - recognition receptors to trigger an inflammatory response .
innate immune cells are an important source of endocannabinoids , and these cells synthesize and metabolize endocannabinoids .
tlr - mediated activation of the innate immune cells enhances their endocannabinoid levels ( figure 3 ) . in the absence of an infection
, tissue damage produces damps perceived as danger signals via tlrs to activate innate immune cells . indeed , local endocannabinoid production increases in response to tissue damage during disease progression and infections .
abundant evidence demonstrates that cannabinoids have anti - inflammatory activity , which is the desired consequence during sterile inflammation . considering that 2-arachidonyl glycerol behaves as a chemoattractant [ 129 , 145 ]
, locally enhanced endocannabinoid levels may recruit immune cells to the site and mitigate further tissue damage .
thus , innate immune cells may play a role in regulating endocannabinoid homeostasis , and , in turn , the endocannabinoid system modulates local inflammation .
tlr signals also alter cannabinoid receptor expression by innate immune cells , which affects their sensitivity to cannabinoids . during progression of an inflammatory disease
, cells may become refractory to cannabinoid immune suppression despite elevated endocannabinoid levels , and the inflammatory response continues and may intensify .
therefore , the final outcome may be enhanced clearance of an infection , facilitation of tissue healing , or exacerbation of tissue damage .
although definition of the link between cannabinoid and tlr signaling pathways awaits further studies , identification of promising molecular targets may provide insights into therapeutic modalities to control injurious inflammation . | since the discovery of the endocannabinoid system consisting of cannabinoid receptors , endogenous ligands , and biosynthetic and metabolizing enzymes , interest has been renewed in investigating the promise of cannabinoids as therapeutic agents .
abundant evidence indicates that cannabinoids modulate immune responses .
an inflammatory response is triggered when innate immune cells receive a danger signal provided by pathogen- or damage - associated molecular patterns engaging pattern - recognition receptors .
toll - like receptor family members are prominent pattern - recognition receptors expressed on innate immune cells .
cannabinoids suppress toll - like receptor - mediated inflammatory responses .
however , the relationship between the endocannabinoid system and innate immune system may not be one - sided .
innate immune cells express cannabinoid receptors and produce endogenous cannabinoids .
hence , innate immune cells may play a role in regulating endocannabinoid homeostasis , and , in turn , the endocannabinoid system modulates local inflammatory responses .
studies designed to probe the interaction between the innate immune system and the endocannabinoid system may identify new potential molecular targets in developing therapeutic strategies for chronic inflammatory diseases .
this review discusses the endocannabinoid system and toll - like receptor family and evaluates the interaction between them . |
louis pasteur 's epochal germ theory of disease triggered the fastest advance of medicine ever .
the great host of infectious diseases , with their aetiology at last discovered , was soon substantially conquered .
additionally , freed of inevitable wound infection , modern surgery was made possible , led by lister . in stark contrast to
coombs and gell 's classification of harmful immune responses was preautoimmunity , antediluvian , based on the misapprehension that paul ehrlich 's horror autotoxicus
, ehrlich wrote that possible failure of the internal regulation of immune processes might be the explanation of many disease phenomena .
the independent demonstration of autoimmunity in four different laboratories [ 59 ] made coombs and gell 's classification obviously wrong , yet it lingered on for decades as an inhibition to clarity of thought .
table 1 shows a valid classification based on awareness of autoimmunity and awareness that allergy is a defect of immune defence against worms and other parasites , being caused by the occurrence of b lymphocyte clones that secrete ige antibodies with specificity for nonparasitic foreign antigens .
autoimmune diseases are caused by defects of immune specificity , which is determined by the amino acid sequence of the variable regions of the receptors for antigen on b and t lymphocytes , not by cytokines nor dendritic cells .
niels jerne won the nobel prize for his epochal realization that antibodies are not formed on a template of antigen but are preformed , in myriad diversity , awaiting contact with an antigen that fits , the selection theory .
macfarlane burnet realised that it is not antibodies that are selected but the cells that make them and that these cells are lymphocytes , whose unit of specificity is the lymphocyte clone , a subset of lymphocytes with identical receptors for antigen , hence , burnet 's clonal selection theory of acquired immunity , today in general acceptance .
however , widely neglected is burnet 's equally important forbidden clone theory , postulating that autoimmune disease is caused by somatic mutation in lymphocytes multiplying in response to a microbial antigenic stimulus . in 1838 ,
robert graves , in dublin , identified the syndrome of tachycardia , goitre , weight loss and exophthalmos , which bears his name as graves ' disease . in 1933 , charles harington isolated thyroxine , the first hormone to be chemically isolated , and found the molecule to contain iodine atoms , explaining the cause of endemic goitre , which is due to deficiency of dietary iodine . in 1956 , adams and purves [ 17 , 18 ] in dunedin , using radioactive iodine for a bioassay of thyroid - stimulating hormone in baby guinea pigs , discovered a long - acting thyroid stimulator ( lats ) .
this proved to be an autoantibody that causes thyrotoxicosis by accidentally reacting with the thyroid gland receptor for thyroid - stimulating hormone from the pituitary gland , the conductor of the endocrine orchestra .
affinity chromatography of thyroid - stimulating autoantibodies from individual patients shows that they contain only one of two possible antibody light chain types , k or , but never both .
therefore , these autoantibodies originate from single b lymphocytes in which a v gene has mutated .
furthermore , the variation from patient to patient of reactivity with the slightly differing tsh receptors of various nonhuman animals shows the random element in the somatic mutations forming the thyroidstimulating autoantibodies . in 1971
allison et al . postulated that lack of suppressor t cells causes the autoimmune diseases . in 1975
nachigal et al . proposed that on meeting antigen , mature t cells become helpers and immature t cells become suppressors .
knight and adams disproved claims that lack of suppressor t cells causes autoimmune disease in the new zealand mice . in 1999 shevack , in paul 's fundamental immunology , reported that the autoimmune diseases are not caused by lack of suppressor t cells . in 2010
adams has postulated that the pericyte loss is the cause of the vascular collapse of diabetic retinopathy .
furthermore , adams proposes that the pericytes are destroyed by forbidden clones of cytotoxic t cells , antigenically related to those that cause type l diabetes by destroying the pancreatic islet cells that make insulin .
this seems to be an example of cross - tissue t cell autoimmunity , analogous to the cross - tissue b cell autoimmunity of graves ' disease , where thyroid cells and orbital fat cells can both be targets , causing thyrotoxicosis and exophthalmos .
therapeutically , this new understanding suggests that ineffective tight glycaemic control should be replaced by immunosuppression with low - dose prednisone for treatment of diabetic retinopathy .
the main reason is for defence against the explosive speed of replication of infecting viruses . in influenza virus infection , for example , each virus - infected cell bursts open after 18 hours to release about a thousand virions , each of which infects a new cell , which in turn produces another thousand virions and so on , with death of the patient in 3 or 4 days .
they found that peptides , from a virus infecting a cell , are extruded to the cell surface on a histocompatibility antigen molecule .
there , the viral peptide is available for reaction with the antigen receptor of a cytotoxic t - cell clone , if one with complementary antigen specificity exists .
this was first explained in the lancet in 1987 , which shows that the mhc presentation is necessitated by the explosive speed of virus replication .
this huge number of virions would swamp the cytotoxic t cells , muffling their antigen receptors and so preventing them from destroying the virus factories that the infected cells become .
it follows that to prevent virus infections from being universally lethal , the defending cytotoxic t - cell clones must be reactive with the conjoint mhc - virus antigen on the surface of the infected cells , rather than reactive with the free virions .
the forbidden clone theory , postulating somatic mutations in lymphocytes as the cause of the autoimmune diseases , does not explain the familial aggregation , which indicates involvement of germline genes
. when the immunoglobulin v ( variable region ) genes , coding for the antigen specificity of lymphocyte clones were discovered , they seemed likely to be germline genes that influence the risks of autoimmune diseases .
however , the discovery that the autoimmune disease lupus nephritis occurs in f l hybrids of two inbred strains of mice , but not in either parental strain , afforded an opportunity to analyse the parental contributions to this autoimmune disease . in dunedin ,
bielschowsky 's nzb bl inbred strain of mice had been found to develop autoimmune haemolytic anaemia , spontaneously . to study the genetics , howie and helyer crossed nzb mice with the normal nzw strain and found that the f l hybrids spontaneously develop lupus nephritis .
backcross studies showed involvement of three genes in the lupus nephritis , one from the nzb strain and two from the nzw strain .
linkage studies showed none were the expected v genes , one being in the mhc and two being in the neighbourhood of minor histocompatibility antigen genes . combining the fields of transplantation genetics and autoimmunity , adams and knight produced the h gene theory , which states that histocompatibility antigens , major , minor , and hy , delete nascent lymphocyte clones with complementary antigen receptors and so dictate the immune repertoire , altering the risks of autoimmune diseases .
additionally , germ - line v genes , which provide the initial immune repertoire , influence the risk of autoimmune disease .
necessary for the h gene theory is explanation of how histocompatibility antigens , by deleting complementary clones , can add to the immune repertoire .
it was postulated that microbes bear multiple antigens and that a high - affinity clone for one antigen will preempt reactivity by lower - affinity clones for other antigens on the same microbe .
hence , histocompatibility antigens , by deleting certain clones , can cause others to be added to the immune repertoire and developed by somatic mutation , an alternative clonal development concept [ 11 , 25 , 36 ] . as described below , ebringer and his colleagues , in the course of discovering the microbial triggers of rheumatoid arthritis and ankylosing spondylitis , have confirmed the h gene theory , by finding that these bacterial triggers have two antigens , one resembling the predisposing histocompatibility antigen and one resembling the autoantigen attacked .
working on graves ' disease , knight realized that schizophrenia has many similar aetiological features .
accordingly , he postulated that forbidden clones of b lymphocytes develop by somatic mutation and make autoantibodies that act on neuronal cell receptors to affect the function of the brain limbic system .
knight proposed that : the familial aggregation is due to h genes and v genes , and the wait for v gene mutation explains the 50% discordance of monozygous twins and the juvenile grace gap ( relative absence in children ) .
kety ' s adoptive studies , remission and relapse , the rheumatoid arthritis / schizophrenia discordance .
strong confirmation comes from harrison and owen 's discovery of predisposition by mhc and b - cell light chain v genes , which are immune response genes . in the light of the forbidden clone and h gene theories , the genetics of autoimmune disease changes from mystery to elegant simplicity .
the key is the random element imposed on the genesis of an autoimmune disease by the need for unlucky somatic mutations in lymphocyte v genes .
table 2 shows that autoimmune disease provides a fourth category to go with dominant , recessive , and sex - linked diseases in mckusick 's catalogue of mendelian inheritance in man .
rheumatic carditis and streptococcibefore the advent of penicillin , rheumatic fever with lesions of the heart and joints was a frequent autoimmune complication of infections by -haemolytic streptococci of lancefield group a. this was because of an antigenic similarity between components of the streptococcus and heart tissue , found by kaplan and meyeserian . today , with such infections therapeutically aborted by penecillin , rheumatic heart disease , once common , has virtually disappeared .
before the advent of penicillin , rheumatic fever with lesions of the heart and joints was a frequent autoimmune complication of infections by -haemolytic streptococci of lancefield group a. this was because of an antigenic similarity between components of the streptococcus and heart tissue , found by kaplan and meyeserian . today
, with such infections therapeutically aborted by penecillin , rheumatic heart disease , once common , has virtually disappeared .
reactive arthritisthis has been observed after enteric infection with shigella , salmonella , yersinia , and campylobacter and genital infection with neisseria gonorrhea .
this has been observed after enteric infection with shigella , salmonella , yersinia , and campylobacter and genital infection with neisseria gonorrhea .
rheumatoid arthritis ( ra ) and proteus mirabilis multiple studies over three decades have found high titres of antibodies against this bacterium in a total of 1,375 ra patients , but not in other diseases or healthy controls , in studies by independent groups in 15 different countries .
there is evidence that the upper urinary tract is the main source of proteus infection in ra .
multiple studies over three decades have found high titres of antibodies against this bacterium in a total of 1,375 ra patients , but not in other diseases or healthy controls , in studies by independent groups in 15 different countries .
there is evidence that the upper urinary tract is the main source of proteus infection in ra .
ankylosing spondylitis ( as ) and klebsiella in worldwide studies involving 1330 as patients and 1191 healthy controls , the as patients showed significantly increased antibody titres to klebsiella
there is evidence that the gut is the main site of klebsiella infection in as .
in worldwide studies involving 1330 as patients and 1191 healthy controls , the as patients showed
there is evidence that the gut is the main site of klebsiella infection in as .
schizophrenia and virus infectionacute schizophrenia has been observed to follow upper respiratory tract virus infections , and g. knight et al
. have assembled much evidence indicating that schizophrenia is an autoimmune disease caused by autoantibodies that react with neuronal receptors influencing the limbic system .
seeking antigenic triggers and their corresponding autoantigen , with excellent technology at the nih , laing et al .
immunized rabbits with neurotropic strains of influenza virus , inducing autoantibodies to a brain - specific 37 kda protein .
this needs further exploration , as does the whole field of psychotic aetiology , until the presumptive forbidden clones have been demonstrated with the clarity obtained for graves ' disease and myasthenia gravis .
acute schizophrenia has been observed to follow upper respiratory tract virus infections , and g. knight et al .
have assembled much evidence indicating that schizophrenia is an autoimmune disease caused by autoantibodies that react with neuronal receptors influencing the limbic system .
seeking antigenic triggers and their corresponding autoantigen , with excellent technology at the nih , laing et al .
immunized rabbits with neurotropic strains of influenza virus , inducing autoantibodies to a brain - specific 37 kda protein .
this needs further exploration , as does the whole field of psychotic aetiology , until the presumptive forbidden clones have been demonstrated with the clarity obtained for graves ' disease and myasthenia gravis .
details of the development of the methods used for successful determination of the amino acid sequences of antigens on the autoimmune disease - triggering bacteria , proteus mirabilis and klebsiella pneumoniae , are recorded in the book rheumatoid arthritis and proteus by ebringer .
this research provides experimental confirmation , at the molecular level , of the h gene theory of the inheritance of the autoimmune diseases , described above , in confirming the speculated presence of multiple antigens on triggering bacteria and alternative clonal development causing the autoimmune disease .
figure 1 shows space - filling models of the amino acid sequences of the histocompatibility antigen , hla - dr1/4 , and the proteus mirabilis haemolysin antigen .
this means the immune tolerance imposed by the histocompatibility antigen will extend to this proteus antigen , preventing immune reaction with it .
figure 2 shows space - filling models of the amino acid sequences of the proteus mirabilis urease antigen and type 11 collagen , an autoantigen attacked in rheumatoid arthritis .
the urease antigen is completely different from hla - dr1/4 , so it will not be protected from immune reaction , being free to stimulate development of a forbidden clone reacting with the closely similar type 11 collagen molecule , an autoantigen attacked in rheumatoid arthritis .
figure 3 shows space - filling models of the amino acid sequences of the histocompatibility antigen , hla - b27 , and two antigenic peptides on the bacterium klebsiella pneumoniae .
the klebsiella nitrogenase antigen closely resembles hla - b27 , so it will be covered by the tolerance induced by hla - b27 , but the bacterium pullanase peptide is different and able to stimulate development of a forbidden clone , attacking the spine causing ankylosing spondylitisn .
it is now apparent that , in the present state of knowledge , there are 4 laws of autoimmunity , as detailed in table 3 . recognition of the universality of microbial triggers is a major advance as it shows that all autoimmune diseases are potentially preventable by vaccinations against their microbial triggers .
a new zealand example occurred in 1938 and was reported in the press and observed by adams , trapped in a boarding school at masterton .
an epidemic of leg paralyses occurred in christchurch and spread progressively north , from town to town , to picton , wellington , featherstone , then carterton , the town next to masterton , engendering great fear .
then , the boy in the bed next to adams complained of a stiff neck , was taken away , and reported to have polio .
these events make it probable that the leg paralyses of poliomyelitis were a rare autoimmune complication of virtually universal virus infection , the paralyses probably caused by forbidden clones of cytotoxic t cells which attacked anterior horn neurons , hence the name , acute anterior poliomyelitis . the salk ( killed ) and sabin ( attenuated ) polio vaccines have both been brilliantly successful in preventing the polio leg paralyses .
this exemplifies how autoimmune diseases in general can be prevented by finding and vaccinating against their microbial triggers .
ebringer has succeeded in this with rheumatoid arthritis and ankylosing spondalitis , he has pioneered this new field of medical research , developing a whole new technology which needs to be copied in other diseases , especially schizophrenia .
systematic studies of other autoimmune diseases , with collaboration between clinicians and microbiologists , are needed .
the american association of microbiologists would be an ideal organization for providing this urgently needed knowledge .
some disease associations are cross - tissue autoimmunity , for example the proptosis of graves ' disease , caused by variants of the thyroid - stimulating autoantibodies that react with receptors on orbital fat cells and diabetic retinopathy , probably caused by destruction of retinal pericytes by antigenic variants of the t - cell forbidden clones that destroy the pancreatic islet cells to cause type 1 diabetes .
immunotherapy , by radiological or chemical immune ablation with immune reconstitution by autologous bone marrow cells , pioneered by farge et al .
, can be used to save the lives of patients with dangerous autoimmune diseases , such as systemic scleroderma .
selective destruction of forbidden clones could be achieved by isolating their autoantigen ( such as the tsh receptor of graves ' disease ) and attaching it to a cytotoxic moiety , such as bungarotoxin or iodine ( emitting short - range beta particles ) , then administering the molecular complex intravenously to destroy the pathogenic clones of plasma cells .
prevention is better than cure , so finding and countering antigenic triggers of autoimmune diseases is the ideal .
it shows how the diseases can be prevented by finding and vaccinating against the triggers .
ebringer has led the way by discovering two major triggers and developing the technology for finding others . | schizophrenia is of mysterious causation . it is not infectious , not congenital , but shows familial aggregation , the mendelian genetics indicating involvement of multiple codominant genes with incomplete penetrance
.
this is the pattern for autoimmune diseases , such as graves ' disease of the thyroid , where forbidden clones of b lymphocytes develop , and cause thyrotoxicosis by secreting autoantibodies that react with the thyroid gland 's receptor for thyroid - stimulating hormone from the pituitary gland . in 1982 , knight postulated that autoantibodies affecting the function of neurons in the limbic region of the brain are a possible cause of schizophrenia .
today , this is even more probable , with genes predisposing to schizophrenia having being found to be immune response genes , one in the mhc and two for antibody light chain v genes .
immune response genes govern the immune repertoire , dictating the genetic risk of autoimmune diseases .
the simplest test for an autoimmune basis of schizophrenia would be trial of immunosuppression with prednisone in acute cases .
the urgent research need is to find the microbial trigger , as done by ebringer for rheumatoid arthritis and for ankylosing spondylitis .
this could lead to prophylaxis of schizophrenia by vaccination against the triggering microbe . |
stroke is one of the leading causes of long - term disability in the world , and in the united states , an average of one stroke occurs every 40 seconds . by this estimate , 795,000 new or recurrent strokes occur each year , with annual direct and indirect cost of cardiovascular disease and stroke in the united states being estimated at $ 312.6 billion .
in addition to the physical disabilities that often follow stroke , the risk factors that make individuals prone to stroke and cerebrovascular disease also place them at risk for potentially devastating cognitive impairments [ 2 , 3 ] . among stroke survivors ,
about 64% exhibit cognitive impairment and up to a third develop dementia [ 57 ] .
wagle and colleagues found that estimates of cognitive functioning taken 2 to 3 weeks poststroke strongly predicted patients ' practical functioning , measured by modified rankin scale scores , after 13 months of poststroke recovery .
in addition , cognitive impairment measured 3 months after first - ever stroke has been associated with increased risks of death and disability 4 years later .
the cognitive impairments associated with stroke are heterogeneous , depending on the location and severity of individual ischemic lesions , preexisting microvascular lesions , and the patient 's premorbid cognitive capacity , among other considerations .
accordingly , there is significant within - group variability in even the best - established cognitive syndromes associated with stroke occurring in discrete vascular territories .
in addition , a wide range of psychiatric symptoms can arise after stroke , related to both the ischemic lesions themselves and the significant psychosocial adjustments patients and their families often have to make after stroke .
these psychiatric comorbidities have a powerful influence over both quality of life and functional outcomes . in a sample of 51,119 ischemic stroke patients , those patients diagnosed with depression or other mental health concerns had an increased risk of death 3 years poststroke , even after the influences of other chronic conditions were statistically controlled .
these points highlight the importance of proactively monitoring stroke patients ' cognitive , psychiatric , and functional outcomes during outpatient follow - up . indeed ,
a holistic , comprehensive , interactive approach of an interdisciplinary team has been regarded as the hallmark of stroke rehabilitation .
in such a model , domain - specific neuropsychological assessments are integral to supporting stroke patients ' quality of life and increasing subsequent patient satisfaction with stroke - related healthcare services . acknowledging this ,
recent harmonization standards called for routine , standardized cognitive and mood assessment in both clinical practice and research with stroke patients . however , there is scant literature addressing the feasibility of multidisciplinary stroke care models incorporating cognitive , neurobehavioral , and neuropsychiatric outcomes . in the available literature ,
it is clear that ( a ) neuropsychological assessments remain underutilized in pertinent settings and ( b ) there is little standardization in the methodology of such assessments . in a recent review of stroke research conducted from january 2000 to october 2011 ,
only 488 of the 8826 relevant studies ( 6% ) included cognitive or mood assessment scales .
within that 6% , assessment methods were highly variable , as researchers used 367 different assessment measures among these studies .
moreover , clinical services that hope to integrate neuropsychological assessment into poststroke care may find its practical implementation challenging .
given the time - sensitive nature of stroke , timely data transfer between providers is crucial not only in achieving the highest patient care standards , but also in promoting patient and provider satisfaction .
however , due to operational incompatibilities between traditional neuropsychology clinics and stroke neurology services in most settings , routine neuropsychological assessments are rarely available in the acute poststroke period , and outpatient follow - up appointments are often substantially delayed .
these factors often limit provision of neuropsychological assessment in poststroke care , and timely data transfer often remains an ideal rather than a practical , achievable standard . at our tertiary care center , inpatient neuropsychological assessment services were not regularly available for stroke patients , and the outpatient referral system for neuropsychological testing was cumbersome .
patients ' quantitative cognitive performance data from neuropsychological testing were seldom available at the time of their stroke clinic posthospitalization follow - up neurology appointments .
these systemic barriers brought to our attention the need to implement a streamlined , interdisciplinary model of care that directly incorporated neuropsychology into standard poststroke care .
this project aimed to enhance patient services by implementing a multidisciplinary model of poststroke care that more closely integrated neuropsychology and neurology services .
we anticipated that such a service would enhance acute poststroke care by ( a ) making inpatient neuropsychological services part of the standard of care .
this would allow for tailored discharge recommendations based on efficient , systematic examination of patients ' poststroke cognitive limitations .
we also predicated that the benefit of the model would extend to outpatient follow - up practice by ( b ) providing quantitative measurements of poststroke cognitive recovery , ( c ) improving patient satisfaction with their poststroke care by mitigating logistical burdens , and ( d ) improving provider satisfaction with the neurology - neuropsychology collaboration by promoting direct communication and rapid data transfer between providers .
we aimed to successfully develop and implement this care model within a 12-month timeframe , thus ( e ) providing preliminary data regarding the tolerability , feasibility , and utility of the neuropsychological assessment protocol for vascular cognitive impairment proposed by the international harmonization standards for poststroke care endorsed by the national institute of neurological disorders and stroke and the canadian stroke network ( ninds - csn vci protocol ) .
the multidisciplinary stroke clinic was developed and implemented via collaboration between the neuropsychology and stroke neurology services within the department of neurology at the university of wisconsin hospital and clinics ( uwhc ) .
financial support for the multidisciplinary stroke service was provided both through the development of a postdoctoral fellowship in stroke neuropsychology , funded by uwhc , and provision of the ambulatory care innovation grant , an in - house quality improvement program funded by physicians plus insurance company and the university of wisconsin medical foundation .
total cost for the project , including support of the stroke neuropsychology postdoctoral fellow and purchase of required materials , was under $ 100,000 .
after initial implementation , the neuropsychology section of the stroke multidisciplinary team became self - sustaining with service - based revenues .
to incorporate neuropsychology into inpatient poststroke care , the stroke neuropsychology postdoctoral fellow was embedded within the inpatient stroke service 's morning rounds on two mornings each week .
patients were identified as appropriate for neuropsychological testing on the basis of the fellow 's clinical observations , in cooperation with the attending or resident neurologist , occupational therapist , speech - language pathologist , and nurses , who made rounds with the neurology team .
patients so identified were then administered the half - hour ninds - csn vci neuropsychological assessment protocol .
results from this brief inpatient battery ; accompanied by salient recommendations for patient supervision , return to school , work , and driving , and medicolegal decision - making capacity , among other considerations ; were provided to the stroke team verbally and then in report form via the hospital 's electronic medical record system .
these recommendations were incorporated into the patient 's discharge plan at the discretion of the attending stroke neurologist . as part of the transition of care from discharge to outpatient follow - up , stroke patients were engaged in communication and treatment planning in two ways .
first , patients were engaged in a three - way conference call between the inpatient unit clerk and the stroke program coordinator in order to schedule the multidisciplinary poststroke follow - up appointment , and this appointment was included in the inpatient stroke service discharge instructions .
further , informational resources , including letters , brochures , and instructions , were mailed to patients in order to again provide a brief description of the follow - up appointment , the nature of stroke , the purpose and nature of neuropsychological assessment , and resources for additional care . upon discharge from the inpatient stroke service ,
an outpatient follow - up appointment was scheduled for each patient for 8 to 12 weeks from discharge during neurology clinic time designated for multidisciplinary stroke services .
each of these follow - up appointments was scheduled as a 2-hour block , with neuropsychological assessment scheduled 1 hour prior to the patient 's follow - up appointment with the stroke neurologist . during the neuropsychology portion of the visit ,
a brief interval history was taken via clinical interview with the patient and any available family members or caregivers , and the ninds - csn vci protocol was repeated in order to provide quantitative measurements of interval cognitive change .
scoring and preliminary interpretation of the test data were performed on - site in the clinic .
results were then provided immediately to the attending neurologist prior to the neurology appointment time , using a side - by - side graphical representation of time 1 and time 2 test results .
the neurologist then incorporated the results of the neuropsychological testing in their counseling and treatment plan for the patient , often focusing on issues related to capacity for independent living and return to usual activities , such as work and driving .
after a pilot year , modified press - ganey scales , industry - standard 18-item surveys , were used to assess patient satisfaction with the multidisciplinary stroke clinic model .
specifically , the press - ganey scales assessed patient satisfaction with the clinic 's appointment logistics , including the scheduling process , access to providers , and communication with care providers . in addition , a 10-item survey assessing stroke neurologists ' satisfaction with the new multidisciplinary clinic was administered 12 months after implementation .
this survey evaluated providers ' satisfaction with clinic scheduling procedures both before and after implementation of the multidisciplinary clinic , data turnaround time both before and after clinic implementation , perceived value of the service to patients , perceived patient satisfaction with their care , and estimation of the clinic 's added value to the comprehensive stroke program as a whole .
institutional review board was consulted , and deidentified data use was authorized given that the nature of the data assessed clinical quality improvement .
the first goal of the multidisciplinary stroke clinic project was to integrate neuropsychology into the standard of care for stroke patients in our care center . during the 12-month pilot implementation of the multidisciplinary stroke clinic ,
second , we hoped to extend the benefit of neuropsychology 's integration to outpatient follow - up care by improving patient satisfaction with poststroke care , in part , by mitigating logistical burdens and improving provider satisfaction with the neurology - neuropsychology collaboration by promoting direct communication and rapid data transfer between providers .
of the 114 stroke patients seen for inpatient cognitive evaluations , 67 patients returned for outpatient follow - up in the multidisciplinary stroke clinic . remaining 47 patients initially seen were not followed up for multiple clinical reasons , for example , death , out of state residence , being cognitively normal deemed at initial evaluation prior to discharge , or being profoundly impaired with no clinical need for further evaluation .
provision of this service required 67 appointment slots in the multidisciplinary stroke clinic , rather than requiring 134 ( 67 2 ) separate time slots divided between the outpatient neuropsychology and stroke neurology services .
this scheduling benefit required patients to travel to the hospital once for their follow - up appointments rather than making separate trips for neuropsychology and stroke neurology appointments .
this represents significant mitigation of the logistical burden on patients , reducing travel costs and increasing feasibility for significantly disabled patients and their caregivers .
prior to the implementation of the multidisciplinary stroke clinic , patients ' mean wait time for a poststroke neuropsychological assessment was 5 to 10 months after their discharge from inpatient care .
this delay was reduced from 1 to 3 months after implementation of the multidisciplinary stroke clinic , decreasing mean wait time by 7.26 months , a 78.32% decrease in patient waiting time .
the 1 to 3 months wait time was also deliberately implemented to gauge poststroke cognitive recovery correlating with the first 90 days of anticipated poststroke improvement .
survey results indicated exceptionally high patient satisfaction with the multidisciplinary stroke clinic , as measured via the press - ganey scales .
composite responses to the press - ganey scales indicated exceptional patient satisfaction with all domains of service within the multidisciplinary stroke clinic , including logistics and scheduling , patient / provider interactions , patients ' perceptions of care quality , and patient confidence in providers .
as measured on a self - report likert scale ranging from 0 to 5 , with 0 representing the worst possible rating and 5 representing the best possible rating , patients rated their multidisciplinary stroke clinic care experience in all press - ganey assessed domains as at least a 4 out of 5 ( range = 4.36 to 5.00 ) . to review patient satisfaction ratings for all domains , see table 1 .
data are limited to those only collected during the project time frame and do not represent the total number of patients seen in clinic .
accordingly , at the time of data analysis , sample size was small ( n = 16 ) .
responses to a 10-item survey of provider satisfaction indicated high provider satisfaction across domains assessed , including providers ' perception of improved efficacy of clinic operations and interdepartmental data transfer time after implementation of the multidisciplinary stroke clinic model .
provider survey results also indicated high provider satisfaction with neuropsychology 's added value to the comprehensive stroke program .
again , providers rated their satisfaction with the multidisciplinary stroke clinic care model in all assessed domains as at least a 4 out of 5 ( range = 4.25 to 4.75 ) . to review provider satisfaction ratings , see table 2 .
stroke neurologists noted practical benefit from the incorporation of neuropsychology into post - troke services , based on enhanced triage capacities , efficient communication between providers , and rapid data transfer regarding patient outcomes .
as neuropsychology was directly embedded into the comprehensive stroke service , and the stroke neuropsychology fellow was available to provide direct input into patient referrals , making it more likely to choose those patients who could best tolerate the ninds - csn vci battery and whose neuropsychological data would likely augment clinical decision making .
effective communication was greatly facilitated , as providers had in - person contact during rounds two mornings a week and in multidisciplinary clinic one afternoon weekly .
in addition , for the patients served during the 12-month pilot program , both inpatient and outpatient neuropsychological evaluation data were available during patients ' acute stroke admissions and outpatient follow - up appointments .
this allowed providers to make recommendations regarding return to school , work , driving , and other usual activities at least in part based on quantitative measurements of patients ' cognitive capacities rather than relying on clinical judgment alone .
the stroke neuropsychology fellow and attending neuropsychologists commented favorably on the general clinical utility of the ninds - csn vci half - hour protocol .
first , the protocol could be completed in approximately one hour by most patients , including a focused clinical interview .
in addition , the protocol was easily customized , if necessary , in order to individualize assessments as needed without unduly taxing stroke patients ' limited tolerance for testing .
examiners did note that patients with significant expressive and receptive aphasias and hemiplegia / hemiparesis involving their dominant hands were often quite limited in their capacity to complete the battery , which heavily relies on verbal and written responses .
finally , the protocol provided a sufficient breadth of useful data across cognitive domains , including cognitive efficiency , speech production , basic visuospatial / constructional skills , verbal learning and memory , and executive functioning .
although the depth of this data did not fully address all clinical issues for every patient , it significantly enhanced clinical understanding , thereby allowing neuropsychology providers to recommend other necessary services , including comprehensive neuropsychological assessments , behind - the - wheel driving evaluations , in - home safety evaluations , skilled nursing placements , and guardianship proceedings .
poststroke cognitive impairment has been demonstrated to be a key predictor of patients ' functional outcomes and risks of disability and mortality after stroke .
furthermore , systematic assessment of patients ' cognitive capacities is necessary for using cognitive profiles in patient care due to the significant heterogeneity in poststroke cognitive outcomes [ 10 , 11 ] .
however , operational barriers often preclude the incorporation of neuropsychological services into routine poststroke care in many clinical settings . in this large tertiary care center ,
the comprehensive stroke program and neuropsychology service collaboratively implemented a multidisciplinary stroke clinic to address the systemic obstacles that hindered effective , collaborative communication between stroke neurology and neuropsychology providers in their poststroke care efforts . as a result ,
multiple follow - up visits were condensed into one , reducing travel - related and other associated burdens on patients and reducing stroke patients ' wait time between neurology and neuropsychology appointments by over 75% . instead of requiring weeks or months for data transfer between providers , stroke neurology and neuropsychology providers were able to communicate directly regarding patient 's cognitive outcomes in a single clinic appointment .
after the first implementation year , the multidisciplinary clinic was associated with high satisfaction ratings from both providers and patients . the half - hour neuropsychological assessment protocol for vascular cognitive impairment , recommended by the 2006 ninds - csn international harmonization standards for poststroke care , demonstrated good clinical feasibility and provided an efficient method for the provision of focused neuropsychological services in a clinical setting that often prohibits full , traditional cognitive assessments . in future studies
, we hope to use the resulting data to provide benchmark evidence - based outcomes regarding the actual validity and reliability of the protocol itself and multidisciplinary stroke care overall .
the current study , however , does not aim to address validity and reliability beyond the purpose of the ninds - csn 's initial proposed recommendations . given suggested clinical feasibility , additional studies are encouraged to explore validity and reliability of the protocols .
the multidisciplinary stroke clinic is a low - cost , easily implemented model of care that quickly becomes financially self - sustaining and is generalizable to both poststroke care teams in other hospitals and other healthcare groups that require multiple disciplines to provide patient care in a streamlined , efficient manner . such significant increases in provider and patient satisfaction , as well as marked improvements in patient
wait time and increases in available patient base , may be anticipated in other services that could utilize neuropsychological services on a regular basis , as well as associated disciplines such as physical therapy , occupational therapy , social work , and health psychology . | as a significant number of stroke victims exhibit cognitive impairment , neuropsychological assessments can enhance poststroke management by identifying pertinent cognitive sequelae and providing salient care recommendations .
however , due to operational differences between traditional neuropsychology and stroke services , neuropsychological assessments often remain underutilized in stroke care .
we developed a novel care model that incorporated neuropsychological testing into a comprehensive stroke program using the modified vascular cognitive impairment ( vci ) half - hour assessment protocol proposed by the national institute of neurological disorders and stroke
canadian stroke network ( ninds - csn ) .
the test batteries were administered during the patients ' acute admissions and then again upon follow - up in the multidisciplinary stroke clinic . patient and provider satisfaction was then evaluated .
surveys revealed high provider satisfaction with improved clinic efficacy , improved data turnaround time , and with value neuropsychology services added to the comprehensive stroke program .
results from the 18-item industry standard press - ganey surveys showed all scores above 4.4/5.0 for patient satisfaction .
this clinic garnered high provider and patient satisfaction after the first year .
the ( modified ) ninds - csn vci assessment protocol demonstrated clinical feasibility , suggestive of an efficient method of providing focused neuropsychological services in a clinical setting that otherwise prohibits traditional , comprehensive cognitive assessments . |
in response to jev activity , as evidenced by human clinical cases or the seroconversion of sentinel pigs , adult mosquitoes were collected on badu island during 1995 , 1998 , and 2003 ( 6,8 , and this article , respectively ) .
badu island is located at 1007s and 14209e in the central western region of the torres strait and is a granite island of 101 km ; its ecology has been described ( 68 ) .
the mosquitoes were collected with centers for disease control ( cdc ) miniature light traps ( model 512 ; john w. hock co. , gainesville , fl , usa ) baited with either co2 alone or in combination with 1-octen-3-ol .
mosquitoes were killed on dry ice and placed in liquid nitrogen dry shippers or on dry ice in insulated containers for transport to cairns for storage at 70c .
mosquitoes were placed on a refrigerated table for species or taxonomic group identification before being placed in pools of < 200 mosquitoes and sent to queensland health forensic and scientific services or the university of queensland , brisbane , for jev detection .
the virus isolation protocols used in 1995 and 1998 have been described ( 6,8 ) . in 2003 ,
virus rna was detected by using a real - time taqman reverse transcription pcr ( 9 ) . to facilitate the comparison of virus distribution on badu island
, trap locations were grouped into 3 general areas : within 1.2 km of the piggery , within the area of human habitation ( the community ) , and at a rubbish dump located 1.5 km from the community and 4.0 km from the piggery ( figure 2 ) .
dumps are a potential focus of jev activity because mosquitoes , feral pigs , and wading birds congregate at them ( 10 ) .
maximum - likelihood estimation of mosquito infection rates with 95% confidence intervals were calculated for each of these general areas by using the pooledinfrate statistical software package ( 11 ) .
aerial photograph of badu island showing the location of the community , piggery , and rubbish dump . because of the difficulty in morphologically separating the members of the cx .
sitiens subgroup , data for the 3 members of the group found in australia cx .
sitiens were pooled for analysis . however , pcr restriction fragment length polymorphism analysis of a subsample of 135 cx .
annulirostris was the dominant member of this group on badu island and comprised 94.1% of polymorphic specimens processed ; the other 4.4% and 1.5% were identified as cx .
sitiens subgroup mosquitoes were processed for detection of jev ; 2,871 , 24,592 , and 16,865 were processed from 1995 , 1998 , and 2003 , respectively ( table ) .
sitiens subgroup mosquitoes ; the highest maximum - likelihood estimation of mosquito infection rate was obtained from mosquitoes collected at the dump in 1998 . despite removal of the domestic pigs ,
sitiens subgroup mosquitoes collected within the community in 2003 . however , the point estimates of infection rates were lower than those obtained in 1995 and 1998 , when domestic pigs were present within the community , although the slight overlap in 95% confidence intervals indicates that this difference in infection rate was not significant .
* mosquito infection rates determined by maximum - likelihood estimation ; 1995 , apr 89 and 2021 , 30 trap nights ; 1998 , mar 56 , 25 trap nights ; 2003 , mar 1319 , 92 trap nights ; ci , confidence interval ; ns , mosquitoes not sampled from this location during the year of collection .
japanese encephalitis virus positive pools detected by virus isolation or taqman reverse transcription pcr .
we demonstrated that although removing domestic pigs from areas of human habitation may reduce contact between amplifying hosts and vectors ( 7 ) , it does not eliminate the presence of jev - infected mosquitoes .
indeed , evidence for low - level virus transmission to humans is provided by hanna et al . , who found a low level ( 32% ) of natural boosting immunity in badu residents who had received an inactivated mouse brain
as has been observed elsewhere in the absence of pigs ( 13 ) , mosquitoes may have become infected by feeding on viremic herons and egrets , populations of which are found on badu island ( 14 ) .
analysis of host feeding patterns demonstrated that birds accounted for 23% of blood meals of cx .
data ) . alternately , feral pigs in the community and at the dump are a potential source of virus for mosquitoes , although < 1% of mosquito blood meals were from swine at these locations .
mosquitoes could become infected by feeding on viremic pigs at the piggery and then disperse to other areas on the island .
annulirostris is 4.4 km ; some females traverse up to 12 km ( 15 ) , which is considerably farther than the 2.5 km between the piggery and the community on badu island .
solomon recommends that domestic pigs be moved > 5 km from human habitation to limit jev transmission to humans ( 3 ) .
indeed , if infected mosquitoes were flying from the piggery to the community , then the data from our study support this recommendation .
however , we suggest that domestic pigs be removed far enough away from human habitation to encompass the flight range of the local culex vectors . | to determine whether relocating domestic pigs , the amplifying host of japanese encephalitis virus ( jev ) , decreased the risk for jev transmission to humans in northern australia , we collected mosquitoes for virus detection .
detection of jev in mosquitoes after pig relocation indicates that pig relocation did not eliminate jev risk . |
extensive soft tissue defects accompanied by infectious spondylitis are rare clinically . in these cases ,
medical / surgical therapy that involves debridement of the dead tissue and treatment with antibiotics can take a long time to carry out and a considerable length of time may elapse before the patient recovers , with this lengthy period of recovery adversely influencing the clinical outcome of patients .
in addition , management of massive soft tissue defects and chronic wounds are major challenges .
various reports have proposed techniques of managing complex back wounds1,8,10 ) . published methods for treating the complex back wound of paraplegia patients
it is even hard to find a case of a complex back wound of a paraplegic patient that is associated with a spinal infection in the literature . in this case , to treat the complex back wound that involved an extensive soft tissue defect associated with spinal infection , we tried a muscle flap technique that is mainly used in the plastic surgery field and not in neurosurgery in this hospital .
the reverse latissimus dorsi muscle flap ( rldmf ) was our final choice for this patient .
this muscle flap technique allowed us to fill up the extensive soft tissue defect , thereby reducing the dead space , and maintain the paraspinal vasculature .
the objective of this report was to evaluate the efficacy of filling up the defect using rldmf in the treatment of severe infectious spondylitis .
we observed that 6 months after the operation , the patient showed no sign of recurrence of infection and the back wounds had completely healed without any complication or sequela .
a 68-year - old woman with a previous history of paraplegia after traumatic spinal epidural hemorrhage from a ruptured l3 arteriovenous fistula ( avf ) was admitted to our institute with high fever above 38 and severe back pain .
about 10 days earlier , she suffered a superficial burn to her back and was admitted into the local hospital because of acute renal failure due to rhabdomyolysis . on neurologic examination ,
examination of the motor system revealed intact muscle strength in the upper extremity and flaccid paraplegia with areflexia .
an examination of the sensory system revealed that all sensory modalities had decreased below the t10 dermatome level .
an abdominal computed tomography scan demonstrated a suspicious pyogenic spondylitis of multiple lumbar spines with multiple perivertebral abscess formation and fluid collections around the pyriform muscles and parasacral area .
a drainage catheter was inserted by a radiologist and the patient was admitted into the infectious diseases ward for antibiotic therapy . empirical antibiotic therapy with ampicillin and sulbactam
drug fever developed after nafcillin was given , so the antibiotic was changed to cefazolin .
this antibiotic was maintained until the culture of the draining catheter was identified . on culture of the draining catheter ,
after 14 days on that regimen , the antibiotic was changed to teicoplanin because the patient developed a skin rash . despite conservative therapy using the appropriate antibiotics for 3 months and surgical drainage
we thought that the fluid collection in the dead space between the intra-/inter - muscular area and the scar of the previously embolized avf was the main source of infection recurrence ( fig .
we believed that by filling the dead space , we would be able to control the spondylitis .
first , we performed multiple posterior screw fixations from l1 to the sacrum with wide debridement of infected soft tissue and obliteration of dead space using rldmf ( fig .
2 ) . second , we performed anterior column reconstruction with an expandable mesh cage via an anterolateral reptroperitoneal approach to correct deformity and bone fusion .
after the staged operation and antibiotic therapy , the multiple infectious spondylitis completely resolved without recurrence ( fig .
conservative management of pyogenic spondylitis is the traditionally used approach5 ) . in certain pyogenic spondylitis cases ,
surgical treatment is strongly indicated , but invasive surgeries often cause various problems . when the patient 's general condition is poor , less invasive treatment is required .
the standard management of infectious spondylodiscitis with spinal epidural abscess has been surgical debridement with or without operative stabilization , followed by prolonged antibiotic treatment7 ) .
nevertheless traditional surgical treatment for pyogenic spondylitis has the disadvantage of increased morbidity caused by the extensive exposure of the wound in the presence of infection .
percutaneous suction aspiration with drainage is another effective alternative for not only mild to moderate spondylodiscitis but also for early - stage pyogenic spondylodiscitis .
nagata et al.12 ) , indicated that the main advantages of the technique were its reduced invasiveness compared with open surgery , and the almost immediate symptomatic relief it provided .
indications for this technique are considered to include the following : 1 ) no improvement in the condition of the patient despite use of antibiotic agents and immobilization for at least 1 month ; 2 ) the presence of only a single affected intervertebral disc ; 3 ) the location of the involved vertebra in the lower thoracic or lumbar spine ; 4 ) the absence of any major neurological deficit ; and 5 ) the absence of radiographically documented marked destructive changes in the affected vertebral body . but this treatment is not indicated in severe pyogenic spondylodiscitis in which abscess formation or marked bone destruction or extensive soft tissue defect is associated with a potential risk of neurological complications , such as existed in our case .
in addition , the procedure failed to improve symptoms in one patient in whom marked destructive changes were apparent on radiography , and open surgery involving an implantation was necessary in that case . in the presence of instrumentation ,
providing preemptive soft tissue reconstruction at the time of the initial spinal surgery can help minimize potentially serious wound complications .
for management of wound complications that have developed , aggressive debridement and coverage with well - vascularized tissue can allow for expedient wound healing while maintaining stabilized instrumentation .
in extensive soft tissue defects accompanied by infectious spondylitis , such as in our case , the conventional approach using serial wound debridement and antibiotic administration could take a considerable period to complete , and is usually only successful in cases of mild infections . in more severe cases of infection with an extensive soft tissue defect , marked skin and muscle reactive fibrosis adjacent to the wound makes using paraspinous muscle flaps to close the defect difficult , if dissection is possible in the first place .
moreover , even if they can be dissected , they often are of insufficient volume to fill these wide and deep wounds . in this situation ,
pedicled latissimus dorsi muscle flaps have proven to be the most reliable method of treatment .
generally , the muscles of the back can be classified into 3 groups : superficial , intermediate , and deep groups .
the superficial muscles such as the latissimus dorsi muscle are often valuable sources for a secondary flap in paraspinous muscle reconstruction as soft tissue defects are usually deep and extensive or require a skin island4 ) .
the pedicled latissimus dorsi flap proved to be a valuable and reliable tool to treat large and deep back wound infections with extensive soft tissue defects .
it can be used as a musculocutaneous advancement or rotation flap when laterally based ( thoracodorsal vessels ) , or as a muscular turnover flap , when medially based ( paraspinous perforators ) .
this versatility enabled the latissimus dorsi flap arc of rotation to reach midline spine defects from high thoracic through lower lumbar levels . and as the procedure is simple and the blood circulation stable during this operation , it is thought to be an effective reconstruction method for extensive soft tissue defects accompanied by infectious spondylitis .
it is true that literature has been published on methods of operative defect closure in the case of complex back wounds .
however , since the data available is limited and the numbers of cases are relatively small in these studies , any comparison between the use of the pedicled latissimus dorsi flap and other methods such as the use of free flaps2,6 ) , perforator flaps9 ) , or tissue expansion13 ) , combined methods3 ) , paraspinous muscle flaps10 ) and superior gluteal muscle flaps15 ) are precluded .
use of the rldmf , which was attempted in our patient , is a reliable method of treatment of infectious spondylitis with an extensive soft tissue defect11 ) .
we experienced good surgical and clinical outcome using rldmf in treating intractable spinal infection . hence , we recommend rldmf in the case of infectious spondylitis with an extensive soft tissue defect or dead space and uncontrolled recurrent spinal infection . | spinal infection is an inflammatory process around the vertebral body , and it can extend to the epidural space , posterior elements and paravertebral soft tissues . infectious spondylitis is a rare infectious disorder , which is often associated with significant neurologic deficits and mortality .
when an extensive soft tissue defect is accompanied by infectious spondylitis , effective infection control and proper coverage of soft tissue are directly connected to successful outcomes .
however , it is not simple to choose the appropriate treatment methods for infectious spondylitis accompanied by a soft tissue defect
. herein , we report a case of severe infectious spondylitis that was accompanied by an extensive soft tissue defect which was closed with a reverse latissimus muscle flap after traumatic spinal epidural hemorrhage . |
breast cancer is the most common malignancy in women , and metastasis is the number one cause of mortality in breast cancer .
invasion depends in part on degradation of extracellular matrix ( ecm ) and interaction with molecules formed in the process .
recently , increasing attention has been paid to chemokines that may modify breast cancer cells and the surrounding matrix to facilitate metastasis .
furthermore , there is evidence that chemokines , vegf - c , and heparanase are interrelated in the process of invasion [ 46 ] .
increased expression of the lymphangiogenesis factors and heparanase has been correlated with progressive disease in certain cancers .
peritumoral lymphangiogenesis involves the secretion of specific glycoproteins designated vascular endothelial growth factor c ( vegf - c ) and ( vegf - d ) that act on lymphatic endothelium , and are components of an established signaling system for tumor lymphangiogenesis .
increased lymph node metastases are correlated with increased expression of vegf - d and vegfr3 by immunohistochemistry in invasive breast cancer .
heparanase is an endo--glucuronidase that cleaves heparan sulfate ( hs ) side chains of heparin sulfate proteoglycan ( hspg ) .
heparanase activity has been correlated with cell invasion associated with breast cancer metastasis , a consequence of structural modification of hs that alters the extracellular matrix [ 9 , 10 ] . however
, classical mammalian heparanase is an intracellular enzyme and is not specific to metastatic sites [ 1113 ] .
cxcl7 is a member of the cxc subfamily of chemokines which can be further subdivided on the basis of the presence of the tripeptide motif glutamate - leucine - arginine ( elr ) .
all elr - cxc chemokines act through cxc chemokine receptor type1 or type2 ( cxcr1 and cxcr2 ) .
cxcl7 , has dual functions of heparin binding and is a ligand to the g - protein linked receptor cxcr2 .
until recently , cxcl7 gene expression was thought to be restricted to cells within the megakaryocytic lineage [ 16 , 17 ] , as well as to neutrophils and lymphocytes .
recent reports have suggested that other cell types may produce this chemokine as well [ 1823 ] . despite studies on increased expression of cxcr2 in breast cancer ,
earlier studies from our laboratory have shown that the malignant breast cancer cells express more cxcl7 than premalignant mcf10at cells .
cxcl7-transfected mcf10at breast cells have much higher heparanase activity than premalignant mcf10at cells . cxcl7-transfected mcf10at cells are as invasive as malignant breast cancer cells .
in addition , the expression of vegf c and d is increased in these transfectants .
heparanase and the lymphangiogenesis factors vegf - c and vegf - d are two important markers closely related to the metastasic capabilty of breast cancer . in this paper , we hypothesize that cxcl7 increases the expression of vegf - c , vegf - d , and heparanase , and increases cell invasion via cxcr2 signaling , all linked to tumor lymphangiogenesis and metastasis .
the mcf10at cells were cultured in dulbecco 's modified eagle 's medium ( dmem)/f 12 ( 1 : 1 ) containing 5% horse serum , supplemented with 10 g / ml bovine insulin , egf ( 20 ng / ml ) , hydrocortisone ( 0.5 g / ml ) , and cholera toxin ( 100 ng / ml ) .
all cell culture reagents were obtained from gibco ( grand island , ny ) .
approximately , 1 10 cells / well were plated in 6-well plates in medium containing 5% horse serum to grow overnight to 60%70% confluency .
transfection of the plasmid was performed by using lipofectamine 2000 ( invitrogen , usa ) .
the cells were divided into blank control group , negative control group , and the test group .
the cells were transfected with the mixture of plasmid and lipofectamine 2000 ( 1 : 3 ) in 2 ml serum - free medium . at 24 hours after transfection , the medium was replaced by normal medium containing 5% fbs and antibiotics up to 72 hours post - transfection . since the mcf10at cells were transfected cells , g418 could not be used for the selection of the stably transfected cell line .
therefore , we submitted the cells for the flow cytometry sorting by using the egfp antibody .
briefly , biocoat matrigel invasion chambers ( becton - dickinson , bedford , ma ) were rehydrated according to the manufacturer 's instructions .
cell suspensions ( 2.5 10 cells per 2 ml serum - free medium ) were added to the top chamber , and complete medium in the lower chamber . for control ,
the cells were allowed to invade the matrigel at 37c in 5% co2 for 48 hours .
the noninvading cells on the upper surface of membrane were removed from the chamber by gentle scrubbing with a cotton swab , and the invading cells on the lower surface of the membrane were stained with the quick - diff stain kit ( becton - dickinson ) .
the number of invading cells was expressed as a percentage by the following : the mean number of the cells invading through the matrigel insert membrane divided by the mean number of cells migrating under control insert membrane conditions multiplied by 100 .
74,804 ; qiagen , valencia , ca ) , including treatment with dnase i to prevent genomic dna contamination using rurbo dna - free kit ( catalog no . am1907 ; ambion , foster city , ca ) according to the manufacturer 's instructions .
total rna ( two micrograms ) was reverse - transcribed to cdna by using the superscript iii first - strand synthesis system ( catalog no . 18080 - 051 ; invitrogen , carlsbad , ca ) , according to the manufacturer 's protocol .
the primer sequences used for the reactions are in table 1 along with expected products and genbank accession numbers .
the thermocycler parameters were as follows : an initial step at 95c for 10 min .
, 40 cycles of 95c for 20 sec . , 58c for 30 sec . , and
72c for 20 seconds . the cycle threshold values were used to calculate the normalized expression of vegfc / d against -actin .
qrt - pcr was performed in abi 7500 sequence detection system using a sybr green detection system ( catalog no .
170- 8880 ; bio - rad laboratories ) . by identifying the threshold cycle ( ct ) for expression of mrna , the ct for vegf - c
/ d was calculated and compared to vector transfected control cells ; cxcl7 transfected cells and sb225002-treated cxcl7 transfected cells .
the ct was converted into a ratio ( target a / target b = 2 ) describing the comparison of the relative expression of vegf - c / d ( target a ) to -actin ( target b ) for each of the lines . the delta - delta model was used for comparison of relative expression rt - pcr results for vegf - c / d .
vegf - c and vegf - d protein concentrations in conditioned media were measured by enzyme - linked immunosorbent assay ( elisa ) using human vegf - c or vegf - d elisa development system ( catalog no .
measurements were done at least in duplicate for 2 dilutions . the optical density at 570 nm and 450 nm was determined for each well using the plate reader .
then the reading at 570 nm was subtracted from the reading at 450 nm for each well .
the protein concentration of the serum - free conditioned medium was measured by the bradford assay ( biorad , richmond , ca , usa ) .
the heparanase activity of the conditioned medium was assessed as heparan sulfate degrading enzyme activity using the heparan degrading enzyme assay kit ( catalog no .
one unit of heparanase activity defined the activity which degraded 0.063 ng of biotinylated heparin sulfate in 1 min . at 37c and
the detection limit of this assay was 0.1 u / ml . each value of heparanase activity was normalized by protein concentration ( u / g protein ) .
briefly , the cells were harvested in pbs , counted , and lysed in the ripa buffer ( catalog no .
protein concentration was determined for all samples using the bio - rad protein assay ( catalog no .
the equal - volume samples ( 50 g ) were separated by sds - page on a 10% polyacrylamide gel and transferred onto nitrocellulose membrane ( catalog no . 162 - 0114 , bio - rad , richmond , ca ) using transfer tank .
9101 , cell signaling technology ) and erk1/2 ( catalog no.9102 , cell signaling technology ) , then developed by ecl ( catalog no . prn 2106 , ge healthcare , waukesha , wi ) and was photodocumented .
results are expressed as mean standard error of the mean ( sem ) , unless indicated otherwise . for statistical analysis , one - way anova was used , and significance was defined as p < .05 .
graphs were generated using graphpad prism ( graphpad software , san diego , ca ) .
to further verify cxcl7 function in cell invasion , a selective nonpeptide cxcr2 antagonist sb225002 was used to check whether it can inhibit the invasive ability of mcf10at cells transfected with cxcl7 .
, cells were added into the matrigel invasion chamber ; meanwhile , sb225002 ( 1.1 m ) was also added into the chamber .
several concentrations of sb225002 were tested ( 0.5 , 0.7 , 0.9 , 1.1 , 1.3 , and 1.5 m ) and 1.1 m had the best inhibitory effect similar to that reported by levashove et al . .
the decrease in the percent invasion of matrigel by the cells is shown in figure 1 .
treatment with sb225002 resulted in 18.06% 0.76% invasive cells compared with 55.7% 1.4% in nontreated cxcl7 stable transfected mcf10at cells .
thus , the cxcr2 antagonist blocked the invasive ability of cxcl7 stably transfected mcf10at cells . to investigate the effect of sb225002 on expression and secretion of vegf - c and vegf - d by stable cxcl7-transfected mcf10at cells , we cultured the cells with and without sb225002 ( 1.1 m ) .
expression of mrna for lymphangiogenic factors vegf - c and vegf - d was determined by quantitative real - time polymerase chain reaction ( qrt - pcr ) assay .
the expression levels of mrnas for vegf - c , vegf - d , are shown in figure 2(a ) .
compared with vector - transfected mcf10at cells , cxcl7-transfected mcf10at cells showed 11-fold higher vegf - c and 18-fold higher vegf - d expression .
administration of sb225002 antagonist resulted in a significant ( p < .01 ) inhibition of vegf - c ( 3.5-fold ) and vegf - d ( 3-fold ) mrna expression .
next , we examined the secretion of the vegf - c and vegf - d by elisa assay .
the secretion levels of vegf - c , vegf - d are shown in figure 2(b ) .
both vegf - c and vegf - d secretion from the cxcl7-transfected cells were significantly ( p < .01 ) increased compared with the vector - transfected control group ( vegf - c , 3-fold ; vefg - d , 2.5-fold ) .
our data also showed a significant ( p < .05 ) inhibition in vegf - c and vegf - d secretion by sb225002-treated cells compared with control treated cells .
thus the cxcr2 antagonist decreases vegf - c and vegf - d mrna and protein expression .
next , we examined whether cxcl7 sirna inhibited the elevated heparanase activity of cxcl7-stably transfected cells .
cells were transfected with cxcl7 sirna or control sirna , and after 48 hours , the cm was collected .
compared with control sirna treatment , cxcl7 sirna - transfected cells showed significant ( p < .01 ) inhibition of heparanase enzymatic activity ( figure 3(a ) ) .
the same results were obtained after using a microcon filter to remove molecules less than 30 kd ( which includes cxcl7 ) from cm , verifying by elisa that cxcl7 is not present after filtering .
and then , retesting for heparanase activity were performed . to further confirm the increased invasion after transfection by cxcl7
the decrease in the percent invasion of matrigel by the cells is shown in figure 3(b ) .
treatment with sirna resulted in 16% 1.2% invasive cells compared with 37% 2.5% by mcf10at cells transfected with cxcl7 .
thus , cxcl7 sirna inhibits the invasion of mcf10at cells transfected with cxcl7 . to further confirm the increased expression and secretion of lymphangiogenesis factors vegf - c and vegf - d after transfection by cxcl7
, we used the sh - ppbp to silence the cxcl7 signal . to investigate the effect of sh - ppbp on expression and secretion of vegf - c , vegf - d of stable cxcl7-transfected mcf10at cells , we cultured the cells transfected with sh - ppbp or control ( sh - con ) .
expression of the mrna of lymphangiogenic factors vegf - c and vegf - d was determined by quantitative real - time polymerase chain reaction ( qrt - pcr ) assay .
the expression levels of mrnas for vegf - c , vegf - d , are shown in figure 4(a ) .
compared with vector - transfected mcf10at cells , cxcl7-transfected mcf10at cells showed 11-fold higher vegf - c and 18-fold higher vegf - d expression .
the sh - ppbp transfection resulted in a significant ( p < .01 ) silencing of vegf - c ( 66% ) and vegf - d ( 68% ) mrna expression .
next , we examined the secretion of the vegf - c and vegf - d by elisa assay .
the secretion levels of vegf - c , vegf - d are shown in figure 4(b ) .
both vegf - c and vegf - d secretion from the cxcl7-transfected cells were significantly ( p < .01 ) increased compared with the vector - transfected control group ( vegf - c , 3.3-fold ; vefg - d , 4.4-fold ) .
our data also showed a significant ( p < .05 ) silencing in vegf - c ( 65% ) and vegf - d ( 68% ) secretion by sh - ppbp transfected cells compared with sh - con transfected cells . the map kinase pathway is important for growth , differentiation , and migration , and is considered a dominant signaling pathway for cxcr2 [ 27 , 28 ] . to investigate the mechanism by which the cxcl7-cxcr2 axis is involved in the stimulation of lymphangiogenic factors vegf - c , vegf - d toward invasion by human breast cancer cells , we investigated the involvement of erk1/2 map kinases by western blotting .
stable cxcl7-transfected mcf10at cells did not induce erk1/2 phosphorylation in comparison with the vector transfected control cells , and there is no difference in erk1/ 2 map kinase expression in stable cxcl7 transfected mcf10at cells compared to vector controls ( figure 5 ) .
although many molecules have been implicated in cancer metastasis , the detailed mechanism of tumor metastasis is still not completely understood .
recently , the interest in chemokines in cancer research has been increasing as new chemokines are being identified and investigated .
working along with many other molecules , the chemokines and their receptors expressed by both the cancer and its stroma influence growth , dormancy , angiogenesis , and invasion [ 3 , 6 , 29 ] .
the cxcr4/cxcl12 ( sdf-1 ) axis was the most common interaction that has been shown to be involved in many different human malignancies , including breast cancer , ovarian cancer , and prostate cancer [ 30 , 31 ] .
however , cxcr4 interactions alone did not completely explain the pattern of metastasis of cancer .
there are 4 families of chemokines ( c , cc , cxc , and cx3c ) based on the arrangement of cysteine residues near the n terminus [ 32 , 33 ] .
the cxc group can be further subdivided on the basis of presence of the tripeptide motif glutamate - leucine - arginine ( elr ) adjacent to the cxc motif .
all elr - cxc chemokines act through cxc chemokine receptor type 1 or type 2 ( cxcr1 or cxcr2 ) , which are transmembrane g - protein - coupled receptors .
secretion of stromal cell - derived factor-1 ( sdf-1)/cxcl12 and expression of cxcr4 have been identified to be associated with breast cancer metastasis . although cxcr4 was more highly expressed in the breast cancer cells tested bymller et al .
than the other cxcrs , the expression of cxcr2 was also increased compared with the remaining receptors .
cxcl7 , which is a member of the elr - cxc chemokines , binds with cxcr2 receptors , stimulating angiogenesis and association with neutrophils and other immune components .
inhibition of cxcr2 function on endothelial cells has been shown to inhibit tumor growth , for lung cancer and renal cell cancer models [ 39 , 40 ] .
the use of a small selective antagonist for cxcr2 ( sb225002 ) represents an attractive targeted therapeutic approach .
previous work in our laboratory showed that breast cancer cells coexpress cxcl7 and cxcr2 , which may act as a potential autocrine mechanism in breast cancer .
the malignant cell line mcf10ca1a.cl1 strongly expressed cxcl7 and has much higher invasive ability than mcf10at .
thus , cxcl7-transfected mcf10at cells were as invasive as malignant cells , suggesting that cxcl7 may have a role in the invasion process . therefore , targeting its receptor , cxcr2 , seemed an obvious choice . by using cxcl7-stably transfected mcf10at cells treated by sb225002
, invasion was decreased significantly compared with cxcl7-transfected cells , which might involve cxcl7 autocrine activity with cxcr2
. the present study did not examine whether sb225002 could block the invasive ability of isogenic malignant cells ( mcf10ca1a.cl1 ) or other malignant cell lines , which would be an interesting focus for future study .
lymphangiogenesis refers to the formation of new lymphatic vessels that may occur in normal developing tissues or in tumors .
overexpression of vegf - c or vegf - d can lead to lymphangiogenesis , intralymphatic tumor growth and formation of lymph node metastases [ 41 , 42 ] .
vegf - c and vegf - d are ligands for vegfr-3 ( also termed fms - like tyrosine kinase 4 , flt-4 ) , a tyrosine kinase receptor that is expressed predominantly in lymphatic endothelial cells .
the breast cancer cells secrete vegf - c , vegf - d which directly interact with the receptor .
this paracrine relationship may lead to further changes in the breast cancer cells leading to invasion .
chemokine and vegf - c interactions have been reported in a model of cross - talk for lymphatic endothelial cells and melanoma cells . in our study
, both the selective cxcr2 antagonist and sh - ppbp suppresses the elevated expression and secretion of vegfc / d by the cxcl7-transfected mcf10at cells .
these results support the notion that the cxcl7/cxcr2 axis plays an important role in cancer cell lymphangiogenesis . the chemokine connective tissue - activating peptide ( ctap - iii ) , which is an n - truncated derivative of cxcl7 , has been reported to have heparanase activity [ 15 , 44 ] .
heparanase activity is also responsible for the egress of metastatic tumor cells and other blood - borne cells from the vasculature .
recently , expression of vegf - c was shown to be induced by heparanase in prostate cancer cells , epidermoid cancer cells , breast cancer cells , and melanoma . in this study ,
heparanase activity in the cm of cxcl7-transfected mcf10at cells was tested , and it was determined that the cxcl7-transfected cells demonstrated increased secreted heparanase activity .
in addition , silencing cxcl7 inhibited the invasive ability of cxcl7-transfected mcf10 at cells which further elucidates its role in invasion .
the expression of vegf - c and vegf - d mrna and protein was also decreased in these transfectants by shrna , thus linking the expression of the chemokine cxcl7 to vegf - c and vegf - d , heparanase expression , and invasive ability .
the mcf10 model of progressive breast disease provides isogenic cell lines with increasing malignant potential to study the steps of metastasis .
additional breast cancer cell lines and breast cancer tissue can be tested specifically for cxcl7 expression and effects on lymphangiogenesis , heparanase expression and invasion in the future .
lymphangiogenesis in human breast cancer samples can be correlated with clinical parameters and cxcl7/cxcr2 staining . to explain the mechanism by which the cxcl7-cxcr2 axis is involved in the stimulation of lymphangiogenic factors vegf - c
, vegf - d , increased invasion and heparanase expression in human breast cancer cells .
we demonstrated that cxcl7-stable transfected mcf10at cells do not activate erk1/2 map kinase signaling .
we did not observe a difference of erk1/2 kinase in cxcl7-stable transfected mcf10at cells compared with control cells .
this result suggests the involvement of different pathways other than map kinase signaling for the cxcl7-cxcr2 axis activation .
in general , the activation of g - protein coupled receptors ( gpcrs ) like cxcr2 leads to the dissociation of the subunit from the ,-dimer when gdp is replaced by gtp .
both subunits can activate many signaling pathways including phospholipase c and adenyl cyclase . when specifically considering cxcr2 , information from cxcl8 might shed some light on other potential pathways for cxcl7 since these two chemokines have 48% identity in their amino acid sequences and both bind cxcr2 .
cxcl8 binding to cxcr2 activates the rac / pi3k , rho and ras pathways .
therefore , the cxcl7/cxcr2 axis may activate pathways other than the map kinase pathway , including rac / pi3k , rho and ras pathways .
in the present paper we showed that both the selective cxcr2 antagonist sb225002 and sh - ppbp suppress vegf - c , vegf - d expression and secretion in cxcl7-transfected mcf10at cells . furthermore , we also observed that both sb225002 and cxcl7 sirna reduced the invasion of cxcl7-stably transfected mcf10at cells , confirming the role of the cxcl7/ cxcr2 axis in cell invasion , possibly through the receptor 's signaling mechanism .
however , this does not involve the map kinase signaling pathway as has been described for other elr cxc chemokines like il-8 .
it has also been shown that cxcl7 sirna knocked down heparanase activity in transfected mcf10at cells , suggesting an important role for cxcl7 in heparanase expression . taken together , these data would support that the cxcl7/cxcr2 axis may be important in breast cancer metastasis .
therapeutics aimed at antagonizing cxc chemokine action , including cxcl7 , may be beneficial in preventing invasion and thus the spread of breast cancer . | increased expression of lymphangiogenesis factors vegf - c / d and heparanase has been correlated with the invasion of cancer .
furthermore , chemokines may modify matrix to facilitate metastasis , and they are associated with vegf - c and heparanase . the chemokine cxcl7 binds heparin and the g - protein - linked receptor cxcr2 .
we investigated the effect of cxcr2 blockade on the expression of vegf - c / d , heparanase , and on invasion .
cxcl7 sirna and a specific antagonist of cxcr2 ( sb225002 ) were used to treat cxcl7 stably transfected mcf10at cells .
matrigel invasion assays were performed .
vegf - c / d expression and secretion were determined by real - time pcr and elisa assay , and heparanase activity was quantified by elisa .
sb225002 blocked vegf - c / d expression and secretion ( p < .01 ) .
cxcl7 sirna knockdown decreased heparanase ( p < .01 ) . both sb225002 and
cxcl7 sirna reduced the matrigel invasion ( p < .01 ) .
the map kinase signaling pathway was not involved .
the cxcl7/cxcr2 axis is important for cell invasion and the expression of vegf - c / d and heparanase , all linked to invasion . |
mammary analogue secretory carcinoma ( masc ) is a recently described pathological entity arising in the salivary glands . in 2010 , skalova et al .
reported a case series comprising 16 cases of this salivary gland tumor showing identical histological as well as molecular features to breast secretory carcinoma .
the fusion gene encodes a chimeric tyrosine kinase , which has potential transformation activity and plays a major role in carcinogenesis [ 2 , 3 ] .
histopathologically , masc is a distinctive entity , and histology in combination with appropriate immunohistochemical analysis is sufficient for a diagnosis in most cases .
however , several histopathological features of masc overlap with those of other salivary gland tumors , such as acinic cell carcinoma ( acicc ) , adenocarcinoma not otherwise specified ( adc - nos ) , and low - grade mucoepidermoid carcinoma [ 1 , 2 , 4 ] . in the first reported case series by skalova et al . , most tumors ( 13/16 cases ) arose in the parotid gland with 3 cases originating in the minor salivary glands . since that seminal paper ,
some retrospective studies and case reports have been published [ 5 , 6 , 7 , 8 ] .
masc arose in the parotid gland in the majority of cases , followed by the submandibular gland and the oral cavity ( soft palate , buccal mucosa , and lip ) [ 2 , 8 , 9 , 10 ] . in this paper , we present the first reported case of masc presenting as a cervical lymph node metastasis of unknown primary site together with a brief review of the literature and discussion of possible appropriate treatments .
a 74-year - old male presented with a 2-month history of a painless lump in the left neck .
he had a past medical history of hypertension , and his family history was not significant . on physical examination
there was a 2 2 cm firm swelling present in the left upper neck , the mobility of which was slightly restricted .
sonograms showed that the mass was hypoechoic , 16 14 20 mm in size , and with a relatively regular border .
computed tomography showed an enhanced lesion in the left upper neck in contact with the carotid bifurcation and the jugular vein ( fig .
1c ) . based on the fine needle aspiration cytological findings , a possible malignant tumor , such as epithelial - myoepithelial carcinoma or basal cell adenocarcinoma , was suspected , but no definitive diagnosis was given . positron emission tomography / computed tomography ( pet - ct ) revealed fdg avidity in the left - sided neck at a level ii lymph node , the size of which was 21 16 mm . there was no evidence of a primary lesion , including the parotid and submandibular glands , on pet - ct ( fig .
further , no tumorous lesions were detected in the mammary gland on pet - ct .
therefore , with a provisional diagnosis of suspected cancer of unknown primary site , he underwent left modified radical neck dissection .
intraoperatively , the level ii lymph node invaded the internal jugular vein and superior thyroid artery , and these vessels were sacrificed .
no random biopsy was performed as there was no possibility that it was a squamous cell carcinoma .
the gross resected specimen was yellow - white in color and formed lobular nodules ( fig .
histopathological examination revealed a metastatic tumor in the left - sided neck at a level ii lymph node .
the tumor consisted of tubular structures containing secretion , papillary - cystic structures , and a solid proliferative component ( fig .
the papillary - cystic lesion showed many microcystic structures in the component cells ( fig . 2c ) .
immunohistochemical staining showed that the neoplastic cells were negative for p63 and dog1 ( fig .
3c ) . a fluorescence in situ hybridization ( fish ) study with an etv6 ( 12p13 )
no other pathologically positive nodes were detected at other levels in the neck , and there was no tumor in the submandibular gland .
we , therefore , made the final diagnosis of a cervical lymph node metastasis of masc from an unknown primary site .
no adjuvant therapy was administered as our case showed solitary lymph node metastasis and complete resection was achieved .
no local recurrence or metastatic disease has been detected during a follow - up of 9 months .
from the literature , masc involves the parotid gland in about 70% of cases , the submandibular gland in about 7% of cases , and other sites , such as the soft palate , buccal mucosa , base of the tongue , and lip , less commonly [ 2 , 810 ] .
the tumor presented herein is the first documented case of masc presenting as a cervical lymph node metastasis of unknown primary site .
the salient histologic features of masc include solid , microcystic , tubular , and papillary - cystic patterns in varying proportions [ 8 , 1012 ] , and these findings were displayed by the tumor presented herein .
in addition , intraluminal and/or intracellular colloid - like secretions that react positively with periodic acid - schiff ( pas ) are often observed histologically in masc [ 6 , 8 , 13 ] .
immunohistochemically , masc characteristically shows positive staining for s100 protein and mammaglobin [ 1 , 10 ] .
masc may also express gata3 , pancytokeratin , ck7 , ck8 , ck18 , ck19 , epithelial membrane antigen , vimentin , muc1 , muc4 , stat5a , gcdfp15 , and adipophilin [ 1 , 10 ] .
masc is typically negative for high - molecular - weight keratin and basal cell / myoepithelial markers , such as calponin , sma , ck5 , ck6 , ck14 , and p63 [ 4 , 10 ] .
the most common malignancy reclassified as masc was adc - nos ( 14/37 ) , followed by acicc ( 11/89 ) .
there are considerable overlapping histological features between acicc and masc , as both exhibit acinar differentiation and intercalated duct - type cells .
a major point of distinction is the presence of pas - positive cytoplasmic zymogen granules , which are observed in acicc [ 8 , 12 ] .
in addition , masc stains more reliably for s100 protein , and acicc is not positive for mammaglobin , whereas dog1 stain is positive in the majority of cases of acicc [ 6 , 10 ] .
similar to those in previous reports , our case displayed positive staining for s100 protein and gata3 but negative staining for p63 and dog1 .
the defining cytogenetic characteristic of masc is the presence of the t(12;15)(q13;q25 ) etv6-ntrk3 translocation , demonstrated by either fish or pcr [ 1 , 8 , 11 ] .
we performed a fish study with an etv6 break apart probe for molecular genetic analysis and revealed split signals in 95% of the nuclei . in the study by skalova et al .
, all but one of the examined cases ( 13/14 ) were positive for the t(12;15 ) ( etv6-ntrk3 ) fusion transcript , and all examined cases ( 11/11 ) were positive for etv6 gene rearrangement on fish .
recently , skalova et al . reported that , in some cases of masc whose profile was ntrk3 split - negative and etv6 split - positive , unknown ( non - ntrk ) genes appeared to fuse with etv6 ( etv6-x fusion ) .
, of the 91 reported cases of masc , only 4 cases of death from disease have been reported , although survival data were variably reported and follow - up was minimal .
. however , some cases of masc have the potential for regional and distant metastasis .
some authors reported that the risk for regional lymph node metastasis at diagnosis may also be higher in patients with masc than in patients with acicc [ 2 , 8 ] . to date
, there is no conclusive evidence that masc should be treated any differently to other low - grade malignant salivary gland cancers . in the review by sethi et al .
, the treatment modalities mentioned in the literature included details for 86 patients : 26% of the patients underwent neck dissections , and 20% were given postoperative radiotherapy ( port ) , while 2% received port with chemotherapy . as our case showed a solitary regional metastasis without a detectable primary lesion and complete resection was achieved , we did not choose port and adjuvant chemotherapy .
fortunately , the patient presented herein has been free from disease for 9 months . to our knowledge , this is the first case report of masc presenting as a cervical lymph node metastasis of unknown primary site and suggests new properties of masc . as previously described , masc has a capacity for an aggressive course , and the etv6-ntrk3 translocation might provide a potential therapeutic target [ 2 , 15 ] .
further investigation is needed to translate targeted molecular therapies into therapeutic options for salivary grand malignancies including masc .
we encountered the first case of masc presenting as a cervical lymph node metastasis of unknown primary site .
no adjuvant radiotherapy was administered , and no local recurrence or metastatic disease has been detected during the 9-month follow - up period .
written informed consent was obtained from the patient for publication of this case report and any accompanying images .
none of the authors have any conflicts of interest to declare in association with this study . | backgroundmammary analogue secretory carcinoma ( masc ) is a pathological entity arising in the salivary glands first described by skalova et al .
[ am j surg pathol 2010;34 : 599608 ] . here
, we report the rst case of masc presenting as a cervical lymph node metastasis of unknown primary site together with a brief review of the literature.case reportwe present a 74-year - old male with a painless lump in his left neck . based on the fine - needle aspiration cytological findings ,
a possible malignant tumor was suspected .
no evidence of a primary lesion was observed using imaging modalities including positron emission tomography / computed tomography .
the patient underwent an ipsilateral modified radical neck dissection .
immunohistochemical staining showed that the neoplastic cells were positive for s100 protein and gata3 .
a rearrangement of the etv6 gene was noted during uorescence in situ hybridization , and the final histopathological diagnosis was masc.conclusionwe encountered a masc presenting as a cervical lymph node metastasis of unknown primary site .
no adjuvant therapy was administered , and no local recurrence or metastatic disease has been detected during a follow - up period of 9 months .
this is the first case report of masc presenting as a cervical lymph node metastasis of unknown primary site and suggests the new properties of masc . |
a 68-year - old male with a history of palpitations was referred to our hospital for further evaluation .
baseline ecg showed sinus rhythm and frequent premature ventricular extrasystoles ( pves ) with a relatively narrow qrs complex ( 115 ms ) , an inferior axis ( tall r waves in leads ii , iii , and avf ) , a qr pattern in lead v1 with no transition zone , and absence of s wave in lead v5 or v6 ( figure 1(a ) ) .
an electrophysiological study was then carried out , where a detailed activation mapping of the pves was performed using a 3-dimentional electroanatomical mapping system ( carto 3 , biosense webster , inc . ,
diamond bar , calif , usa ) . left ventricular ( lv ) mapping was performed using a 4 mm tip deflectable catheter ( navistar , biosense webster ) .
the earliest local activation site of the pves ( preceding the onset of the surface qrs by 40 ms ) was identified at the aortomitral continuity of the basal lv ( figure 1(b ) ) .
( 12 of 12 leads ) with the morphology of the pves ( figure 1(c ) ) .
the first radiofrequency ( rf ) energy application ( target temperature of 60c , 30 w , 60 s , and impedance drop of 510 ohms ) abolished all the pvbs arising from the aortomitral continuity ( figure 2 ) .
additional rf applications ( total ablation time up to 180 s ) were delivered at this region in order to ensure a long - term success ( red dots in figure 2 ) .
lv outflow tract ventricular arrhythmias originating from the aortomitral continuity , the left coronary cusp , the superior basal septum , and the epicardial left ventricular summit display common electrocardiographic and electrophysiological features , probably due to the close proximity of those locations [ 13 ] .
data regarding ventricular arrhythmias arising from the aortomitral continuity are limited and conflicting [ 13 ] .
have shown that pves arising from the aortomitral continuity display narrow qrs complexes , tall r waves in inferior leads ( particularly in lead ii ) , predominantly positive forces in lead i ( where superior and lateral basal sites display negative forces ) , and a qr pattern in lead v1 .
have demonstrated the occurrence of a qr pattern in lead v1 and s wave in lead v5 or v6 in 50% of patients with pves arising from aortomitral continuity .
the absence of s wave in lead v5 or v6 favours the diagnosis of pves originating from the left coronary cusp or the epicardial left ventricular summit . in a different study , kumagai et al .
have shown that all patients with pves arising from the aortomitral continuity exhibit monophasic r waves without s waves in almost all precordial leads .
these investigators failed to demonstrate a qr pattern in lead v1 or s wave in lead v6 . | left ventricular outflow tract arrhythmias originating from the aortomitral continuity , the left coronary cusp , the superior basal septum , and the epicardial left ventricular summit display common electrocardiographic and electrophysiological features , probably due to the close proximity of those locations .
catheter ablation of these arrhythmias can be challenging .
the case of a 68-year - old male with frequent premature ventricular extrasystoles arising from the aortomitral continuity of the basal left ventricle is described .
the electrocardiographic and electrophysiologic characteristics of this arrhythmia are discussed . |
it is inherited in an autosomal recessive manner and is caused by mutations in the ctns gene , which is located on chromosome 17p13.1 these mutations cause intralysosomal cystine accumulation in cells throughout the body . today , over 100 pathogenic mutations are known to cause cystinosis .
the most frequent mutation found in western europe is a large deletion of 57,257 base pairs.2 ctns encodes the lysosomal cystine transporter cystinosin , which is a 367 amino acid lysosomal membrane protein , with seven transmembrane domains .
cystinosin is a proton - cystine cotransporter , coupling the excretion of both cystine and protons out of the lysosome .
protons are carried back into the lysosome by h - atpase.3 besides the cystinosin that is localized on the lysosomal membrane , a second isoform exists due to alternative splicing of ctns messenger rna ( mrna ) .
this isoform lacks one of the two lysosomal targeting motifs , consists of the amino acid sequence gydql , and was named cystinosin - lkg ( the letters lkg representing its last three amino acids ) .
cystinosin - lkg is localized at the plasma membrane in lysosomes and several other organelles , including the golgi apparatus and the endoplasmic reticulum .
its exact function remains unclear ; however , it is known to facilitate the transport of cystine in an acidic environment , just like its counterpart cystinosin.4 cystinosis has an incidence of one per 100,000200,000 live births , and in suspected cases , the diagnosis can be confirmed by measuring white blood cell ( wbc ) cystine levels and/or by ctns gene analysis .
three clinical forms of cystinosis can be distinguished , the most frequent being infantile nephropathic cystinosis ( omim 219800 ) .
patients generally present before the age of 12 months with polyuria , polydipsia , and failure to thrive caused by generalized proximal tubular damage , also known as renal fanconi syndrome .
it is characterized by increased urinary excretion of several small molecules , including glucose , salts , amino acids , and low molecular weight proteins .
photophobia is caused by corneal cystine crystals , which can be recognized by an experienced ophthalmologist from the age of 12 months.5 further , several other organs can be involved during progression of the disease .
these include the thyroid gland ( hypothyroidism),6 gonads ( hypogonadism , azoospermia),7,8 pancreas ( exocrine and endocrine insufficiency),9,10 muscles ( hypotrophy , swallowing difficulties),11 and the peripheral and central nervous systems.11 late - onset or juvenile nephropathic cystinosis ( omim 219900 ) is characterized by the same symptoms as infantile cystinosis , but with a disease onset mostly after the first decade of life and a slower rate of disease progression .
in contrast , patients with the ocular form of cystinosis ( omim 219750 ) only have photophobia due to corneal cystine crystals while other organs remain spared .
the latter two forms of cystinosis are very rare ; over 95% of all cystinosis patients are diagnosed with the infantile form.5
cysteamine ( -mercaptoethylamine ) was first introduced as a possible therapeutic agent to treat cystinosis in 1976,12 and until now has been the only available treatment for the disease .
after its introduction as a treatment for patients with cystinosis , cysteamine was first prescribed in the form of cysteamine hcl . because of the low bioavailability of the hydrochloride salt , it was replaced by phosphocysteamine in 1980.13 in 1994 , cysteamine bitartrate ( cystagon , mylan pharma , morgantown , wv , usa ) was introduced14 and this is currently the most widely used preparation of cysteamine .
recently , an enteric - coated preparation of cysteamine bitartrate ( procysbi , raptor pharmaceuticals inc . , novato , ca , usa ) was approved by the us food and drug administration and european medicines agency for the treatment of cystinosis .
cysteamine uses an unknown transporter to enter the lysosome and subsequently breaks the disulfide bond in cystine .
this leads to formation of cysteine , which leaves the lysosome using the cystine transporter , and cysteine - cysteamine disulfide , which leaves the lysosome using the pqlc2 transporter ( figure 1).5,15 once in the cytosol , cysteine can be further metabolized to form glutathione , one of the cell s most potent redox buffers .
indeed , in vitro experiments showed that the altered redox status demonstrated in cystinotic cells was corrected by administration of cysteamine , thereby increasing intracellular glutathione levels.16 one hour after ingestion of cysteamine , the drug s plasma levels reach their maximum while wbc cystine levels are at their minimum , followed by a gradual decline in plasma cysteamine levels and a gradual increase of wbc cystine levels .
both reach their original values around 6 hours after ingestion.17 this underscores the importance of a dosing regimen in which cysteamine is administered every 6 hours ; ideally , it should also be administered during the night.18 understandably , such a dosing scheme causes a great burden in terms of compliance with cysteamine therapy .
treatment of cystinosis patients with cysteamine is further hampered by the side effects of the drug .
when cysteamine was first prescribed to patients with cystinosis , several of them developed hyperthermia , lethargy , and rash .
however , all of these side effects were reversible after the treatment was discontinued , and none of these adverse events are noted today since it has become common practice to introduce cysteamine at a low dose with a subsequent gradual increase over several weeks.19 gastrointestinal complaints are due to increased gastric acid secretion after ingestion of cysteamine,20 and thus concomitant use of proton pump inhibitors is advised.21 after administration of cysteamine , a small amount is metabolized into the volatile sulfur compounds methanethiol and dimethylsulfide , causing halitosis.22 recently , eight cystinosis patients were found to have new adverse events associated with cysteamine , consisting of bruise - like lesions on the elbows , skin striae , and severe bone and muscular pain .
skin biopsies taken from the bruise - like lesions showed reactive angioendotheliomatosis and alterations in collagen fiber diameter.23 the reactive angioendotheliomatosis could be attributed to increase survival of dermal microvascular endothelial cells after exposure to cysteamine,24 while the collagen fiber abnormalities could be the result of a synergistic effect of cysteamine and copper deficiency ( which was found in all patients identified as having these side effects ) on inhibition of collagen cross - linking.25 the newly reported side effects of cysteamine resulted in new recommendations regarding dosing . instead of using body weight to calculate the dose of cysteamine ( 50 mg / kg / day , with a maximum of 90 mg / kg / day ) it
is now advised to use body surface area ( 1.30 g / m / day , with a maximum of 1.95 g / m / day ) .
this difference in calculation method is particularly important in growing children , in whom body weight increases faster than body surface area .
this may lead to higher ( potentially toxic ) doses when using body weight to calculate the daily cysteamine dose , since 90 mg / kg may exceed 1.95 g / m.23 despite its side effects and four times daily dosing regimen , cysteamine remains the cornerstone of treatment for cystinosis and adequate administration of cysteamine significantly reduces the rate of progression towards end - stage renal disease , and postpones or even prevents the occurrence of extrarenal complications.2629 hypothyroidism , myopathy , pulmonary dysfunction , and diabetes mellitus ( caused by pancreas insufficiency ) occur less frequently in cystinosis patients who have been treated with cysteamine for a prolonged period of time.27,30 further , while children with cystinosis show more pronounced growth retardation than children with other renal diseases , adequate treatment with cysteamine significantly improves growth parameters.29,31,32 nonetheless , most children with cystinosis also require treatment with recombinant human growth hormone to achieve a final height higher than two standard deviations below the mean.29,32 unfortunately , not all complications of cystinosis are delayed by administration of cysteamine .
cysteamine does not influence the incidence of primary hypogonadism in male patients with cystinosis , nor does it improve delayed puberty . also
, male infertility due to azoospermia is not ameliorated by adequate treatment with cysteamine.7,8 further , systemic administration of cysteamine does not prevent formation of corneal cystine crystals,33 indicating a need for topical cysteamine eye drops which should be applied several times daily .
recently , a new gel formulation of cysteamine ( cystadrops , orphan europe , montluon , france ) was tested for its ability to diminish the formation of corneal cystine crystals , with better effects and less applications needed per day compared with the usual cysteamine hydrochloride formulation.34
the most promising results with regard to treatment of cystinosis without use of cysteamine come from the field of stem cell transplantation . in a ctns mouse model treated with wild - type allogeneic bone marrow and hematopoietic stem cells , the mice that received a bone marrow graft showed better renal function after 4 and 15 months , as well as decreased cystine content in a number of tissues , including the brain , eyes , heart , kidneys , liver , skeletal muscle , and spleen.35,36 however , despite these promising results , tissue cystine levels remained higher than those in asymptomatic heterozygous carriers , so administration of cysteamine may still be warranted .
in addition to bone marrow transplantation , gene therapy was also studied in the ctns mouse model in order to minimize the use of immunosuppressive agents , along with the risks of rejection and development of graft versus host disease .
hematopoietic stem cells from ctns mice were transduced with the human ctns complementary dna ( cdna ) sequence followed by transplantation into ctns mice . again
, tissue cystine levels were significantly decreased after 4 and 8 months , including in the brain , eyes , kidneys , liver , muscle , and spleen , but were still higher than those after wild - type bone marrow transplantation.37 prodrugs are under development in order to diminish the bad taste and smell of cysteamine and to increase its half - life .
several prodrugs of cysteamine have been tested in vitro;38,39 however , none are as effective as cysteamine in decreasing intracellular cystine levels , and none have been tested in vivo .
the observation that administration of cysteamine directly in the small intestine resulted in higher plasma cysteamine levels with a higher area under the curve when compared with gastric administration of the drug40 led to development of the enteric - coated formulation of cysteamine bitartrate ( procysbi ) .
in contrast with the immediate - release formulation of cysteamine bitartrate , this new delayed - release formulation can be administered every 12 hours instead of every 6 hours.41 the delayed - release formulation consists of capsules containing small granules than can be administered through a feeding tube .
this is important , given that most small children with cystinosis require a lot of fluids and electrolyte supplements , and thus have feeding tubes that are also used for administration of medication . in a phase iii trial testing the noninferiority of the delayed - release formulation ,
41 nontransplanted patients with cystinosis were randomized to treatment with either immediate - release or delayed - release cysteamine bitartrate for 3 weeks and then switched to the other formulation for a further 3 weeks .
the mean difference in wbc cystine levels after treatment with the delayed - release formulation compared with the immediate - release formulation was 0.08 nmol cystine / mg protein ( 95.8% confidence interval 0.000.16 ) , meaning that the primary endpoint of noninferiority was met.42 interestingly , the average total daily steady - state dose of the delayed - release formulation was 82% of the previously established incoming dose of the immediate - release formulation,42 indicating that similar results for depletion of wbc cystine can be achieved with a slightly lower daily amount of cysteamine . in theory , this could result in fewer side effects .
an early pilot study was conducted comparing the excretion of volatile sulfur compounds in four patients after ingestion of immediate - release or delayed - release cysteamine bitartrate ( as part of the crossover phase iii clinical trial ) .
this study showed a trend towards a lower area under the curve for dimethylsulfide excretion in all four tested patients after ingestion of the delayed - release formulation .
however , this trend did not reach statistical significance , patient numbers were small , and the measured levels of dimethylsulfide remained above those that cause halitosis at all time points.43 a long - term study is presently underway using delayed - release cysteamine bitartrate in cystinosis patients with renal fanconi syndrome and in those who have undergone renal transplantation .
again , as part of this study , excretion of volatile sulfur compounds in breath will be measured in a subgroup of patients , comparing the levels at study onset when patients are treated with the immediate - release formulation and again a few months after treatment with the delayed - release formulation .
the final results of this study are pending , but preliminary data show that dimethylsulfide breath levels again remained persistently above the threshold that causes halitosis ; however , further analysis should determine whether the area under the curve is influenced by the delayed - release formulation .
previous reports regarding the long - term treatment of two cystinosis patients with the delayed - release formulation show a good effect on the mean trough wbc cystine level , with no deterioration of renal , thyroid , or liver function observed during a treatment period of 6 years.44
overall , the quality of life for patients with cystinosis has improved significantly since the early 1980s . since the use of cysteamine to treat cystinosis and the introduction of renal replacement therapy in pediatric populations ( either through dialysis or by renal transplantation )
, patients with cystinosis are increasingly surviving into adulthood , whereas before the introduction of cysteamine as a possible treatment for cystinosis they generally died from end - stage renal disease by around the age of 10 years.27 this increased life expectancy has caused an increase in the number of longer - term complications of the disease , that were not noticed in younger patients .
this increased interest and recognition of longer - term complications of cystinosis prompted a search for a possible treatment for these complications .
for instance , male infertility due to azoospermia was first described only a few years ago , awareness of which has enabled patients to seek further treatment options , such as testicular sperm extraction , which could open the possibility for male patients to produce their own offspring.8 another example is the severe growth retardation seen in patients with cystinosis . since this is now treated more effectively ( eg , by improving nutritional and electrolyte status , prescribing cysteamine , and administration of recombinant human growth hormone ) , today most cystinosis patients can reach a final height within the normal range,29,32 which further improves their quality of life . apart from better recognition and treatment of ( late - term ) complications of cystinosis , better treatment of the disease also improves the long - term outcome for the patient .
cystinosis is now more frequently recognized at a young age , resulting in more patients being adequately treated with cysteamine at a young age .
this is very important , since early initiation of cysteamine therapy can significantly postpone or even prevent complications of the disease , including deterioration of renal function , hypothyroidism , and myopathy.2629 however , one of the most important burdens regarding cysteamine therapy is the need to administer the drug every 6 hours in order to ensure adequate wbc cystine depletion.18 development of the new delayed - release formulation can overcome this problem , which will likely improve patient compliance with therapy , resulting in fewer long - term complications of cystinosis and improving patient life expectancy even further .
it remains uncertain to what degree potential new therapies for cystinosis ( including bone marrow transplantation and/or gene therapy ) will further improve the long - term outcomes .
although impressive results have been documented in the ctns mouse model , there are as yet no results of such treatment in humans .
the advent of cysteamine has significantly improved the outcome for patients with cystinosis , as have the better recognition and treatment of the complications of the disease .
new treatment options , such as bone marrow transplantation are promising , but are not currently performed routinely in humans .
further , although intracellular cystine levels in a number of tissues in ctns mice decrease significantly after bone marrow transplantation derived from their wild - type litter mates , cystine levels remain above those of asymptomatic heterozygous carriers.35,36 thus , even after bone marrow transplantation , there may still be a need for low doses of cysteamine to decrease the remaining accumulation of cystine .
this indicates a need to further improve the treatment of cystinosis with cysteamine . in this regard ,
the new delayed - release formulation of cysteamine bitartrate is of importance to improve compliance with cysteamine therapy . | cystinosis is an autosomal recessive inherited lysosomal storage disease .
it is characterized by generalized proximal tubular dysfunction known as renal fanconi syndrome and causes end - stage renal disease by the age of about 10 years if left untreated .
extrarenal organs are also affected , including the thyroid gland , gonads , pancreas , liver , muscle , and brain .
treatment consists of administration of cysteamine , resulting in depletion of cystine that is trapped inside the lysosomes .
since cysteamine has a short half - life , it should be administered every 6 hours .
recently , a new delayed - release formulation was marketed , that should be administered every 12 hours .
the first studies comparing both cysteamine formulations show comparable results regarding white blood cell cystine depletion ( which serves as a measure for cystine accumulation in the body ) , while a slightly lower daily dose of cysteamine can be used . |
most bladder squamous cell carcinomas have been described in paraplegics or in patients with spinal trauma .
they are also known to occur in patients with long - term indwelling catheters and during chronic inflammatory states associated with frequent irritation and persistent infection . here
we report a case study of a patient who developed a squamous cell carcinoma involving the bladder mucosa from a well - healed suprapubic cystostomy scar after 9 years .
a 56-year - old male patient who had a suprapubic foley catheter for 9 years after the formation of an urethrocutaneous fistula presented with a spontaneous suprapubic urinary leak from a well - healed suprapubic cystostomy tract scar .
he originally had a urethral stricture that required multiple surgical corrections for 16 years beginning at the age of 28 years .
nineteen years later ( at the age of 47 years ) , a urethrocutaneous fistula occurred spontaneously and a suprapubic cystostomy tube was eventually required for urinary diversion .
on the computed tomographic ( ct ) scan of the abdomen , a tumor mass surrounding the suprapubic cystostomy tract was clearly visible ( fig .
histological examination of the biopsy from the bladder mass revealed squamous cell carcinoma , moderately differentiated , probably originating from the vesicocutaneous fistula tract involving the bladder mucosa ( fig .
2 ) . the patient did not want to undergo aggressive surgical excision of the complete bladder and the abdominal wall and underwent radiation therapy .
although a ct scan of the abdomen after radiation therapy showed partial remission ( fig .
3 ) , the patient died of lung metastasis and pneumonia at 6 months after the start of radiation therapy .
squamous cell carcinoma is a rare form of carcinoma of the bladder that accounts for only 5% of such carcinomas .
it is usually caused by chronic irritation from urinary calculi , long - term indwelling catheters , chronic urinary infections , or bladder diverticula .
as many as 80% of paraplegics with chronic infections or indwelling catheters experience squamous changes in the bladder , and about 5% develop squamous cell carcinoma .
cigarette smoking is also significantly associated with an increased risk of bladder squamous cell carcinoma .
male predominance is far less striking in squamous cell carcinoma ( male to female ratio , 1.3:1 to 1.7:1 ) .
there are only four case reports of suprapubic catheter tract squamous cell carcinoma ( two of them involving the bladder ) in the english literature [ 5 - 8 ] .
the case reported here is the third case of suprapubic catheter tract squamous cell carcinoma extending into the bladder .
we present the published cases of suprapubic catheter tract squamous cell carcinoma in table 1 .
the prolonged presence of a tube with continuous irritation of the tract site leading to metaplasia and carcinoma provides a possible mechanism for carcinoma . in the literature
, a 10% incidence of squamous cell carcinoma of the bladder has been noted in patients who have had indwelling catheters for more than 10 years .
. found that spinal cord injury patients who have had indwelling catheters for more than 10 years have a 20% prevalence of squamous cell carcinoma of the bladder , and those authors advocated for periodic cystoscopies combined with random bladder biopsies for this category of patients . because this type of cancer is extremely rare , insufficient experience exists to postulate the etiology in cases that present several years later in a well - healed suprapubic catheter tract site .
persistent discharge is often attributed to recurrence of stricture , for which cystostomies are frequently performed , leading to missed malignancies .
these lesions are usually localized and are best treated by radical excisional surgery because of the rapid death of conservatively treated patients .
bladder involvement generally dictates surgical management because long - term survival benefits resulting from radiotherapy or chemotherapy have not been reported to date .
however , the patient in our study chose not to undergo planned aggressive surgical excision of the complete bladder and the abdominal wall and was instead treated with radiation therapy .
he died 6 months after the start of radiation therapy owing to lung metastasis and pneumonia .
several reports suggest that the stage - by - stage prognosis of squamous cell carcinoma is comparable to that of transitional cell carcinoma .
the prognosis of suprapubic catheter tract squamous cell carcinoma is comparatively poor because most patients have advanced disease at the time of diagnosis . in conclusion ,
persistent discharge from a well - healed suprapubic catheter scar site several years after a cystostomy should be grounds for suspicion of this rare clinical entity , and an excisional biopsy should always be performed , especially when no stricture or bladder pathology is detectable on cystoscopy .
this case study underscores the need for close monitoring of patients with any type of long - term indwelling catheter . | herein we report a case of a squamous cell carcinoma of a well - healed suprapubic cystostomy tract scar involving the bladder mucosa in a 56-year - old man .
he presented with a spontaneous suprapubic urinary leak from a suprapubic cystostomy tract scar .
he had a history of urethral stricture and failed urethroplasty .
preoperative cystoscopy suggested a bladder mass .
transurethral biopsy of the bladder mass revealed a squamous cell carcinoma confined to the suprapubic cystostomy tract involving the bladder mucosa .
the patient died 6 months after the start of radiation therapy after lung metastasis and pneumonia . |
despite advances in plastic surgery , reconstruction of soft tissue defects with exposed tendons on the hand and foot remains a challenge .
there are several reconstruction options , including skin grafts , local flaps , distant flaps and free flaps.1 , 2 , 3 however , the experience over the past 20 years has demonstrated that tissue reconstruction with flap would lead to adhesion of tendon and functional impairment at the recipient site.4 , 5 , 6 the posterior rectus sheath - peritoneal free flap , developed by salgado et al .
they proposed that the peritoneum interposition might reduce adhesion formation in surgery of exposed extensor tendons of the hand and foot .
traditionally , the peritoneal free flap was retrieved by making a paramedian incision under direct vision . considering underlying tendon excursion
this flap was supplied by the peritoneal branches of the deep inferior epigastric artery . here ,
a 30-year - old woman was involved in a machine accident , with extensive soft tissue defects .
after debridement and repair of tendons , the patient received vacuum sealing drainage ( vsd ) treatment .
extensor tendons , metacarpal bones and carpus were exposed ( fig . 1 ) .
the existing defect of the dorsal hand was 17 cm 12 cm .
the peritoneal flap measuring 22 cm 15 cm retrieved by laparoscopic surgery was transplanted to cover the defect ( fig . 2 , fig . 3 ) .
the peritoneal flap survived two weeks postoperatively and no postoperative complications were observed ( fig .
the remaining wound received a skin graft and the abdominal wall demonstrated a satisfying appearance ( fig .
the function of the injured hand recovered partially within the first year follow - up ( fig .
after radical debridement , repair of extensor tendons and vsd treatment , the size of the defect measured 17 cm 12 cm ( fig . 1 ) .
by using the laparoscopic system , pneumoperitoneum was created through an optiview trocar placed in the subumbilical position .
a laparoscope was introduced through this port and two other trocars were placed in the right and left lower abdominal wall .
the bilateral pedicles of the flap were divided through laparoscopic procedures using the ultrasonic - activated scissors .
the bilateral deep inferior epigastric artery was carefully isolated , double clipped and then transected at its origin to maximize pedicle length and diameter .
the peritoneal free flap was transplanted to cover the defect with exposed tendons , metacarpal bones and carpus .
the deep inferior epigastric artery and its accompanying veins , as pedicles of the peritoneal free flap , were anastomosed to the radial artery and cephalic vein .
after survival of the peritoneal flap , the skin defect was covered by a split - thickness skin graft .
reconstruction of extensive soft tissue defects accompanied by exposed tendons and/or skeleton on the hand and foot continues to be a challenging issue for plastic surgeons .
the skin grafts , local flaps , distant flaps and free flaps have become the most popular procedures to cover soft tissue defects .
these above mentioned treatment regimens will inevitably involve severe tendon adhesion.2 , 5 , 8 although various biochemical and physical methods have been adopted to decrease adhesion formation , most of the results are unsatisfactory.9 , 10 , 11 a thin flap that easily supports a skin graft and provides a gliding surface for tendon coverage represents an ideal option .
the peritoneum acts as a physiological barrier in prevention of abdominal adhesion , and thus , a similar mechanism may apply to tendon coverage .
salgado et al firstly introduced the posterior rectus sheath - peritoneal free flap for reconstruction of hand and foot .
the totally laparoscopic peritoneal flap with bilateral pedicles allows a larger size , across median line .
a more cosmetically acceptable result was obtained in both the donor site and the recipient site .
creating one - stage reconstruction without following skin grafting might be achieved by introducing the concept of a chimeric flap , which can resurface the exposed tendons and provide cutaneous wound coverage simultaneously . in conclusion ,
the totally laparoscopic peritoneal free flap is a good choice for reconstruction of the hand and foot with exposed tendons .
however , larger clinical trials and long - term follow - up are required to support the recommendation of this flap .
this work was supported by grants from the foundation of science & technology department of zhejiang province , p.r.china ( no . 2012c13020 - 3 ) and from the foundation of health & family planning commission of zhejiang province , p.r.china ( no . | management of defects on the hand and foot with exposed tendons remains a major challenge for plastic surgeons . here
, we present a case of hand reconstruction with a totally laparoscopic peritoneal flap .
the anterior rectus sheath was preserved in situ .
the peritoneal free flap supplied by peritoneal branches of the deep inferior epigastric artery was retrieved by laparoscopy to cover the soft tissue defect of the hand .
the defect of the dorsal hand was 17 cm 12 cm .
the peritoneal flap measuring 22 cm 15 cm survived completely without any complications .
a following split - thickness skin graft offered the successful wound closure .
motor and sensory function improved gradually within the first year follow - up .
the totally laparoscopic peritoneal free flap is a good choice for reconstruction of the soft tissue defects accompanied by exposed tendons on the hand and foot . |
a 40-year - old male patient came with the complaint of pain and swelling in the anterior vestibular region for the past 2 weeks .
history revealed recurrent episodes of pain and swelling and spontaneous remission following nasal discharge . on examination ,
the patient had a well - defined , fluctuant swelling obliterating the labial vestibule from 12 to 22 .
the swelling also extended in the midpalatal region between two central incisors posteriorly up to the premolar level [ figure 1 ] .
the central incisor exhibited mild grade 1 mobility buccopalatally without any movement in the corono - apical region .
patient had poor oral hygiene and inflamed gingiva with 5 mm of gingival pocket in both central incisors .
the periapical radiograph showed large triangular - shaped radiolucent lesion measuring about 2 cm diameter was present between the two maxillary central incisors [ figure 3 ] .
the lesion had a thin radio - opaque margin with displacement of both the roots in a distal direction [ figure 4 ] .
mild root resorption was observed in 21 root and the lesions also involved the periapical region of the two lateral incisors .
the central incisors had bone only on the cervical region , and the remaining portion was completely in the radiolucent lesion .
occlusal view radiograph revealed an oval radiolucent lesion from the central incisor to the 2 premolar region in the midpalatal region measuring about 3 cm 2 cm lesion . due to the proximity of the lesion to the nasal floor ,
cone - beam computed tomography ( cbct ) scan was taken to plan for the surgical procedure which clearly showed the complete breach of both the nasal floor and hard palate with hollowing in the anterior maxillary region [ figure 5 ] .
based on the clinical and radiographic findings , provisional diagnosis of nasopalatine duct cyst was made .
preoperative intraoral swelling preoperative ( a ) intraoral periapical radiograph , ( b ) occlusal radiographs orthopantamograph showing distal displacement roots of upper central incisors ( a and b ) cone beam computed tomography images of the lesion showing complete anterior palatine bone loss the patient was advised about the nature and severity of the lesion , and the complete bone loss of anterior palatine bone was explained .
the significance of surgical enucleation of the lesion was explained to the patient and also advised for bone graft placement .
the patient gave consent for the cyst enucleation but was not willing for bone grafting procedure . before the surgery
, root canal treatment of the involved teeth was done , and complete scaling was done to improve the oral hygiene . before surgery , all preliminary investigations were done , and the results were within normal limit . due to the mild mobility of the central incisors , splinting of the teeth with arch bar
the possibility of tooth avulsion or extractions of incisors were explained to the patient , and informed consent was obtained . as the cystic wall was found attached to the nasal mucosa and palatal mucosa , both labial and palatal approach
were used [ figure 6 ] . the cyst was carefully dissected and enucleated , without breaching the nasal mucous membrane or the palatal mucosa .
intraoperatively , the cyst was found to be a capsuled lesion which was adherent in the nasopalatine region of palatal mucosa but was not attached to the root or periodontal ligament .
the flap was closed , and palatal acrylic plate was placed to support the palatal mucosa .
histopathologically , it was found to have fibrous capsule with stratified squamous epithelial lining , and final diagnosis of nasopalatine duct cyst was confirmed [ figure 7 ] .
intraoperative picture showing attachment of the cystic wall with ( a ) nasal mucosa , ( b ) palatal mucosa histopathological picture showing stratified squamous epithelium ( h and e , original magnification , 10 )
nasopalatine duct canal develops as a right and left side canal but generally fuses into single canal .
however , three anatomical variations have been reported ; wherein , it sometimes appears as two parallel canals or single canal or a y - shaped canal . depending on the anatomical structure , the nasopalatine cyst appears as a midline cyst or paramedian cyst when it involves one side of the canal only . however , midline nasopalatine duct cyst is more common than the other types ( stafne 1969 ) .
it has been postulated that localized trauma such as previous endodontic treatment , implant placement or infection or mucous retention may initiate the cellular proliferation and may give rise to nasopalatine duct cyst .
it is not been associated with any habits such as smoking or alcoholism . in our study
the cyst arises from the epithelial lining of nasopalatine duct or from the vomer nasal organ of jacobson or mucous glands present in the nasal mucosa .
the epithelium close to the nasal floor has ciliated columnar epithelium , and the one closer to the oral cavity has squamous epithelium and the one in the middle has cuboidal epithelium .
hence , the nasopalatine cyst also exhibits the mixed nature of the epithelial lining . in our case ,
it appears as either asymptomatic which is recognized on routine imaging or may present with pain or swelling or with other symptoms such as burning sensation , tooth displacement , nasal obstruction , or discharge into the nasal cavity or oral cavity .
the size of the swelling is variable in size but most often it is < 2 cm in diameter .
however , occasional cases of large and aggressive lesion up to 52 mm have been reported with complete destruction of labial and palatal bony plates .
when the nasopalatine duct cyst present at higher level , there is not much effect over the tooth .
the nasopalatine duct cyst when it is present at lower level always displaces the tooth and often considered characteristic of the lesion .
however , it has been reported in association with nonvital tooth and periodontally compromised tooth . in our case , both the incisors were nonvital , and the patient had chronic periodontitis with poor oral hygiene .
root resorption is uncommon ; however , shear and speight have reported cases with root resorption .
radiographically , it appears as a round , ovoid , pear - shaped , or heart - shaped radiolucent lesion in the middle of the palate .
bone expansion is uncommon , and usually , it causes resorption of bone . in our case ,
the cyst completely eroded the palatal bone and the nasal floor , and the swelling was fluctuant in nature .
the average size of the normal nasopalatine fossa is 3 mm in anteroposterior , transverse , and in vertical direction .
however , shear and speight have reported that the mean size of the nasopalatine fossa was as high as 10.19 + 3.24 mm anteroposteriorly and 4.79 + 1.33 mm in width .
it is advised that if the radiographic size of nasopalatine fossa was < 10 mm without any signs and symptoms , it should be re - radiographed periodically . however , bodin , isaacson , and julin advised for surgical exploration if the size of the radiolucency exceeds 8 mm , or if it shows asymmetric bulging . aspiration of the radiolucent lesion will help to differentiate a normal nasopalatine fossa from the cyst .
cbct is useful when planning for the surgical enucleation of the lesion as it clearly shows the proximity of the lesion with the nasal floor and the posterior extent in the hard palate .
the differential diagnosis of the lesions includes odontogenic cysts and tumors such as dentigerous cyst , lateral periodontal cyst , cystic ameloblastoma , keratocystic odontogenic tumors , and periapical granuloma .
however , marsupialization is considered in large lesions followed by second stage enucleation can be done .
recurrence is uncommon following enucleation ; however , recurrence rate of 1130% has been reported in the literature . in our study , we used both palatal and vestibular approach as the cyst was large in size extending from nasal floor to palatal mucosa .
paresthesia of the anterior palatal region has been reported in occasional cases following the surgery .
apart from odontogenic cysts such as radicular cyst , dentigerous cyst , and odontogenic keratocyst , the nonodontogenic origin of cyst such as nasopalatine duct cyst also should be considered in the differential diagnosis of the radiolucent lesions in the anterior maxillary region . as per the literature review , nasopalatine duct cyst is considered a rare pathology , simple enucleation surgery is often considered curative .
| nasopalatine duct cyst is the nonodontogenic developmental cyst , frequently occurring in the midline of the anterior maxillary region .
the clinical presentation of the cyst is often varied and presents a diagnostic difficulty and frequently misdiagnosed as developmental or inflammatory odontogenic cystic lesion .
this paper represents a large infected nasopalatine duct cyst presenting with complete destruction of anterior palate and pyriform rim . |
the development of analytical tools for rapid analysis and identification of expressed protein profiles in cell , tissue or organism is currently an important area in biological research [ 13 ] .
although two - dimensional gel electrophoresis ( 2de ) is a classical technique that monitors and distinguishes multiple forms of proteins with differences in molecular mass or pi values , it can face difficulties with proteins of extreme mass ( e.g. , > 200 kda or < 10 kda ) or pi values [ 46 ] . in addition , 2de is not readily amenable to automation .
liquid - phase separation methods such as size - exclusion chromatography , affinity chromatography , and ion - exchange chromatography exhibited practical difficulties due to the lack of the isoelectric ( pi ) information and limited labeling efficiency [ 79 ] .
alternatively , the proteomelab pf2d platform ( beckman coulter , usa ) that can be used for the separation / fractionation , as well as quantitative comparisons of various biological and clinical samples , works in full automation combining chromatofocusing separation and hydrophobic fractionation . during the first - dimension chromatofocusing of pf2d ,
proteins are separated by their pi and separated proteins with a ph gradient are collected using a fraction collector [ 11 , 12 ] .
subsequently , fractions collected from the first dimension are separated using reversed phase chromatography in the second dimension , which separates on the basis of hydrophobicity .
separated fractions are monitored with uv detection to observe changes in the proteome [ 1315 ] .
although pf2d system offers high loading capacity and improved detection limit with lower abundance proteins [ 16 , 17 ] , its protein recovery efficiency during the chromatofocusing step will be low when the standard protocol recommended by the manufacturer is used .
reported that the inclusion of 20% isopropanol in the isoelectric focusing ( ief ) buffer increased the number of proteins they could identify in the serum .
they demonstrated improved recovery of protein , but purified bsa was used instead of complete serum .
this buffer 's ability to improve the recovery of all proteins thus remains unclear .
the columns used with pf2d require the use of nonionic detergents such as triton x-100 for the separation of proteins .
triton x-100 is a low - cost mixture of octylphenol ethoxylates , with an average of about 9 - 10 ethylene oxide units per molecule . in search of an alternative method that can increase the recovery of a wide range of proteins ,
buffers recommended by beckman coulter 's proteomelab pf2d protocol was replaced by triton x-100 during protein profiling of escherichia coli k12 , in which its recovery efficiency was determined at various triton x-100 concentrations .
subsequently , the elution accuracy of triton x-100 at its optimized condition was confirmed by the control protein mixture of ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , and cholecystokinin ( cck ) .
the eligibility of triton x-100 as a replacement for buffers used for proteomelab protein liquid chromatography was determined with escherichia coli k12 substrain w3110 and control mixture of proteins ( ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , and cholecystokinin ( cck ) ) .
e. coli k12 was cultivated at 37c in 200 ml lb medium ( 1% bacto tryptone , 0.5% bacto yeast extract , 0.5% nacl , ph 7.2 ) , to an od600 of 1.5 .
the cells were harvested by centrifugation at 6000 g for 10 min at 4c and washed in 10 mm tris - hcl buffer ( ph 8.0 ) . after being resuspended in 10 ml of ice - cold lysis buffer ( 7 m urea , 2 m thiourea , 4% 3-[(3-cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) , 65 mm dithiothreitol ( dtt ) , 1.25 mm protease inhibitor ) , cells were sonicated on ice 30 times for 10 s each . obtained cell lysates were centrifuged at 6000 g for 10 min at 4c , and the final protein concentration in the supernatant was determined using the bradford protein assay ( biorad , usa ) .
proteomelab pf2d protein test mix , which included 5 proteins ( ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , and cholecystokinin ( cck ) ) , was purchased from beckman coulter ( ca , usa ) . before the chromatofocusing , cell extracts
were exchanged to various start buffers using a pd-10 column ( ge healthcare life sciences , usa ) , and the first 3.5 ml fraction was collected . while the start buffer included in the proteomelab kit was designated as the start buffer a ,
start buffer b , c , and d were prepared with the following : 0.1% , 0.15% , and 0.2% triton x-100 in distilled water ( emd chemicals , inc . ,
usa ) at ph 8.4 for b , c , and d , respectively ; 6 m urea ; 25 mm bis - tris ; 1 m ammonium hydroxide .
protein concentration was estimated using quant - it protein assay kits ( invitrogen , usa ) .
all samples were diluted with each start buffer to obtain a final protein concentration of 1.5 mg / ml , and 2 ml of e. coli protein was injected into the chromatofocusing column .
all protein samples were filtered through 0.2 m pes membrane filters ( millipore , usa ) .
the chromatofocusing was performed using the proteomelab pf2d ( beckman - coulter , usa ) with an hpcf-1d column ( 250 mm 2.1 mm , eprogen , usa ) that was loaded with each starting buffer ( ph 8.5 0.1 ) for 120 min .
each starting buffer was then equilibrated to the initial ph 8.5 , and protein sample was loaded at a flow rate of 0.2 ml / min for 45 min .
the protein sample elution was initiated with a linear gradient of various elution buffers ( ph 4.0 0.1 ) that took ~60 min to complete .
eluent buffer included in the proteomelab kit was labeled as eluent buffer a. eluent buffer b , c , and d were prepared with the following : 0.1% , 0.15% , and 0.2% triton x-100 in distilled water at ph 4.0 , respectively ; iminodiacetic acid ; 6 m urea ; 10% v / v polybuffer 74 ( ge healthcare , usa ) .
proteins were eluted and collected by their isoelectric point ( pi ) in 4.08.5 range with 0.2 ph intervals into a 96-deep - plate well using the fc / i module .
remaining protein samples were finally eluted by washing the column with 1 m nacl for 40 min .
the column was then rinsed with 10 column volumes of distilled water before the next sample injection .
the entire chromatofocusing step was operated at 20c with a flow rate of 0.2 ml / min , and elution profiles were monitored at 280 nm by beckman 166 uv detector ( beckman coulter , usa ) . and the second - dimension separation was performed using hprp column ( 33 mm 4.6 mm , 1.5 m nonporous ods - iiie c18 silica beads , eprogen , usa ) at 50c with a flow rate of 0.75 ml / min .
a 200 l from the first chromatofocusing fraction was injected into the column and eluted with a 0100% linear gradient of solvent a ( 0.1% w / v tfa in distilled water ) and solvent b ( 0.08% w / v tfa in acetonitrile ) for 35 min . at the end of second - dimension run ,
proteins were detected by a beckman 166 uv detector ( beckman coulter , usa ) at 214 nm .
protein profiles obtained using uv detection were analyzed by proteovue 2d ( beckman coulter , usa ) . to investigate the elution accuracy of 0.15% triton x-100
, the mixture of five proteins purchased from beckman coulter ( usa ) was injected into the hpcf-1d column that allows the elution of proteins in ph range of 4.08.5 .
the first- and second - dimension protein separations were achieved following the standard procedure described in section 2.2 using start buffer c ( ph 8.4 ) and eluent buffer c ( ph 4.0 ) .
protein profile data obtained using uv detection were collected and analyzed by proteovue 2d software ( beckman coulter , ca , usa ) .
the eligibility and efficiency of triton x-100 as an alternative to the buffers suggested for proteomelab ( beckman coulter , california , usa ) pf2d were evaluated with e. coli , using the standard proteomelab buffer as well as solutions prepared with 0.1% , 0.15% , and 0.2% triton x-100 .
the first - dimension chromatofocusing separates proteins to differences in pi values , and the absorbance profiles created by each solution are shown in figure 1 .
proteins were eluted in the order of decreasing pi values , and all four solutions eluted its first peak during the first 20 min of sample loading period .
as the hpcf-1d column used in this study provides limited elution efficiency with proteins whose pi values are in 8.54.0 range , the first peak corresponds to protein unbound to hpcf-1d column because of its pi value being greater than 8.5 .
the details of protein separation and ph gradient formation varied among four solutions . at first
, e. coli chromatofocusing was performed using the buffer suggested for beckman coulter 's proteomelab platform .
as shown in figure 1(a ) , first unbound protein was eluted during the first ~20 min , and the ph gradient started forming at 50 min .
the ph gradient which started forming at 50 min ( from ph 8.1 ) lasted until 105 min ( to ph 3.5 ) , with a slight downward angle created at 92 min . during the ph gradient
acidic proteins which remained in hpcf-1d column were eluted after 130 min as a result of washing the column with 1 m nacl . under given conditions , beckman coulter 's standard
triton x-100 were prepared and used in place of standard buffers to perform first - dimension protein profiling of e. coli .
when the solution that included 0.1% triton x-100 was used the first protein peak created in ~20 min was inverted , giving a negative au280 reading ( figure 1(b ) ) .
this peak was followed by another inverted peak that was created during 4858 min .
when ph gradient formed during 50130 min , proteins of low pi values ( 5.0 > pi ) were eluted indistinctively .
after 130 min , remnant protein in hpcf-1d column was eluted with column washing and created a large peak area .
figure 1(c ) of 0.15% triton x-100 showed significantly improved protein profiling results : an unbound protein was eluted at < 20 min , small amounts of proteins were eluted during 2060 min ( ph 8.57.74 ) while the sample was being loaded , and a ph gradient was created during 60110 min ( ph 7.743.95 ) .
unlike 0.1% triton x-100 , 0.15% triton x-100 created a linear ph gradient , an indication that the capacity is even throughout its ph range . chromatofocusing that took place over the ph gradient
finally , 0.2% triton x-100 was used to determine whether increasing the concentration of triton x-100 would bring further improvements for chromatofocusing of e. coli .
interestingly , after the first unbound protein was eluted at < 20 min , no significant protein elution was observed until the start of the ph gradient at 50 min ( figure 1(d ) ) . as the ph gradient for 0.2%
triton x-100 was created over the shortest time frame ( during 5080 min , ph 7.113.89 ) , a high volume of proteins were eluted abruptly during ph gradient thus creating unreliable profiling data ( figure 1(d ) ) .
the performance of each buffer was judged upon its ability to achieve a well - defined ph gradient as well as an accurate pi - based protein separation at a given ph gradient range .
indistinct chromatofocusing results and negative au280 readings made 0.1% triton x-100 inadequate for pf2d . while the performance of 0.15% triton x-100 was comparable to that of standard buffers suggested for proteomelab pf2d , 0.2% triton x-100 created a protein profile whose pi values were dubious as it happened in a shorter time frame .
however , au280 readings of 0.15% and 0.2% triton x-100 were as much as 9 - 10 times higher than those of beckman coulter 's .
such high au280 readings obtained using 0.15% triton x-100 were investigated further during the second - dimension separation that was performed on 16 first - dimension protein fractions generated using the standard and 0.15% triton x-100 solution .
second - dimension of pf2d fractionates in the order of increasing hydrophobicity . as shown in figure 2(a ) , 16 protein fractions in ph 7.923.95 obtained using beckman coulter buffers were fractionized , and a thick protein band indicating a large quantity of proteins was seen around ph 5.014.70 .
the second - dimension result of 0.15% triton x-100 that began from 16 first - dimension fractions ( ph 7.743.95 ) showed distinctive bands for wider pi ranges .
this novel methodology that utilizes 0.15% triton x-100 enhances protein recovery efficiency by at least tenfold .
first- and second - dimension chromatography results shown in this study confirmed protein recovery can be increased in the presence of 15% triton x-100 during pf2d analysis of e. coli , but its reliability with regard to accurate pi separation is yet to be judged without comparing the results with the standard proteins whose pi values are known .
once again , the elution profiles of five - protein test mixture ( ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , cholecystokinin ( cck ) ) were generated using 0.15% triton x-100 ( figure 3(a ) ) .
multiple peaks were eluted in high resolution during its ph gradient . the accuracy of such protein elution was to be determined using the second - dimension protein chromatogram with a mixture of 5 proteins whose theoretical pi values are known ( figure 3(b ) ) .
the theoretical pi value of ribonuclease a is pi > 8.5 but the actual elution took over pi 8.1 - 8.0 ( shown with the protein band e in figure 3(b ) ) , possibly due to a limited ph elution range set by the hpcf-1d column .
the elution intervals of insulin , -lactalbumin , trypsin inhibitor , and cck ( shown by protein bands d , c , b , and a , resp . ) were similar to their theoretical ranges .
the detailed comparison of the experimental elution intervals of five control proteins with regard to theoretical values is summarized in table 1 .
triton x-100 is a common nonionic surfactant , and the experimental results of this study affirmed that 0.15% triton x-100 can be applied towards pf2d of a protein .
not only can 0.15% triton x-100 greatly increase amount of protein recovery from chromatofocusing column , but it also enables pf2d analysis of protein with low pi . combining the beneficial qualities mentioned thus far , 0.15% triton x-100 for pf2d system can be exploited for further analyses of metaproteome originating from various sources . | one of the main challenges in proteomics lies in obtaining a high level of reproducible fractionation of the protein samples .
automated two - dimensional liquid phase fractionation ( pf2d ) system manufactured by beckman coulter provides a process well suited for proteome studies .
however , the protein recovery efficiency of such system is low when a protocol recommended by the manufacturer is used for metaproteome profiling of environmental sample . in search of an alternative method that can overcome existing limitations ,
this study replaced manufacturer 's buffers with triton x-100 during the pf2d evaluation of escherichia coli k12 .
three different triton x-100 concentrations0.1% , 0.15% , and 0.2%were used for the first - dimension protein profiling .
as the first - dimension result was at its best in the presence of 0.15% triton x-100 , second - dimension protein fractionation was performed using 0.15% triton x-100 and the standard buffers .
when 0.15% triton x-100 was used , protein recovery increased as much as tenfold .
the elution reliability of 0.15% triton x-100 determined with ribonuclease a , insulin , -lactalbumin , trypsin inhibitor , and cholecystokinin ( cck ) affirmed triton x-100 at 15% can outperform the standard buffers without having adverse effects on samples .
this novel use of 0.15% triton x-100 for pf2d can lead to greater research possibilities in the field of proteomics . |
ivemark syndrome is a heterotaxy syndrome affecting multiple organs of the body , often presenting with congenital asplenia and cardiac problems [ 1 , 2 ] .
commonly seen as right isomerism , typically there are two right - sided lungs and right - sided atria .
the heart contains septal defects with right ventricular hyperplasia . additionally , the pulmonary venous return is irregular , long , and tortuous as it winds back to the heart .
life expectancy is heavily reduced with 10% of cases dying within the first 24 h and 80% dying within the first year of life .
these cardiac problems may include obstruction of pulmonary venous connections , seen in 48% of patients with heterotaxia and total anomalous pulmonary venous return ( tapvr ) . here , we discuss an unusual case of tapvr causing bronchial stenosis using cardiac ct angiogram to produce 3d functional figures displaying the anomalous pulmonary venous back to the left atrium .
a 3-month - year - old male with total anomalous pulmonary venous connection , a double - outlet right ventricle , congenital stenosis of the pulmonary artery valve and heterotaxy syndrome with asplenia is one of twins who presented clinically stable .
his right - sided pulmonary veins use an alternate route back to the heart as they drain into a right - sided vertical vein which passes under the carina and then back over the left main bronchus , joining the left lower lobe pulmonary vein to empty into the left atrium ( figs 1 and 2 ) . as a result ,
also , there is systemic venous communication with the left - sided vertical vein , left subclavian vein and the hemiazygos system ( figs 5 and 6 ) .
the left venous vertical vein is shown passing over the left mainstem bronchus ( yellow ) as it enters the heart .
multiple pulmonary vein branches are shown conjoining together to form the left - sided vertical vein ( white arrow ) .
it dips under the carina and loops back over the left mainstem bronchus ( yellow ) .
the left venous vertical vein is shown passing over the left mainstem bronchus ( yellow ) as it enters the heart .
multiple pulmonary vein branches are shown conjoining together to form the left - sided vertical vein ( white arrow ) .
it dips under the carina and loops back over the left mainstem bronchus ( yellow ) .
additionally , there is a clockwise rotation of the aortic root , but the coronary arteries still originate from their respective sinuses ( figs 3 and 4 ) .
there is no aortic insufficiency or stenosis , nor is there any aortic coarctation ( figs 5 and 6 ) .
he also has both a large ventricular septal defect ( vsd ) with a septum primum atrial septal defect ( asd ) , with a double - outlet right ventricle and left ventricle hypoplasia ( figs 5 and 6 ) .
figure 3:ct 3d showing anterior pulmonary arterial circulation ( blue ) . both left and right branches are nearly identical , characterized by the two right - sided lungs .
the left - sided subclavian vein ( light blue ) brings blood to the right atrium ( light blue ) .
the right ventricle ( dark purple ) provides outflow to both the aorta ( red ) and the pulmonary arteries ( dark blue ) .
the multiple branches of the pulmonary veins ( pink ) can be seen coming together and wrapping over the left mainstem bronchus .
the hypoplastic left ventricle ( white arrow ) can be seen small and shrunken slightly to the left of the large right atrium .
this view focuses primarily on the anomalous branching and route of the pulmonary venous flow in relation to other vessels and airways nearby .
the left - sided vertical vein can be seen traveling over the left bronchus ( white arrow ) .
both left and right branches are nearly identical , characterized by the two right - sided lungs .
ct 3d showing anterior heart and circulation . the left - sided subclavian vein ( light blue ) brings blood to the right atrium ( light blue ) .
the right ventricle ( dark purple ) provides outflow to both the aorta ( red ) and the pulmonary arteries ( dark blue ) .
the multiple branches of the pulmonary veins ( pink ) can be seen coming together and wrapping over the left mainstem bronchus .
the hypoplastic left ventricle ( white arrow ) can be seen small and shrunken slightly to the left of the large right atrium .
this view focuses primarily on the anomalous branching and route of the pulmonary venous flow in relation to other vessels and airways nearby .
the left - sided vertical vein can be seen traveling over the left bronchus ( white arrow ) .
ivemark syndrome is a unique form of heterotaxy , taking the right - sided form and thus resulting in no spleen development .
many times it is associated with anomalous pulmonary venous return , with the newly oxygenated blood traveling a long or ambiguous route back to the heart .
unfortunately , this kind of anomalous pulmonary venous return is different patient - to - patient contributing to the need for cardiac ct angiogram and subsequent 3d functional figures .
the concurrent septum primum asd with corresponding vsd makes this issue very difficult to correct .
due to the double - outlet right ventricle , there is a largely hypoplastic left ventricle and hyperplastic right ventricle .
both the pulmonary and systemic circulation falls upon the pumping action of the right ventricle , when typically the left ventricle handles the systemic circulation and the right ventricle handles the pulmonary circulation .
subsequently , the disuse of the left ventricle puts additional pressure on the right , increasing the pressure at which the right ventricle pumps out blood . typically , the pulmonary circulation has lower blood pressure due to the nature of the small capillaries the blood passes through .
the left ventricle usually pumps harder in order to provide circulation throughout the entire length of the body . with the right ventricle now serving as pumps for both types of circulation
, there is the possibility of pulmonary edema and poor blood perfusion throughout the body . without efficient oxygenation of all of the blood and the adequate circulation of oxygenated blood out of the heart
, the patient will likely suffer from chronic poor perfusion and shortness of breath , as well . additionally , the pulmonary veins curve over the left mainstem bronchus causing it to narrow compared to the right mainstem bronchus . with
the heterotaxy seen in this patient , both lungs are right - sided causing the same slight angle of entry into the lungs by both mainstem bronchi
. the slight narrowing of the left mainstem bronchus could lead to problems with breathing and physical activity .
as of yet , there have not been many attempts in permanently correcting these issues in ivemark patients .
apart from a bidirectional glenn operation to shunt blood from the vena cava to the pulmonary arteries and therefore bypass the right side of the heart , these patients must be kept under close observation for visible distress due to the heart condition .
| abstractivemark syndrome is a heterotaxy syndrome which affects multiple organs and affects roughly 1 in every 6000 deliveries .
specifically , it can cause total anomalous pulmonary venous return and cardiac defects , which ultimately lead to decreased life expectancy . in order to better understand the nature of cardiac structures ,
ct angiogram has been heavily relied upon as it also allows for 3d reconstruction and optimal visualization of those features .
this specific case presents with an anomalous venous return accompanied by multi - organ right isomerism that was reconstructed with 3d ct angiogram to better visualize and understand the cardiopulmonary system , as well as contribute to a fund of knowledge in hopes of discovering a solution to this condition . |
in the last few years , there has been a shift in interest within the asthma community from focusing on what features all asthmatics have in common , to the differences that set them apart.1 we now speak routinely of asthma as a heterogeneous disease which can be categorized in various ways based on such characteristics as demographics ; epidemiology ; natural history ; histopathology ; clinical manifestations ; and response to therapy
. however , some of the most important advances made recently in the characterization of asthma syndromes paint a picture of a common underlying etiology to at least the most prevalent form of the disease in children and perhaps in many adults .
viral asthma , primarily human rhinovirus - induced ( hrv ) , is by far the most common form of active disease in children , accounting for over 80% of acute exacerbations2 and the majority of hospitalizations and clinic visits . in young infants
aged under 2 years , winter viral respiratory infections play a major role in inducing the asthma phenotype.3 it appears to be less well recognized that hrv may also account for most cases of persistent asthma .
several recent studies have shed light on this topic and document that the effects of rhinovirus - triggered asthma attacks can be sustained for weeks to months following the initial upper respiratory infection ( uri).4 evidence of hrv persistence within the airways of patients5,6 and animal models,7 suggest that persistent asthma may also be a consequence of an initial or recurrent hrv - induced airway injury.8 based on these observations , and from a clinical perspective , a case can be made that at any point in time , most children and many adults with symptomatic asthma are somewhere along a continuum of partial recovery or convalescence dating back to their most recent hrv infection , with the extent of recovery being primarily dependent on the robustness of their immune response to the viral injury .
it is possible to conceptualize a cycle of injury - repair over time that would explain variations in clinical severity of the disease , modified somewhat by the effects of anti - inflammatory therapy . in some cases ,
the acute episode does lead to a brisk immune response with quick clearance of the virus and resolution of symptoms .
this would be the clinical pattern we associate with so - called intermittent asthma or children that wheeze only during acute hrv uris .
in others , the innate and adaptative immune response to the initial uri is less than adequate and fails to either clear the virus or successfully repair the damage , which then leads to persist inflammation for many weeks or months , resulting in what has been termed a
prolonged exacerbation.9 to the clinician who encounters this individual for the first time , this pattern would simply look like a multi - trigger asthmatic
if a pre - schooler , or a persistent asthmatic if an adult .
a similar mechanism was proposed some years ago by burrows10 to explain the association between frequency of acute exacerbations of chronic obstructive pulmonary disorder ( copd ) and rate of decline in forced expiratory volume in 1 second ( fev1 ) . since then , several studies have documented both the primary role of hrv in acute exacerbations of copd,11 and the relationship between exacerbations and rate of decline in lung function.12 thus , the natural histories of asthma and copd may both be linked in a sizable percentage of patients by the number and duration of hrv - induced exacerbations . in more severe asthma , we are positing that viral injury leads to the phenotypic characteristics of persistent asthma .
hrv infections tend to reoccur fairly frequently in the pediatric population , particularly during the fall through spring months .
a new uri arriving on top of a partially resolved previous infection could further overburden antiviral defenses or find an already immunologically
airway , leading to a shift in clinical pattern from a mild - persistent to a more moderate or severe one .
it has been our experience over many years of practice that when the fall outbreaks of hrv infections begin , it is typically not the first but the second or subsequent uris that trigger the more serious asthma attacks .
based on recent experimental evidence on the mechanisms of viral - induced airway injury as mediated by pattern - recognition receptors,13 this priming effect of a viral infection could have significant implications for clinical asthma .
another fall out from this paradigm is that it helps to explain why most children with asthma ( even fairly significant persistent asthma ) tend to outgrow the disease as they mature .
it is well known that from childhood into early adolescence , older children do not experience as many uris as their younger counterparts .
not only does the number of acute exacerbations quickly diminish with age , but the intervening period of recovery available between attacks is also extended .
there are also host factors , particularly in terms of viral immunity , that play a significant role in modulating an individual patient s long - term pattern , but the viral etiology of the disease remains primary .
thus the role of hrv infections should not be confined to acute exacerbations but also extended to explain chronicity of the disease .
this is supported by recent evidence from animal models of experimental respiratory viral infections that suggest that airway remodeling and hyperresponsiveness can persist long after the virus is cleared from the airways.14 whatever role other environmental factors such as allergens play in the epidemiology of asthma appears trivial when faced with the accumulating evidence for the viral paradigm.15 even previous distinctions between atopic and nonatopic asthma seem less important in light of recent endobronchial biopsy evidence that fails to distinguish between the histological presentation of atopic and nonatopic asthma.16 knowing that the host s genotype ( such as the ability to mount a robust th2 allergic inflammatory response ) plays a significant role in the viral response to injury , the primary role of hrv in driving the histopathology and clinical presentation in the large majority of asthmatics remains a valid consideration .
this idea is further supported by recent advances in the biology of antiviral th2 immune responses .
the concept of virus as allergens may well lead to more widespread recognition that in most asthmatics , whether atopic or nonatopic , the disease is closely linked to an infectious viral etiology.17
what are the implications of the role that viral infections play on the natural history of asthma for clinicians who treat asthma ? first and foremost is the concept of asthma as a constantly evolving disease , driven by the natural history of viral - induced airway injury and repair . to the clinician
, this translates to asthma being a dynamic disease consisting of periods of acute injury followed by periods of convalescence that are of variable duration and intensity .
this pattern can be further modulated by the effects of environmental triggers or by anti - inflammatory therapy .
predicting which pattern a particular patient will follow remains an elusive goal and should be the subject of further research .
we believe that one of the most important questions that should be routinely asked of patients , that is not currently included in asthma control questionnaires , is how long has it been since your last ( uri - triggered ) exacerbation ? the answer can provide a useful index of how persistent the current exacerbation has been .
the second most important question is how many upper respiratory infections , with and without worsening asthma , have you experienced in the last x number of months ? it is clear that in order to predict correctly outcomes in asthma we need clinical studies that focus on the clinical course and immunopathology of the recovery period or convalescent phase of acute asthma exacerbations .
it has recently been shown that anti - inflammatory therapy with inhaled corticosteroids has limited disease - modifying effects in asthma .
one might well expect this to be the case in a primarily virus - driven condition . in a sense
, viral asthma may be a disease of inadequate inflammation ( less than efficient immune response to viral infection ) in contrast to too much
although its clinical manifestations clearly correlate with the extent of the inflammatory processes within the airways , this response clearly fails to confine the virus to the upper airways .
thus , we are probably more likely to find success in discovering new anti - asthmatic therapies if we concentrate on antiviral agents , or ways to boost the immune response to viral spread , rather than on merely suppressing airway inflammation , whether broadly with corticosteroids or narrowly with single - path antagonists of the immune response . another key component of the concept of asthma as an acute / subacute viral disease has substantial implications for clinical trials . asthma clinical trials , at least those performed for the purpose of gaining regulatory approval , are typically designed around the premise that asthmatics can be neatly classified within a particular category of severity , from mild , to moderate , to severe .
several studies have documented that the patients that populate these categories do not remain static within a particular category for long , being classified as mild on some visits and more moderate or severe at another .
if most asthmatics are constantly evolving over time ( perhaps related to when the last viral - induced exacerbation occurred , as well as the robustness of the host response ) , it makes it difficult during the course of a clinical trial to ascribe changes in symptomatology or objective parameters of disease severity to a particular test treatment or intervention , even when the trial includes a placebo arm ( clearly impossible without one ) . since spontaneous drift in disease severity is the rule and we have no way of randomly assigning treatments based on future patterns of disease , it is not surprising that most asthma trials require large numbers of enrollees in order to minimize the effects of this variability .
this intrinsic within - subject variability may also render ethical issues of using a placebo in asthma trials moot , since no currently available therapy has been shown to alter the course of viral - induced respiratory disease and the effect of inhaled corticosteroid therapy on rate and severity of future exacerbations is modest at best.18 needless to say , patients with unstable disease should not be enrolled in placebo - control trials for obvious reasons .
in conclusion , it is generally accepted that a majority of asthma exacerbations in both children and adults are the result of viral uris .
these acute episodes are followed by a convalescent phase which can be difficult to characterize or predict in any given patient
. the rate of and extent to which recovery occurs determines for the most part the asthma severity scores of the majority of patients . the frequency of viral infections should have a major role in determining disease severity and long - term patterns .
future research can productively focus on ways of characterizing airway response to viral injury , both in the acute and convalescent phases.19 increased efforts should be directed at enhancing antiviral immune response in asthma and discovering effective and safe antiviral agents , with a vaccine against the major respiratory viral pathogens being the ultimate primary prevention strategy , although a humoral - immunity enhancing vaccine may prove futile because of the large number of hrv serotypes . | the natural history of asthma appears to be driven primarily by the timing and duration of viral respiratory infections . from the very high rate of infections in childhood , to the more sporadic pattern seen in adults , the cycle of acute injury followed by an inefficient repair process helps explain the clinical patterns of asthma severity currently recognized by asthma guidelines
. why the asthmatic host responds to viral injury in a particular way is largely a mystery and the subject of intense investigation .
the role of viruses in asthma extends not just to intermittent but to persistent disease , and to both the atopic as well as nonatopic phenotypes .
future therapeutic strategies should include primary prevention via the development of antiviral innate immunity - enhancing vaccines , as well as secondary prevention via the use of antiviral agents , or immunomodulators designed to boost the antiviral response or interrupt the proinflammatory cascade . |
synoptic reporting of all tumor excisions is required by the college of american pathologists ( cap ) .
the cap specifically requires that each element in a synoptic report be reported in a required data element ( rde ) pair consisting of the element and the corresponding response ( cap laboratory accreditation process checklist question anp .
however , recent studies suggest that specific formatting features are associated with not only the accuracy of the information entered into the report , but also user preference , accuracy of information retrieval , and speed of information retrieval .
currently , cap requires that each element of the gleason grading system ( primary pattern , secondary pattern , tertiary pattern , and total score ) be reported on separate lines as separate and distinct rdes , despite the fact that in the biopsy setting these elements are reported on one line as primary pattern + secondary pattern = total score with tertiary pattern on a separate line if and only if it is present . whether these or other formats might affect the reader 's experience is not known .
we sought to determine if such differences in reporting formats would affect the speed or accuracy of information retrieval by nonpathologists reading synoptic reports of prostatectomy specimens .
to test the accuracy and speed of identification of specific data elements in a synoptic report , a python script was written that provided instructions and a test platform for these quizzes .
specifically , the participant is shown a specific phrase that may or may not be in a synoptic report ( with exceptions listed below ) .
when the user presses enter the synoptic report appears on the screen and the timer starts .
the user then examines the report to determine if the phrase is or is not present .
if it is present , they enter the number two , if it is not they enter 1 and then press return .
the program automatically records the time and whether the answer was correct , and this data is then transferred to a comma separated values file for further analysis .
we constructed our synoptic report for a radical prostatectomy using the checklist from cap , [ figure 1 ] for the standard format .
all elements were identical except for changes in the reporting of gleason grade ( primary grade , secondary grade , tertiary grade , and total score ) .
the formats were tested in three different quizzes , each given in sequence and at the same sitting .
the first quiz contained 36 total questions and compared the standard format [ figure 1 ] with a format in which if there was no tertiary grade the line concerning tertiary grade was omitted [ figure 2 ] . in the standard format ,
the response for tertiary grade could be 3 , 4 , 5 , or not applicable .
, where ? could be 3 , 4 , 5 , or not applicable .
standard format synoptic report tertiary grade omitted because it was not applicable / present quiz two contained 32 questions and compared the standard format [ figure 1 ] with a biopsy format , [ figure 3 ] , where gleason grades are reported as primary pattern + secondary pattern = total score on one line and tertiary pattern is on a separate line . although this format is taken from and labeled as
biopsy format , for this quiz , the total score was calculated as it is for prostatectomies where primary plus secondary regardless of the tertiary pattern ( also regardless of whether it was < 5% or > 5% of tissue ) which was reported separately . the question for this part could pertain to any of the 4 elements ( primary pattern , secondary pattern , total score , and tertiary pattern ) , and the answers could be any of 3,4,5,6,7,8,9 , or 10 ( not applicable was not included ) . in order for the participant to know which format to expect , 16 questions with the standard format were presented and then 16 questions with the biopsy format . within each group ,
the order of questions were randomized for each participant , but each participant got the exact same questions for each group .
biopsy format synoptic report quiz three contained 32 questions and compared the standard format [ figure 1 ] with a biopsy format , [ figure 4 ] , where gleason grades are reported as primary pattern + secondary pattern + ( tertiary pattern ) - > total score on one line .
the question for this part could pertain to any of the four elements ( primary pattern , secondary pattern , total score , and tertiary pattern ) , and the answers could be any of 3,4,5,6,7,8,9 , or 10 ( not applicable was not included ) . in order for the participant to know which format to expect , 16 questions with the standard format were presented and then 16 questions with the biopsy format . within each group ,
the order of questions were randomized for each participant , but each participant got the exact same questions for each group .
they were all nonpathologists and included , five cancer registrars , four mds ( all internists ) , four non - md medical personnel ( all laboratory technologists ) , and three nonmedical personnel ( administrative assistants , other professionals ) .
we specifically excluded pathologists from this testing , since we wanted to measure the performance of a user other than a pathologist . similarly , although we have no evidence that different types of readers perform differently on these tests than any other type of reader , like pathologists we did not include urologists to ensure that their particular set of knowledge and experience did not influence the results .
although , previous studies have suggested that in this type of test format there were no significant differences between these different users in terms of accuracy or time to retrieval of information , this was again tested in the current study .
previously we had noted that participants got faster from quiz 1 to quizzes 2 and 3 as they got practice with the test . as a result
, no comparison was made between the three quizzes , and all three quizzes were always taken in the same order .
in addition , there was a wide range of speed for the different users . in order to allow comparison between these uses ,
as a result , the normalized time for the standard format was the control with a normalized time of one , and the time for all other formats was in comparison with that time .
statistical analysis was performed using a t - test or fishers exact test as appropriate with a significance threshold of 0.05 .
as shown in table 1 , the nonmedical group had a significantly higher accuracy rate than the cancer registrars ( p = 0.02 ) .
as a result , any significant findings related to accuracy using grouped data also underwent subset analysis .
there was no significant difference in normalized times between any of the four user groups , [ table 2 ] .
accuracy for different users compared to cancer registrars normalized time for all questions for different users compared to cancer registrars when tertiary grade was omitted whenever the result was not applicable , the accuracy of classification by the readers was significantly lower than when tertiary grade was listed [ 72 vs. 97% , p < 0.001 table 3 ] .
0.001 ) were evaluated as well as when all other users were evaluated ( 70% vs. 97% , p < 0.001 ) .
in addition , when tertiary grade was omitted , the time to answer the question increased significantly ( 63% , p < 0.001 ) [ table 4 ] .
accuracy for different formats compared to standard format normalized time compared to standard format when the standard format ( all four elements each on a separate line ) was compared with the biopsy format ( primary + secondary = total , with tertiary on a separate line ) , there was no difference in accuracy of data extraction ( 98 vs. 97% , p = 0.56 ) , however , the time needed to extract the data decreased 18% with the biopsy format ( p < 0.001 ) . nevertheless , no user preferred the biopsy format . when the standard format was compared with a single line format ( primary
+ secondary + ( tertiary ) - > total ) , the accuracy increased from 97% to 100% ( p = 0.02 ) using the single line format .
subset analysis showed that the group with the biggest difference were the registrars ( 6/7 errors with the standard format ) . the time to extract the data also decreased 24% ( p < 0.001 ) with the single line format .
nine users preferred the standard format and seven the single line format , even though they were more accurate and faster with the single line format .
the data in this report suggest three major conclusions : removing not applicable elements from a synoptic report can reduce both the accuracy and speed of information retrieval , combining all elements of gleason grading on one line significantly improves the accuracy and speed of information retrieval from synoptic reports , and users preferences may not always correlate with either accuracy or speed .
these results were similar across a wide variety of different users , just as they were in our previous studies .
. one of the original intents of synoptic reporting was to have one data element per line , making automated data extraction easier .
however , as the tools available for data extraction have evolved , and as the field has moved to data extraction before generating the pathology report , this restriction may not be as important as it has been in the past .
in particular with gleason grading , the use of + and = signs makes regular expression extraction of multiple pieces of information from a single line both easy and routine . in this context , other factors , such as end users ability to quickly and correctly extract information from the report may be of increased value .
it is not a surprise that removing elements form a synoptic report reduces the speed of identification of such elements .
it has previously been documented that having a reproducible format where all elements are always included in exactly the same order has been associated with faster information identification .
when not applicable elements are missing , the reader has to first ensure that indeed they are missing before they can then conclude that they are not applicable .
however , we were surprised that removing items from the synoptic format also reduced the accuracy of the response .
nevertheless , in constructing synoptic reports , there are many opportunities where omitting not applicable elements would reduce the length of the report , a feature that has previously been shown to be associated with decreased error in constructing the synoptic report . whether the benefit in terms of error by making the report shorter outweigh the reduced accuracy of interpretation by leaving out this information is not clear at this time .
it is also not a surprise that combining all parts of gleason grading into one line is associated with faster information retrieval .
however , in the standard format , the information ( response ) in those four separate lines consists only of a number and the label that explains the number ( rde ) is separated from it on the other side of the page . in discussing this with the participants , they felt that the reason that the single line was faster was not just because it was a single line , but also because they could simply look down the right - hand side of the report ( the responses ) to find the grading section without referring to any rde .
this was because the format of the grading information had a structure that was easily identifiable , unique from all other responses in the report , and conveyed information without having to refer to the listed rde .
thus , having a unique structure to the response and not just the rde is a key part of improving the speed of information retrieval from a synoptic report .
this hypothesis is also consistent with our previous studies that examined the effect of different formats .
nevertheless , the format of reporting gleason patterns / grades and scores has been the subject of considerable discussion previously .
most importantly , the reasons one may prefer one format over another is multifaceted , and the information in the current study is just one aspect of this discussion .
the information we present may be of value in future discussions by pathologists and organizations such as the international society of urologic pathologists who make recommendations using all of the different aspects that affect reporting of gleason grading / scoring . at present , though , current recommendations make a distinction between reporting formats for biopsies and prostatectomies .
specifically , in the biopsy specimen if the tertiary pattern is high grade then that is reported as the secondary pattern rather than the true secondary pattern .
this is done to ensure that the presence of this tertiary pattern is not overlooked by either clinicians or other readers , who may simply omit or skip the presence of a high - grade tertiary pattern if it is only reported in a note .
while the rationale for this practice is clear , it does bring up two issues .
secondary pattern or a high - grade tertiary pattern , this form of reporting is subject to information loss . specifically ,
if one reads a report with a gleason score of 3 + 5 , one can not tell from the report whether there are only two patterns ( 3 and 5 ) present in this biopsy , or whether this is a biopsy with patterns 3 , 4 and a tertiary pattern 5 .
this may in part explain some of the controversy associated with assigning gleason score 3 + 5 cases in the newly proposed gleason groups .
more importantly , there is an assumption in this analysis that the only other way to report tertiary grades is as a separate note . as our study clearly shows , this is not true
this study uses just one of many possible options for reporting all four elements on one line . while the order is intuitive ( primary secondary tertiary and total in that order ) the signs that are used to distinguish between the different patterns are not and
could be changed if different signs were identified that could convey more information than the signs we have used here . obviously using a plus signs and an equal sign implies that the numbers will add up , and in this format that is not true , and is the reason why we put tertiary patterns in parentheses .
using a grade of 0 may also be easier to interpret than using the phrase not applicable or
finally , recent studies have also suggested that the percentage of high - grade tumor should also be reported as this may also be prognostically significant as well as gleason groups . whether and how this information may be incorporated into a single line format is not clear at this time .
nevertheless , the need to begin reporting gleason groups is an opportunity to re - address the most effective format for reporting all elements in gleason grading .
first data recognition and user preference are just a few elements of the user interaction with a synoptic report .
indeed , several of the nonmedical participants noted that while they could perform the study , they really did not understand the meaning of the phrases they were looking for .
this in part explains why we were unable to find a consistent difference in the performance between our different user groups this test measures the users ability to read and identify information , regardless of their understanding of that information .
other measures that focused on comprehension would most likely find significant differences between these different types of users .
different report formats for gleason grading significantly affect users speed , accuracy , and preference ; users do not always prefer either speed or accuracy .
the data and study design we describe may be useful in deciding between different format options .
| context : the format of a synoptic report can significantly affect the accuracy , speed , and preference with which a reader can retrieve information.objective:the objective of this study is to compare different formats of gleason grading / score in synoptic reports of radical prostatectomies.methods:the performance of 16 nonpathologists ( cancer registrars , mds , medical non - mds , and nonmedical ) at identifying specific information in various formatted synoptic reports using a computerized quiz that measured both accuracy and speed.results:compared to the standard format ( primary , secondary , tertiary grades , and total score on separate lines ) , omitting tertiary grade when not applicable reduced accuracy ( 72 vs. 97% , p < 0.001 ) and increased time to retrieve information 63% ( p < 0.001 ) .
no user preferred to have tertiary grade omitted .
both the biopsy format ( primary + secondary = total score , tertiary on a separate line ) and the single line format ( primary + secondary + ( tertiary ) - > total score ) were associated with increased speed of data extraction ( 18 and 24% , respectively , p < 0.001 ) .
the single line format was more accurate ( 100% vs. 97% , p = 0.02 ) .
no user preferred the biopsy format , and only 7/16 users preferred the single line format.conclusionsdifferent report formats for gleason grading significantly affect users speed , accuracy , and preference ; users do not always prefer either speed or accuracy . |
in may 2009 , diarrhea and fever developed in 8 persons living in southwestern france 1 day after they ate a homemade tiramisu prepared with raw eggs . fecal analysis was performed on samples from 5 of the 8 persons .
the isolation was performed by using standard procedures ( i.e. , use of conventional selective media ) .
isolation from the food sample was performed as required by the current international organization for standardization iso 6579:2002 ( i.e. , by 2 selective enrichment media ) ( 9 ) .
enterica 4,5,12::. an investigation at the suspected layer farm was conducted and showed the presence of 11 nonmotile salmonella spp .
isolates ( with the same antigenic formula ) in dust and feces collected from laying - hen houses .
the layer farm , located in northwestern france , is a major farm that produces > 32,000,000 eggs per year .
all 17 isolates ( 5 from humans , 1 from the tiramisu , and 11 from the laying hens ) were pan - susceptible to all antimicrobial drugs tested ( 10 ) .
analysis by pulsenet ( www.cdc.gov/pulsenet/protocols.htm ) standardized xbai pulsed - field gel electrophoresis ( pfge ) showed an indistinguishable profile , xtym-1 , associated with the multidrug - resistant s. enterica serotype typhimurium dt104 clone ( 10,11 ) ( figure a1 ) .
multilocus variable number tandem repeat analysis ( mlva ) typing ( 12 ) showed an unique type 314721311 ( loci sttr956103 , respectively ) in 16/17 isolates and a single - locus variant ( differing by 1 repetition ) for the remaining isolate , which was an environmental isolate collected 1 month after the control measures were implemented ( slaughter of laying hens ) ( figure a1 ) .
the tiramisu isolate 09ceb3100 was also characterized by multilocus sequence typing ( mlst ) for the flic and fljb genes encoding the flagellar antigens ( 13 ) .
the isolate belonged to mlst sequence type 19 , the main sequence type of serotype typhimurium ( http://mlst.ucc.ie/mlst/dbs/senterica ) .
pcr and sequencing identified the flic gene encoding the i antigen and the fljb gene encoding for the 1,2 antigen , confirming that this salmonella spp .
the investigation and molecular data concluded that a nonmotile variant strain of serotype typhimurium , genetically distinct from the emerging monophasic variant described worldwide , has been circulating in laying hens , whose contaminated eggs had likely caused food poisoning . during 20002009 , a total of 108,362 serotyped salmonella spp .
isolates from humans were registered at the fnrc - salm , of which 374 ( 0.3% ) were nonmotile .
such 1,4,,12:: strains have been rarely reported in food , environment or animals with 166 registered isolates collected by the french food safety agency salmonella network during 20012009 ( compared with 21,214 serotype typhimurium and 157,885 salmonella spp .
was circulating , we studied 43 additional s. enterica serotype 1,4,,12:: strains : 22 human and 21 nonhuman strains ( 5 from laying hens , 6 from cattle , 2 from sheep , 2 from partridges , 1 from chickens , 3 from the environment , and 3 from foodstuffs ) isolated during 20012009 .
these strains were selected on the basis of their diversity ( geographic area , year of isolation , and source ) .
all the strains were characterized by antimicrobial drug susceptibility testing , pfge , mlva , and by flic and fljb genes sequencing .
the 60 s. enterica serotype 1,4,,12:: isolates studied ( 17 isolates linked to the food poisoning and 43 additional isolates ) displayed 22 different pfge profiles . only 10 profiles , from 49 isolates ( 81.6% ) , matched with profiles from the fnrc - salm pfge typhimurium database ( which includes 207 profiles generated in routine surveillance on 632 strains isolated during 19812010 ) .
all 49 strains were confirmed as nonmotile variants of typhimurium by the identification of the flic gene encoding the i antigen and the fljb gene encoding for the 1,2 antigen .
the 11 strains whose pfge pattern did not match any typhimurium pfge profile in our database belonged to serotypes typhimurium ( corresponding to 4 new pfge profiles ) , paratyphi b ( n = 3 ) , agona , derby , indiana , and saintpaul ( 1 each ) . among the 49 strains with a xtym profile , 37 ( 75% ) belonged to xtym-1 ( figure a1 ) .
the nonmotile xtym-1 strains were divided into 2 groups regarding their susceptibility to antimicrobial agents : pan - susceptible for those ( 17 ) linked to the food poisoning plus 6 strains isolated before 2009 ( 3 human and 3 nonhuman isolates ) and penta - resistant profile ( resistant to amoxicillin , streptomycin / spectinomycin , sulfonamide , chloramphenicol , and tetracycline ) for the 14 remaining strains .
notably , all 23 nonmotile xtym-1 pan - susceptible strains had a single amino acid substitution in the flic gene ( asp251 encoding an asparagine residue ) compared with those available in public database , including serotype typhimurium reference strain lt2 .
xtym-1 profiles exhibited 7 pfge profiles , suggesting the loss of motility was acquired independently by 7 distinct serotype typhimurium populations .
the mlva type 3147 - 21311 found in the food poisoning strains was not observed in the other strains tested .
a minimum - spanning tree based on the mlva types is shown in the figure .
the strains from the food poisoning clustered at the external extremity of a tree branch , whereas 3 human and 3 laying hen strains ( from the same producer ) isolated earlier ( 20052007 ) clustered at the internal extremity of the same branch .
two laying - hen strains isolated in 2009 ( also from the same producer ) were grouped in the interconnecting node .
this finding , combined with antimicrobial drug susceptibility testing , pfge , and flic sequencing data , suggested that the 2009 strain causing the food poisoning is a derivative of the 2005 strain ; both strains were isolated from the same egg producer during a 4-year interval .
regarding the european directive and the commission regulation on the monitoring and reduction of zoonotic agents ( 14 ) , the french regulation has extended the target for reduction of prevalence of salmonella spp . in poultry producers to include notification of monophasic ( because of the recent emergence in humans ) and nonmotile ( because of this food poisoning ) variants of typhimurium after january 2010 ( 15 ) .
this food poisoning outbreak also highlighted the need for a second selective enrichment media for salmonella spp .
detection not based on the motility in complement to the modified semisolid rappaport - vassiladis medium recommended as a single medium by the european directive .
we report a foodborne outbreak caused by a nonmotile s. enterica 4,5,12:: strain in france .
this strain has been present in laying hens in france for the past decade . despite continuous advances in food safety and disease surveillance , control , and prevention , atypical pathogenic salmonella spp . | we report a food - related outbreak of salmonellosis in humans caused by a nonmotile variant of salmonella enterica serotype typhimurium in france in 2009 .
this nonmotile variant had been circulating in laying hens but was not considered as typhimurium and consequently escaped european poultry flock regulations . |
the metabolic syndrome has become a major public health challenge with an estimated 22% of us adults having this condition .
a consensus group for the international diabetes federation defines metabolic syndrome as central obesity , plus any two of the following : raised triglycerides , reduced high - density lipoprotein ( hdl ) cholesterol , raised fasting plasma glucose , and raised blood pressure .
the consensus group also recommends additional criteria that should be part of further research into the metabolic syndrome including tomographic assessment of visceral adiposity and liver fat , biomarkers of adipose tissue ( leptin , adiponectin ) , and glucose tolerance testing .
the cause of the syndrome remains obscure but the pathophysiology seems to be largely attributable to insulin resistance , excessive flux of fatty acids , and a chronic proinflammatory state .
therapeutics include lifestyle changes ( e.g. , weight reduction and increased physical activity ) and pharmaceutical agents , but prevention would be preferred .
a mounting body of evidence indicates that certain adverse exposures during the perinatal period contribute to the development of the metabolic syndrome .
this early life stage may offer an attractive point in the disease process for prevention and intervention strategies .
in the 1970s forsdahl , using official statistical data on norwegian counties , reported that poverty during adolescence , followed by prosperity , was positively correlated with risk of death from coronary heart disease .
although no biological mechanisms were identified , forsdahl speculated that some form of permanent damage caused by the nutritional deficit may be involved . in 1986 , barker and colleagues began publishing reports on the association between an adverse intrauterine environment , as determined primarily by low birth weight , and an increased risk of coronary heart disease later in life .
these studies involved examining men and women in middle and late life whose body measurements at birth were recorded in the archives and records offices of britain [ 5 , 6 ] . upon further investigation , it was found that the correlation between low birth weight and heart disease was also present between low birth weight and type 2 diabetes with the prevalence of impaired glucose tolerance and type 2 diabetes falling progressively with increasing birth weight .
moreover , in a follow - up study , an association between birth weight and the presence of metabolic syndrome was discovered . in this
study metabolic syndrome was defined as impaired glucose tolerance , hypertension , and elevated triglycerides . in both men and women ,
of the 64-year - old men whose birth weights were 2.95 kg ( 6.5 pounds ) or less , 22% had metabolic syndrome and their risk of developing the syndrome was more than 10 times greater than that of men whose birth weights were more than 4.31 kg ( 9.5 pounds ) [ 8 , 9 ] .
the association between metabolic syndrome and low birth weight prompted the authors to suggest that the metabolic syndrome , also called syndrome x , be renamed the small - baby syndrome .
collectively , these studies generated the forsdahl - barker hypothesis , recognizing forsdahl as the original source of the idea and barker as the developer of the concept .
in the decades following their initial publications , the forsdahl - barker hypothesis has become more well known as the fetal origins hypothesis and has produced a new branch of scientific knowledge and inquiry known as the developmental origins of health and disease ( dohad ) .
historically , investigations on the fetal origins hypothesis have focused on maternal undernutrition and specific nutrient deficiencies .
today , the world faces the dual burden of malnutrition that encompasses both under- and overnutrition .
it is estimated that maternal and child undernutrition is the underlying cause of 3.5 million deaths globally .
while at the same time overweight and obesity are major public health challenges in both developed and developing regions of the world . in 2005 , 33% of the world 's adult population ( 1.3 billion people ) was overweight or obese , and it is estimated that by 2030 , up to 57.8% of the world 's adult population ( 3.3 billion people ) will be overweight or obese . adding to the complexity of the dual burden of malnutrition is the influence of socioeconomic status . by and large , in developed countries lower socioeconomic status is associated with both lower birth weights among the offspring of women and a higher prevalence of obesity among women . as a result ,
failure to control for social position could obscure a relationship between high birth weight and subsequent obesity and obesity - related disorders .
incidentally , although the vast majority of the literature concerning the dohad is focused on the relationship between small size at birth and increased incidence of disease in adult life , it is now recognized that higher incidences of disease occur in both those born small and those born large , thus reflecting a u - shaped curve [ 15 , 16 ] .
this paper provides a brief overview of the putative concepts and mechanisms behind the dohad and assembles thoughts on how alterations in maternal consumption of specific nutrients may irreversibly direct fetal programming of the metabolic syndrome ( figure 1 ) .
developmental plasticity is the ability of an organism to change its phenotype in response to changes in the environment .
if this change or adaptation is permanent , it is considered a programming change and is associated with persistent effects in structure and/or function [ 18 , 19 ] .
gluckman and hanson use the example of the meadow vole ( microtus pennsylvanicus ) to illustrate programming .
meadow moles born in autumn have a thicker hair coat than those born in the spring .
this occurs as a response of the fetus to maternally derived signals for day length .
a thick coat reflects an adaptive programming response to help ensure survival in the cold environment that was predicted while in utero .
humans have similar numbers of sweat glands at birth , but they are essentially nonfunctional . in the first few years of life a proportion of the glands become functional depending on the temperature to which the child is exposed
. the hotter the conditions , the greater number of functional sweat glands . after a few years
programming changes can also be experimentally - induced as demonstrated by examination of adult ( 100-day - old ) female rats that were given a single subcutaneous injection of testosterone at 2 , 5 , 10 , or 20 days of age .
those that were injected between birth and 10 days of age had lasting changes in organ weight , specifically larger adrenal and pituitary weights and smaller ovarian and uterine weights , as well as absent corpora lutea , failure to develop normal female sexual behavior , and permanent sterility [ 22 , 23 ] .
theses aberrations in morphology and physiology were much reduced or absent in those rats injected at 10 or 20 days of age , indicating that the period of plasticity during which rats are androgen sensitive is between birth and 10 days of age . in most cases programming
however , the problem of mismatch occurs when individuals developmentally adapted to one environment are exposed to another . revisiting the functional activity of the sweat glands to provide an example ,
the child who has experienced hot conditions will be better adapted to such conditions in later life because more functioning sweat glands provide the ability to cool down faster .
while the child who has experienced cool conditions faces the problem of mismatch and will be unable to cool down as easily in hot conditions because of fewer functioning sweat glands .
other examples of this mismatch phenomenon include people whose birth weights were towards the lower end of normal that subsequently grow up in affluent societies being at increased risk for hypertension , type 2 diabetes , and cardiovascular disease [ 16 , 25 ] .
the problem of mismatch is thought to be involved in the current epidemic of type 2 diabetes and cardiovascular disease in the young adult and middle - aged populations of india .
several countries , including india , are undergoing swift economic and nutritional transitions , exposing individuals to conditions that promote weight gain .
the current surge in metabolic and cardiovascular disease in india may be being fueled by a combination of undernutrition in early life and overnutrition in later life . in a study of indian children , those born small and where relatively fat and tall by 8 years of age had the most adverse risk profiles for cardiovascular disease , including components of the metabolic syndrome .
the authors commented on the possible importance of preventing childhood obesity in the prevention of disease in the low birth weight members of the indian population .
it is important to note that indians as a race are more prone to the development of the metabolic syndrome , compared to caucasians , as a result of having a phenotype of high fat mass and low lean mass .
the thrifty phenotype hypothesis , put forth by hales and barker , proposes that poor fetal and early postnatal nutrition imposes mechanisms of nutritional thrift upon the growing individual . in conditions of severe intrauterine deprivation
the developing fetus may lose functional and structural units such as pancreatic cells , nephrons , and cardiomyocytes .
such changes have been deemed an adaptive mechanism to ensure the survival of the fetus .
the thrifty phenotype hypothesis is based on a study of 468 men in which the percentage of those with impaired glucose tolerance or type 2 diabetes fell progressively with increasing birth weight and weight at 1 year . from this research
it was hypothesized that poor intrauterine nutrition results in the growth of vital organs , specifically the brain , at the expense of other organs ( i.e. , endocrine pancreas resulting in cell hypoplasia ) .
such adaptations may increase the chance of fetal survival by means of brain sparing but result in difficulty in coping with nutritional abundance as an adult .
fetal salvage hypothesis which offers a different explanation for the insulin resistance seen in those affected by intrauterine growth restriction . in this alternate explanation
it is not hypoplasia of the pancreatic cells that leads to impaired glucose tolerance , but rather it is that the fetus develops peripheral insulin resistance .
it is this peripheral insulin resistance that ensures that adequate amounts of glucose are delivered to essential organs such as the brain with subsequent reduced delivery to nonessential tissues such as skeletal muscle .
support for the fetal salvage hypothesis comes from intrauterine growth restriction studies in rats . in these rodent models ,
glucose transport ( glucose uptake and glucose transporter mrna and protein levels ) is reduced in lung and skeletal muscles of growth - restricted fetuses , whereas glucose transport is unaffected in brain [ 32 , 33 ] .
the kidney is another organ thought to be affected by nutritional thrift . in both human and animal studies
small offspring , due to maternal undernutrition and other causes , have reduced numbers of nephrons which has been strongly linked to hypertension [ 16 , 34 , 35 ] . in the human ,
nephrogenesis is completed in utero , and no new nephrons are formed after birth ; therefore a nephron deficit present at birth persists throughout life [ 29 , 36 ] . in a study of human infants affected by intrauterine growth restriction ,
a similar study found a 20% reduction in nephron number in human neonates with low birth weights as compared to normal birth weight neonates .
additionally , researchers found a positive relationship between weight at birth and the number of glomeruli as well as a negative correlation between weight at birth and glomerular volume .
this reduction in nephrons may reflect an adaptation that has the immediate advantage of energy and resource conservation but no long - term advantages [ 16 , 34 ] .
catch - up growth , also known as compensatory growth , is where children return to their genetic trajectory for size after a period of growth delay or arrest .
it may occur at any stage of growth but is most commonly observed in the first 2 years of life .
studies have found that catch - up growth often results in overcompensation , whereby the organism exceeds normal weight and often has excessive fat deposition .
this rapid and excessive growth has been associated with the development of adult obesity , insulin resistance , metabolic syndrome , and type 2 diabetes [ 3840 ] . in the avon longitudinal study of pregnancy and childhood ( alspac ) birth cohort ,
children who showed catch - up growth , as calculated by specified gains in standard deviation scores , between 0 and 2 years of age , were heavier , taller , and fatter ( body mass index , percentage body fat , and waist circumference ) at 5 years of age than other children .
a study that evaluated glucose tolerance and plasma insulin concentrations in more than 1400 young adults ( 26 to 32 years old ) who had grown up in the city of delhi , india , found an association between thinness in infancy and the presence of impaired glucose tolerance or diabetes in young adulthood .
as a group , the young adults in the study who had impaired glucose tolerance or diabetes were overweight . they were not , however , overweight or obese in childhood . instead , they were characterized by their high rate of gain in body mass after the age of 2 years .
cianfarani and colleagues speculate that the tremendous effort to recover lost growth shortly after birth involves insulin and insulin - like growth factors ( igf ) .
infants affected by intrauterine growth restriction have low concentrations of insulin and igf-1 at birth , and normalization of these parameters occurs in the postnatal period [ 42 , 43 ] .
it is thought that tissues chronically depleted of insulin and igf-1 throughout fetal life and then suddenly exposed to increased concentrations of the two hormones shortly after birth may counteract the actions of insulin by developing insulin resistance .
thus , in this proposed scenario , insulin resistance is serving as a metabolic defense mechanism to protect the organism against hypoglycemia .
in addition to insulin resistance and type 2 diabetes , accelerated weight gain in early life has been associated with elevated blood pressure [ 44 , 45 ] and coronary heart disease .
the implication of catch - up growth is that rapidly enhancing early childhood growth by a nutrient enriched diet may cause harm overtime and that encouraging slower growth rates may actually be beneficial . in a study that provides evidence of the benefits of steady ( i.e. , not accelerated by dietary means ) growth , investigators measured fasting concentrations of 32 - 33 split proinsulin , a marker of insulin resistance , in adolescents born preterm who had participated in randomized intervention trials of neonatal nutrition .
fasting 32 - 33 split proinsulin concentration was greater in those given a nutrient - enriched diet than those given the lower - nutrient diet and was associated with greater weight gain in the first 2 weeks of life .
excessive reactive oxygen species can cause modulation of gene expression and/or direct damage to cell membranes and other molecules at critical developmental windows .
many believe that oxidative stress is the primary link between adverse fetal growth and later elevated risks of the metabolic syndrome , type 2 diabetes , and other disorders .
smoking , hypertension / preeclampsia , inflammation / infection , obesity , and malnutrition are common causes of preterm and/or low birth weight as well as known sources of oxidative stress .
malnutrition can directly lead to a pro - oxidative state by means of creating protein and micronutrient deficiencies .
proteins provide amino acids needed for antioxidant synthesis , such as glutathione and albumin , and many micronutrients themselves are antioxidants , such as vitamins a , c , and e [ 40 , 48 ] .
pancreatic cells are particularly sensitive to reactive oxygen species because they are low in enzymatic antioxidant defense equipment .
it has been demonstrated that oxidative stress can blunt insulin secretion . with the susceptibility of pancreatic cells to oxidative stress
, it is believed that early and ongoing exposures to oxidative insults could result in the eventual manifestations of the metabolic syndrome and related disorders .
elevated oxidative stress has been observed among human infants born small for gestational age ( sga ) as compared to those appropriate for gestational age ( aga ) and among preterm as compared to term infants .
it is important to note that preterm ( birth at < 37 weeks of pregnancy ) infants are generally of low birth weight but are not necessarily growth restricted or small for gestational age .
a study using cord blood to compare the status of oxidative stress between sga infants born to undernourished mothers and aga infants born to healthy mothers found elevated oxidative stress , as determined by increased quantities of malondialdehyde ( one of the major products of lipid peroxidation ) , reduced quantities of the antioxidant glutathione , and decreased activity of the antioxidants superoxide dismutase and catalase in the sga infants as compared to the aga controls .
a human study determined vitamin a , c , and e levels in the umbilical cord blood of term and preterm infants and the blood of their mothers taken at the time of delivery .
maternal vitamin a and e levels were higher than cord values , yet there was a positive correlation between maternal and cord levels of these two vitamins .
a and e , cord vitamin c levels were higher than maternal levels , and no significant correlation between cord and maternal vitamin c levels was present .
in comparing vitamin levels in term and preterm infants , term babies had significantly higher levels of vitamins a , c , and e. the authors concluded that for vitamins a and e , neonatal levels are dependent on maternal levels and that preterm babies have fewer lipid - soluble antioxidant vitamins in their serum compared to term infants thus may be more susceptible to oxidative stress . gestational diabetes , a common cause of macrosomia , is associated with oxidative stress which could have effects on the developing infant .
a study evaluating oxidative and antioxidative status in pregnant diabetic ( gestational diabetes or type 1 diabetes ) women between 26 and 32 weeks gestation demonstrated increased oxidative stress in those who were diabetic .
levels of malondialdehyde were significantly higher , and vitamin a and e levels were significantly lower in all diabetic women than controls . moreover , glutathione peroxidase and superoxide dismutase activities were significantly reduced in diabetic women ; glutathione peroxidase was reduced in both those with gestational and type 1 diabetes , and superoxide dismutase was reduced only in type 1 diabetics .
an increase in circulating glucocorticoids may play a role in early programming of adult disease .
experimental studies in animals and epidemiological studies in humans have suggested an altered set - point of the hypothalamic - pituitary - adrenal ( hpa ) axis as an important long - term change that occurs in association with reduced fetal growth .
pregnant animal models have shown that exposure to a variety of stressors , including nutrient restriction , results in the birth of offspring with elevated basal or stress - induced glucocorticoid secretion [ 54 , 55 ] .
it is thought that maternal exposure to stressors during pregnancy subsequently leads to excessive fetal exposure to glucocorticoids resulting in persistent alterations in hpa axis activity . in support of this hypothesis
are studies conducted in rats in which fetoplacental exposure to maternally administered steroids throughout gestation reduced birth weight and produced hypertensive adult offspring .
in one such study , dexamethasone administration to pregnant rats on days 1520 of gestation resulted in offspring with reduced birth weight , elevated blood pressure , increased basal plasma corticosterone , lower mrna expression of hippocampal neuronal glucocorticoid receptor , and decreased gene expression of hippocampal mineralocorticoid receptor . in a sheep study , dams were treated with dexamethasone over 2 days ; treatment group 1 was treated during 2229 days of pregnancy and treatment group 2 was treated during 5966 days of pregnancy ( term 145 days ) .
offspring from dams that had received dexamethasone during 2229 days gestation , but not days 5966 of gestation , had elevated blood pressures . such studies suggest that excess glucocorticoid exposure at certain developmental stages or windows programs higher blood pressure .
another study of pregnant sheep found that severe brief undernutrition in late gestation altered the function of the hpa axis of adult offspring .
those exposed to gestational undernourishment for 10 days demonstrated altered steroid levels including an increased adrenocorticotropic hormone ( acth ) response as compared to offspring from dams fed ad libitum or offspring from dams undernourished for 20 days .
an epidemiological study in humans involving three populations found that adults who had lower birth weights had elevated fasting plasma concentrations of cortisol and that cortisol concentrations positively correlated with current blood pressure .
the authors concluded that increased activity of the hpa axis may link low birth weight with raised blood pressure in adult life . in a study of over 300 men ,
plasma cortisol concentrations fell progressively with increasing birth weight ; this trend was independent of age and body mass index .
raised plasma cortisol concentrations were significantly associated with higher blood pressure as well as plasma glucose concentrations and insulin resistance , suggesting that programming of the hpa axis may be a mechanism underlying the association between low birth weight and the metabolic syndrome in adult life .
the hypothalamus plays a critical role in the regulation of appetite and body composition by way of responding to cues from neuropeptides .
a series of studies , primarily in rodents , have explored the possibility that maternal nutrition during pregnancy may alter the level of energy intake in the offspring through inducing changes in the hypothalamic circuitry and in the expression , localization , and action of specific neuropeptides .
appetite stimulating neuropeptides include neuropeptide y ( npy ) , agouti - related peptide ( agrp ) , and ghrelin .
conversely , appetite suppressing neuropeptides include cocaine and amphetamine - related transcript ( cart ) , melanocyte - stimulating hormone ( msh ) , serotonin , insulin , and leptin [ 61 , 62 ] . of note
is that the overweight and obese tend to develop resistance to insulin and leptin ; therefore these two neuropeptides are usually elevated in such groups .
the hypothalamus has multiple nerve centers , or nuclei , that are sensitive to a variety of physiologic changes .
hypothalamic nuclei include the arcuate nucleus ( arc ) , paraventricular nucleus ( pvn ) , and ventromedial nucleus ( vmn ) which are involved in a number of biological activities with a fair degree of overlap . in general ,
the arc is a conduit of many diverse signals involved in various functions such as energy homeostasis , while the pvn is associated with blood pressure and stress responses and the vmn with satiety .
each also plays a role in the central nervous system regulation of food intake and body weight .
studies in rats have found that exposure to overnutrition in the fetal or neonatal period can result in permanent changes in body fat mass and in the hypothalamic neuronal circuitry regulating appetite in the adult brain .
drner , plagemann , and colleagues found immunocytochemical and morphometric aberrations in the hypothalamus of offspring of diabetic dams or in those exposed to milk from diabetic dams . in one of their early studies ,
offspring of diabetic dams had permanent hypoplasia of the vmn , decreased insulin responsiveness to glucose , impaired glucose tolerance , and increased susceptibility to diabetes .
a later study examined the effects of exposure to milk from diabetic dams . in this study ,
offspring of control dams cross - fostered to diabetic dams developed early postnatal growth delay and showed structural and functional hypothalamic
exposure to milk from a diabetic dam resulted in an upregulation of the appetite stimulants npy and agrp and a downregulation of the appetite suppressant msh .
although the researchers expected to find changes in the vmn as in their previous study , they found increased total number of neurons in the pvn which , as mentioned previously , is associated with regulation of blood pressure . incidentally ,
their research team performed a human , clinical study and reported an association between the neonatal ingestion of breast milk from diabetic mothers and increased blood pressure during childhood .
leptin has received a considerable amount of attention in regard to early programming of appetite and body composition . in a rodent study to establish whether neonatal ( postnatal day 313 ) leptin treatment can alleviate postnatal obesity and the associated metabolic sequelae that occur in the offspring of undernourished dams , leptin treatment reversed the programmed phenotype .
the benefits of leptin treatment in these rats included a slowing of neonatal weight gain and normalized caloric intake , body weight , fat mass , and fasting plasma glucose and insulin .
studies in leptin deficient ( lep / lep ) mice have demonstrated permanently disrupted neural projection pathways from the arc . in the adult lep
/ lep mice , leptin treatment did not reverse these neuroanatomical defects ; however treatment of neonatal lep / lep mice with exogenous leptin rescued the development of arc projections .
epigenetics refers to all modifications to genes other than changes in the dna sequence itself and includes dna methylation and histone modifications [ 18 , 72 ] .
dna methylation is a post - replication modification that is predominantly found in the cytosines of the dinucleotide sequence cytosine phosphate guanine ( cpg ) .
changes in methylation status ( hyper- or hypomethylation ) have been associated with various health conditions including malignancies .
histones are proteins that permit the packaging of dna into nucleosomes , the fundamental units of chromatin .
histones are subject to a large number of post - translational modifications which are likely to control the structure and/or function of the chromatin fiber
. different modifications may have distinct consequences such as transcriptional activation or dna repair .
every cell in the body has the same genetic information ; what makes cells , tissues , and organs different is that different sets of genes are turned on or expressed .
an increasing body of evidence supports the role of environmentally - induced epigenetic changes in disease susceptibility .
experimental studies in agouti mice suggest a role for maternal diet in inducing epigenetic changes in the offspring [ 75 , 76 ] .
those carrying the dominant agouti alleles a or a develop the complex set of traits collectively referred to as the yellow obese syndrome .
the yellow obese syndrome encompasses the pleiotropic effects of yellow fur , obesity , hyperinsulinemia , hyperglycemia , and increased susceptibility to neoplasia .
female a / a mice fed a methyl - supplemented diet with extra folate , vitamin b-12 , choline , and betaine 2 weeks prior to mating with male a / a agouti mice and throughout pregnancy and lactation passed along the agouti gene to their offspring intact , yet demonstrated a shift in distribution towards having more offspring with the pseudoagouti phenotype .
pseudoagouti mice are brown , lean , healthy , and longer lived than their yellow siblings .
this shift in the distribution in coat color was mediated by cpg methylation at the at the a locus .
another example of environmentally - induced epigenetic changes is reduced pancreatic and duodenal homeobox 1 ( pdx1 ) , also known as insulin promoter factor 1 , in a rat model of intrauterine growth restriction .
pdx1 is a transcription factor necessary for development and function of the insulin producing pancreatic cell .
rats faced with intrauterine growth restriction had permanently reduced expression of pdx1 in cells and developed type 2 diabetes in adulthood [ 78 , 79 ] .
reduced pdx1 transcription was mediated through a cascade of epigenetic modifications characterized by loss of upstream stimulatory factor-1 binding at the proximal promoter of pdx1 , recruitment of the histone deacetylase 1 and the corepressor sin3a , and deacetylation of histones h3 and h4 which culminated in the eventual silencing of pdx1 .
the dutch famine of 1944 has been used by various investigators as an equivalent to an experimental study to investigate the effects of perinatal undernutrition in humans .
the ongoing hunger winter families study contributed empirical support for the hypothesis that early - life environmental conditions cause epigenetic changes in humans that persist throughout life .
individuals who had been exposed to famine during periconception had , 6 decades later , less dna methylation of the insulin - like growth factor ii ( igf2 ) gene compared with their unexposed , same - sex siblings .
the dutch famine cohort has also been used to study the transgenerational effects of famine exposure .
women exposed to famine while in the womb later had offspring with birth weights lower than offspring of women not exposed to famine .
this effect of in utero exposure to famine on birth weight in the subsequent generation persisted after control for potential confounding and intervening variables . in a study of 2 prospective cohorts , godfrey and colleagues used dna extracted from umbilical cord tissue obtained at birth in children who were later assessed for adiposity at 9 years of age to measure methylation status of cpgs in the promoters of candidate genes .
five candidate genes were selected based on a number of criteria which included animal data [ 8385 ] and correlations with overall gene methylation status and adiposity .
methylation of retinoid x receptor- ( rxra ) chr9 : 136355885 + ( in cohort 1 and 2 ) and endothelial nitric oxide synthase ( enos ) chr7 : 150315553 + ( in cohort 1 only ) at birth was correlated with greater adiposity in later childhood .
although the data is correlative and does not prove causality between dna methylation at birth and adiposity in childhood , the observation suggests that epigenetics is involved in fetal programming of later obesity .
a sizable number of experimental studies , predominantly in rats , have explored the effects of protein restriction on fetal growth and later health . as discussed in the section on the thrifty phenotype hypothesis , protein restriction during pregnancy
has been found to produce small offspring that have reduced numbers of nephrons which potentially contributes to the development of hypertension in adulthood [ 16 , 34 , 35 ] .
low protein exposure in fetal rats has also been shown to result in reduced pancreatic cell mass at birth and reduced insulin secretion in later life presumably due to dietary - induced reduction in proliferation rate and increased apoptosis of cells .
weanling ( 26-day - old ) rats exposed to low protein during gestation and lactation were smaller and had reduced serum insulin and increased serum glucose and triglycerides .
in addition , low protein - exposed offspring had increased hepatic triglycerides and hepatic expression of lipogenic enzymes favoring fat synthesis .
the authors suggested that the increased expression of fat - synthesizing enzymes may account for the increased levels of serum and liver triglycerides seen in those exposed to low protein and that these increases may predispose the offspring to excessive accumulation of fat and ultimately obesity and insulin resistance .
rat studies indicate that gestational exposure to low levels of protein due to maternal protein restriction establishes a preference for high fat foods .
when given the choice of selecting high fat , high protein , or high carbohydrate foods , 12-week - old offspring of dams fed a low protein diet throughout gestation consumed significantly more of the high fat food and significantly less of the high carbohydrate food than their control counterparts . at 30 weeks of age , there was no difference in the pattern of food selection between the two groups .
their results suggest that early exposure to undernutrition programs a preference for fatty foods , and thus maternal nutrition may influence pathways involved in control of appetite or the perception of palatability in the offspring .
although the literature on protein restriction and disease risk is quite extensive , it is largely based on rodent models with few having assessed the specific role of protein restriction in human risk of disease .
one study of men and women in scotland sought to determine how diet of the mother in pregnancy influences blood pressure in the 40-year - old adult offspring .
the authors found that the relations between the diet of mothers and the blood pressure of their offspring were complex .
when maternal intake of animal protein was less than 50 g daily , a higher carbohydrate intake was associated with higher blood pressures in the offspring .
conversely , when daily protein intake was above 50 g , lower carbohydrate intake was associated with higher blood pressure .
an additional finding was that increases in blood pressure were associated with decreased placental size .
their conclusions were that blood pressure in adulthood may be influenced by maternal intakes of protein and carbohydrates during pregnancy and that this may be mediated through effects on placental growth .
all dietary carbohydrates can be converted into glucose . in both animal and human studies , intrauterine exposure to high sugar diets and/or
hyperglycemia has been found to increase the risk of the metabolic syndrome . in rats ,
a high fructose diet for 2 weeks resulted in increases in systolic blood pressure as well as plasma insulin and triglyceride concentrations as compared to rats fed a normal chow . in another rat study early - and long - term exposure to a high sucrose diet ( hsd ) was assessed to determine whether such exposure alleviates the detrimental effects of sucrose feeding in later life .
dams were fed either standard or hsd ( 70% calories as sucrose ) starting 1 week before breeding and throughout gestation and lactation . after weaning ,
all male offspring were fed hsd until the age of 20 weeks , then detailed metabolic and morphometric profiles were ascertained .
offspring of sucrose - fed dams displayed higher adiposity and increases in triglyceride liver content together with higher low - density lipoprotein ( ldl ) cholesterol concentrations .
although the significance and mechanisms are not clear , a somewhat perplexing observation was that of substantial increases in the insulin sensitivity of skeletal muscle together with higher concentrations of adiponectin in the offspring of sucrose - fed dams compared with the offspring of standard diet - fed dams .
triglycerides , free fatty acids , overall glucose tolerance , and the insulin sensitivity of adipose tissue were comparable in both groups .
data derived from the camden study of adolescent pregnancy implicated high sugar consumption with a twofold increased risk for delivering small for gestational age infants .
additionally , evaluation of the data based on ethnicity led to the detection of a substantial decrease in the duration of gestation among puerto rican adolescents with high sugar diets , whereas there was no effect of a high sugar diet on gestation duration among black and white adolescents .
the authors commented that other studies involving comparisons among ethnicities have found hispanic women to be more likely to have abnormalities of carbohydrate metabolism during pregnancy as evidenced by abnormal glucose tolerance tests and increased incidence of gestational diabetes [ 9395 ] .
pregnancies complicated by maternal diabetes , in any form , place the offspring at risk for developing obesity and glucose intolerance [ 29 , 96 ] . in a study of 18- to 27-year - old women born to diabetic ( gestational diabetes or type 2 diabetes ) mothers ,
the risk of overweight was doubled in the offspring of diabetic mothers as compared with offspring from a background population .
moreover , the risk of the metabolic syndrome was increased 4-fold for offspring of mothers with gestational diabetes and 2.5-fold for offspring of mothers with type 1 diabetes .
offspring risk of the metabolic syndrome increased significantly with increasing maternal fasting blood glucose as well as 2-hour blood glucose following an oral glucose load .
their findings indicate that intrauterine exposure to hyperglycemia contributes to the pathogenesis of the metabolic syndrome , thus offspring of diabetic mothers are risk groups for this condition . our laboratory has performed multiple experimental studies aimed at determining the role of high fat diet in maternal - fetal health . in one such study ,
mice exposed to a high saturated fat diet during gestation and lactation developed adult obesity , hyperglycemia , insulin resistance , and hypertension , despite being fed a standard rodent diet post - weaning . given
that oxidative stress is a potential mechanism connecting fetal exposures to the development of the metabolic syndrome , a group of pregnant mice were supplemented with quercetin , a powerful antioxidant , which mitigated the detrimental effects of high fat diet exposure .
similarly , bouanane and colleagues used a rat model to determine the effects of a cafeteria diet , that had 42% of energy from fat , on oxidant / antioxidant status as well as variety of metabolic markers .
dams fed the cafeteria diet developed increased body weight , hyperglycemia , hyperinsulinemia , hyperleptinemia , hyperlipidemia , and an imbalance between oxidants / antioxidants favoring oxidative stress .
male and female offspring displayed similar effects , which persisted after birth , suggesting an unrelenting effect of maternal diet on metabolism and body habitus of surviving offspring . in the development of a dietary - induced gestational diabetes mouse model ,
our laboratory demonstrated that consumption of a high saturated fat diet prior to conception and throughout pregnancy can result in insulin resistance and placental vascular damage and that these abnormalities could be a result of oxidative stress .
frias and colleagues , using a nonhuman primate model to determine the effect of chronic high fat diet on pregnancy outcome , found that consumption of such a diet , independent of maternal obesity , led to a significant reduction in uterine blood flow , a rise in placental inflammation , and an increase in fetal risk of nonalcoholic fatty liver disease , as evidenced by increased levels of liver triglycerides and increased hepatic oxidative stress [ 101 , 102 ]
. offspring of high fat diet dams also exhibited elevated hepatic expression of gluconeogenic enzymes and transcription factors .
moreover , reversing the maternal high fat diet to a low fat diet during a subsequent pregnancy improved fetal hepatic triglyceride levels and partially normalized gluconeogenic enzyme expression . the authors concluded that a developing fetus is highly vulnerable to excess lipids , independent of maternal diabetes and/or obesity and that such exposure may increase the risk of pediatric fatty liver disease .
human observational studies have also found high fat intakes to be related to pregnancy and birth outcome as well as long - term health of the offspring . in a case - control study of
912 women admitted to obstetric hospitals for spontaneous abortion , the risk of miscarriage was directly associated with consumption of the main types of fats , butter and oil , while a significant inverse relationship was established between the risk of spontaneous abortion and the consumption of green vegetables , fruits , milk , cheese , eggs , and fish .
results of a comprehensive dietary review of women with a history of gestational diabetes found that women with recurrence of gestational diabetes in a subsequent pregnancy had significantly higher fat intakes as a percentage of total energy than women who did not have recurrence .
lipids and lipid disorders play a central role in the development of the metabolic syndrome and its associated diseases .
fatty streaks in the aorta have been observed in children as young as 3 years old and autopsies of young soldiers killed in the korean and vietnam wars revealed advanced coronary artery lesions [ 108 , 109 ] .
similarly , the pathobiological determinants of atherosclerosis in youth ( pday ) study investigated atherosclerosis in autopsied persons 15 to 34 years old that died from various traumas and observed advanced lesions of atherosclerosis in adolescents and young adults and that serum lipoprotein concentrations were a risk factor .
although micronutrient deficiencies are known causes of several well - characterized diseases , such as scurvy and rickets , the role of micronutrients in the fetal origins of adult disease has yet to be explored in detail .
the section on oxidative stress provided examples on how micronutrients , as antioxidants , may be associated with various short- and long - term effects on the offspring . in relating maternal micronutrient intake with fetal glucocorticoid exposure ,
a mouse model of maternal dietary restriction of copper , zinc , and vitamin e demonstrated reduced activity level of placental 11-hydroxysteroid dehydrogenase-2 , an enzyme that protects the fetus from overexposure to maternal glucocorticoids . as mentioned in the section on the hypothalamic - pituitary - adrenal axis , fetal exposure to glucocorticoids
is associated with small size at birth and insulin resistance and hypertension in adult life .
indeed , in this study offspring exposed to the micronutrient restricted diet had significantly reduced body weight and crown - to - rump length at birth as well as increased systolic blood pressure and insulin levels post - weaning as compared to those exposed to a control diet .
as discussed in the section on epigenetics , experimental studies on the impact of methyl - supplemented diets containing added folate and vitamin b-12 revealed the potential role of these micronutrients in inducing major changes in offspring phenotype , including the ability to impact the development of chronic diseases .
studies on maternal dietary zinc restriction in rats indicate that zinc deficiency during intrauterine and postnatal growth can induce elevations in blood pressure and renal lesions in adulthood [ 114 , 115 ] . concerning the renal alterations identified , early exposure to zinc deficiency resulted in a decrease in glomerular filtration rate which was associated with a reduction in the number and size of nephrons .
these animals also had proteinuria , higher lipid peroxidation end products , and evidence of increased renal apoptosis and fibrosis [ 114 , 115 ] .
few human studies have explored the role of early micronutrient deficiencies in the development of adult metabolic disorders . of the existing reports
, results are often conflicting and the significance is ambiguous . in two randomized controlled trials , both in nepal ,
one found that daily multiple micronutrient supplementation in pregnant women resulted in slightly lower blood pressure in the offspring at 2.5 years of age , while the other found no effects on blood pressure with maternal multiple micronutrient supplementation among 6- to 8-year - old offspring . as part of the pune ( india ) maternal nutrition study ,
higher maternal erythrocyte folate concentrations at 28 weeks gestation were associated with higher adiposity and insulin resistance in children 6 years of age .
conversely , a study in england found that maternal folate intake at 18 or 32 weeks gestation was not associated with any measures of body composition in children at 9 years of age .
clearly more work is needed to disentangle the intricate interactions among micronutrients and to determine how these interactions may be involved in the initiation and progression of chronic disease .
it is now widely accepted that certain chronic diseases of adulthood may have their origins in the womb .
studies discussed in this paper provide evidence that a mother 's diet during pregnancy can exert major effects on the short- and long - term health of her children including programming of the metabolic syndrome .
the challenges at present are to identify common mechanisms and pathways involved in disparate perinatal malnutrition paradigms , deciphering physiological and/or pathological roles of specific nutrients , and to determine which components of the maternal diet may be best modified to optimize maternal health , placental integrity , birth outcome , and lifelong health of the offspring .
the implications of this avenue of research , particularly to obstetric and preventative medicine , dictate that effective interdisciplinary communication and knowledge transfer occur and that the information generated is disseminated to the general public . a recent review provides encouraging evidence that prenatal nutritional advice appears effective in reducing the risk of preterm birth and that balanced energy and protein supplementation seem to improve fetal growth and may reduce the risk of stillbirth and infants born small for gestational age . whilst the evidence in support of the dohad is compelling
, there are many caveats and controversies in the field including species differences , body weight verses body composition , and twin studies .
first , species differences ( e.g. , maternal age , parity , gestation length , fetal number , and variability in fetoplacental development ) should be considered when results derived from animal studies are extrapolated to humans .
rodents are particularly important to reproductive and obstetric studies because of the strong similarities between human and murine placentas [ 121123 ] ; however , there are many differences between rodents and humans particularly relating to early development .
rodents are altricial species , and their organs mature after birth , which explains why postnatal factors may be more pertinent in rodent species , whereas pre- and perinatal factors play a vital role in human development .
human infants of hyperglycemic mothers develop macrosomia , whereas hyperglycemic rodent dams typically have normally sized or small pups .
the more precocious human cells produce fetal insulin prior to birth , thus increasing glucose transport and growth prenatally , which does not occur in the rodent , whose endocrine pancreas becomes functional postnatally [ 62 , 124 , 125 ] .
second , studies relating early exposures to later life risk of disease have been largely based on body weight and/or body mass index ; however , these measurements provide no information on body composition .
more recent studies have included the assessment of body composition , and evidence suggests that lower birth weights reflect increased relative adiposity , while higher birth weights reflect higher lean mass .
the increased use of body composition analysis is likely to offer a more complete understanding as to why small babies are prone to the development of the metabolic syndrome , an association that likely involves increased adiposity and decreased lean mass at birth .
finally , studies in twins , especially monozygotic twins , have caused considerable contention in the field [ 15 , 29 ] .
the fetal origins hypothesis indicates that such individuals should have increased morbidity and mortality from metabolic and cardiovascular disease , but this has not been proven to be necessarily the case [ 15 , 29 , 126 ] .
the dohad is a relatively new field of research and in time such discrepancies may be resolved . with a more complete understanding of the role of maternal health and nutrition in the initiation and progression of the metabolic syndrome and other disorders comes the hope of prevention of chronic diseases at their earliest beginnings . | chronic diseases such as type 2 diabetes and cardiovascular disease are the leading cause of death and disability worldwide .
although the metabolic syndrome has been defined in various ways , the ultimate importance of recognizing this combination of disorders is that it helps identify individuals at high risk for both type 2 diabetes and cardiovascular disease .
evidence from observational and experimental studies links adverse exposures in early life , particularly relating to nutrition , to chronic disease susceptibility in adulthood .
such studies provide the foundation and framework for the relatively new field of developmental origins of health and disease ( dohad ) .
although great strides have been made in identifying the putative concepts and mechanisms relating specific exposures in early life to the risk of developing chronic diseases in adulthood , a complete picture remains obscure . to date , the main focus of the field has been on perinatal undernutrition and specific nutrient deficiencies ; however , the current global health crisis of overweight and obesity demands that perinatal overnutrition and specific nutrient excesses be examined .
this paper assembles current thoughts on the concepts and mechanisms behind the dohad as they relate to maternal nutrition , and highlights specific contributions made by macro- and micronutrients . |
ovarian carcinoma is the leading cause of death from gynecologic malignancy , with an estimated 15 520 deaths in the usa in 2008 .
ovarian cancer is a highly metastatic disease that is rarely detected when disease is confined to the ovary ( stage i ) and 5-year survival is > 90% .
the great majority of ovarian cancer patients are initially diagnosed with disseminated intra - abdominal disease ( stages iii
iv ) and have a 5-year survival of < 20% . clinically , ovarian tumors often involve the ovary and omentum , with diffuse , multifocal intraperitoneal metastases and malignant ascites [ 2 , 3 ] .
the combined factors of late diagnosis and the cellular and molecular heterogeneity of ovarian cancers hamper efforts to effectively treat this disease . for many cancers , including those of the ovary , there is growing emphasis on the identification and development of targeted therapies to disrupt specific molecular pathways contributing to disease progression .
the egf receptor impinges on multiple key hallmarks of cancer defined by hanahan and weinberg and the egf receptor is associated with a gene expression pattern unique to invasive tumor cells
. aberrant expression and activity of the egf receptor is generally recognized to have a deleterious impact on the clinical outcome of cancer patients which has fueled development of targeted therapeutics ( reviewed in [ 712 ] ) .
this paper will discuss potential contributions of egf receptor activation to ovarian cancer pathogenesis and the status of egf receptor inhibitors and egf receptor targeted therapies in ovarian cancer treatment .
the egf receptor is a member of the receptor tyrosine kinase ( rtk ) family of growth factor receptors and the founding member of the erbb subfamily that includes four proteins : erbb1 ( egf receptor ) , erbb2 ( her-2 ) , erbb3 ( her-3 ) , and erbb4 ( her-4 ) .
ligand binding promotes egf receptor homo- and heterodimerization with erbb family members , activation of the intracellular tyrosine kinase domain , and stimulation of numerous downstream signaling cascades associated with cell growth and survival , increased angiogenesis , and tumor metastasis ( reviewed in [ 710 ] , [ 1317 ] ) .
the most common form of ovarian cancer arises from the ovarian surface epithelium ( ose ) .
the ose expresses egf receptors in vivo and egf receptor activity is implicated in gonad development , growth and differentiation of the ovarian follicle , and postovulatory repair [ 1820 ] .
it has been proposed that egf stimulation of the ose contributes to its rapid post - ovulatory proliferation and to epithelial - mesenchymal transition ( emt ) of ose cells within the ruptured follicle .
malfunctions in post - ovulatory repair are believed to contribute to formation of epithelial inclusion cysts , which are the preferential sites of malignant transformation [ 15 , 21 , 22 ] .
the normal ose responds to egf receptor generated signals by displaying a phenotypic plasticity characterized by transition between epithelial and fibroblastic phenotypes , a characteristic usually limited to immature , regenerating , or neoplastic epithelia .
these attributes of the adult ose suggest that this tissue is primed to respond to the egf receptor during tumor development and progression .
in addition to its role in normal ovarian epithelium , there is abundant evidence of aberrant egf receptor and/or ligand expression in ovarian cancer .
a recent review provides an excellent and comprehensive summary of immunohistochemical studies evaluating erbb receptor and erbb ligand expression in malignant ovarian tumors .
briefly , published reports estimate egf receptor expression in 1070 percent of human epithelial ovarian cancer cases ( reviewed in ) .
a smaller subset of studies has examined amplification of the egf receptor gene in ovarian cancer .
an advantage of this approach is the relative stability of dna in archived samples , but because egf receptor overexpression can occur in the absence of gene amplification , these studies may underestimate the frequency of elevated egf receptor protein in tumors . despite this caveat ,
egf receptor gene amplification is detected in ~1020 percent of ovarian cancer cases [ 2426 ] , with low - level gains detected more frequently in 43 percent of tumors .
thus , based on detection of protein or gene amplification , there is strong evidence for elevated egf receptor expression in a significant fraction of ovarian cancer cases .
overall , elevated egf receptor is associated with less favorable disease outcomes in a number of human tumors [ 17 , 2729 ] . despite evidence for egf receptor expression in ovarian tumors , studies on the relationships between receptor and patient outcomes
do not provide a uniform picture on the clinical consequences of elevated egf receptor levels . based on studies with normal tissue reference controls , elevated egf
receptor levels significantly correlated with aggressive disease characteristics and high tumor egf receptor expression was proposed as the most significant prognostic factor for disease - free and overall survival . an overall conclusion that aberrant egf receptor status is a factor in ovarian cancer outcome
is supported by a meta - analysis study revealing a relationship between egf receptor and decreased survival , and the abundant evidence linking egf receptor to poor patient outcome in other cancers of epithelial origin .
a limited number of studies examine activated ( tyrosine phosphorylated ) egf receptor in ovarian tumors and overall , little attention has been given to receptor activation status and disease parameters .
in one study , 11.8 percent of ovarian tumors were positive for phosphorylated egf receptor ( pegfr ) but no clinicopathological parameter or survival differences were noted . in another study , twenty - four heavily pretreated patients with epithelial ovarian cancer all had detectable egf receptor and p - egfr ( y1148 ) , suggesting that egf receptor activation might be more evident in advanced disease .
we conducted a tumor tissue array analysis and found evidence for pegfr in approximately 1/3 of ovarian tumor samples .
egf receptor activation was statistically positively correlated with matrix metalloproteinase ( mmp)-9 expression , a protein associated with tumor invasion and metastasis . together , these in vivo data indicate that activated egf receptor is present in ovarian tumor specimens , likely driving aspects of tumor behavior .
the mitogenic effects of egf receptor activation in ovarian tumor cells are well documented .
egf increases the growth potential of primary ovarian surface epithelial ( ose ) cells in culture and gene expression profiling of normal rat ovarian surface epithelium following egf treatment demonstrates egf - dependent activation of genes involved in cell cycle and proliferation , apoptosis , and protein turnover . in addition , malignant transformation of rat ose cells results in alteration of downstream effectors of the egf receptor pathway .
regarding ovarian tumor cells , numerous studies demonstrate that autocrine and paracrine stimulation of the egf receptor promotes ovarian tumor cell growth ( reviewed in [ 37 , 38 ] ) .
furthermore , blockade of egf receptor activation or signaling inhibits ovarian tumor cell growth in vitro and in vivo ( reviewed in ) .
in addition to fostering cell growth , activation of the egf receptor is associated with stimulation of metastasis - associated cellular responses .
many aspects of tumor metastasis resemble features of epithelial - mesenchymal transition ( emt ) [ 3943 ] .
notably , egf receptor activation is capable of driving emt - associated events in epithelial ovarian carcinoma cells in culture including migration and invasion , disruption of e - cadherin - mediated intercellular junctions , and production of matrix degrading proteinases ( reviewed in [ 37 , 38 , 44 , 45 ] ) .
in contrast to the well - defined events that characterize emt in development , tumor - associated emt is currently viewed as a continuum of phenotypic plasticity and gain of mesenchymal characteristics .
tumor phenotype likely reflects the particular complement of emt regulatory factors expressed in cells or within the tumor microenvironment [ 4245 ] .
the functional consequences of this phenotypic plasticity are not fully understood , but may play a role in modulation of cell survival in suspension ( ascites ) , chemoresistance , and intraperitoneal anchoring of metastatic lesions ( reviewed in [ 42 , 44 , 46 ] ) .
based on the evidence that ( 1 ) ovarian tumors share certain characteristics ( egf receptor overexpression and activation ) with tumors approved for treatment with egf receptor inhibitors , ( 2 ) receptor activation drives tumor - relevant responses in ovarian tumor cells , and ( 3 ) ovarian tumor growth is reduced by egf receptor directed therapeutics in preclinical models , the egf receptor inhibitors have moved forward into clinical trials for ovarian cancer and are discussed in the following section .
with the advent of better understanding of the molecular mechanisms contributing to ovarian cancer , novel receptor targeted therapeutics or
biologic therapeutics either administered alone or in combination with conventional chemotherapy have become a rapidly developing strategy in clinical trials design . based on expression of the egf receptor in ovarian cancer and the known consequences of receptor activation
numerous anti - egf receptor agents are under active development and each compound has subtle differences in target binding , downstream signaling , ease of administration and toxicity profiles . yet despite favorable preclinical studies using egf receptor antagonists , clinical trial outcomes in ovarian cancer have been overall disappointing .
investigations are underway to understand the mechanism of escape from egf receptor blockade as well as to identify clinical predictors of antagonist response .
the following sections will summarize the success and shortcomings of these agents in ovarian cancer trials .
the majority of egf receptor inhibitor agents in clinical trial development fall into two categories : small molecule tyrosine kinase inhibitors ( tkis ) that compete with atp for its binding site in the tyrosine kinase domain or monoclonal antibodies ( mabs ) against the extracellular domain that interfere with ligand binding and/or receptor dimerization .
additional egf receptor directed therapeutic strategies include development of egf vaccines , receptor downregulation by antisense oligonucleotides .
egf receptor dependent targeting of imaging agents , chemotherapeutic agents , and toxins will be discussed later in this paper .
a significant clinical difference between the small molecule tkis and mabs is that the tkis are orally administered and require daily dosing ( especially the reversible inhibitors ) to maintain target blockade whereas the mabs are given intraveneously usually weekly or every 2 weeks .
the tkis and mabs share a toxicity profile which includes fatigue , diarrhea , and a robust acneiform rash .
the cutaneous rash has been described as a clinical indicator of egf receptor blockade due to abrogation of receptor signaling in nontumor tissues such as the skin and gut mucosa .
in addition , hypersensitivity reactions are a concern with mabs , especially the nonhumanized or chimeric agents .
several tkis and mabs are fda approved for treatment of specific solid tumors , yet none have performed well enough in ovarian cancer trials to warrant such approval ( table 1 ) .
additional compounds are under clinical development in ovarian cancer and other solid tumors ( table 2 ) . in clinical trials
generally the trials are conducted in patients with recurrent ovarian cancer , and often patients have been heavily pretreated before receiving the targeted therapeutics .
the common dosing schedules from multiple phase i trials for the oral tkis are shown in table 1 .
gefitinib alone ( 500 mg ) performed poorly in phase ii trials with minimal clinical response for ovarian cancer patients .
the only responder had an activating mutation in the egf receptor catalytic domain similar to the mutations evident in responsive lung cancer patients .
erlotinib alone ( 150 mg ) performed slightly better with 6% of the patients responding based on tumor regression and 44% of patients had stable disease .
gefitinib has been combined with cytotoxic chemotherapy such as carboplatin , paclitaxel , topotecan , oxaliplatin , vinorelbine , and the aromatase inhibitor anastrazole in multiple phases i and ii trials with some patients responding to treatment [ 4 , 47 ] .
eroltinib has been combined with carboplatin , docetaxel , paclitaxel , and the vegfr inhibitor bevacizumab .
several of these trials were performed as front line treatment after cytoreductive surgery demonstrating good clinical and some pathologic complete response rates , but the response rates do not appear dramatically different when compared to historic controls for conventional therapy alone .
the pipeline of egf receptor tyrosine kinase inhibitors continues to expand ( table 2 ) .
a randomized phase ii trial of the irreversible egf receptor inhibitor ci-1033 was performed in a heavily pretreated population of women with recurrent ovarian cancer .
two different oral dose regimens were given ( 50 mg versus 200 mg daily ) for 21 days .
unfortunately there were no responders to single agent treatment and no association between baseline erbb expression and disease stability .
future studies will likely see these new agents in combination with cytotoxic and other biologic agents .
there are many possible reasons to account for the modest responses to egf receptor inhibitors .
the oral tyrosine kinase inhibitors can be difficult to use in this patient population , as advanced disease causes loss of bowel function and potential unreliable absorption of drug .
another significant concern is the lack of validated biomarkers for response to these tkis . to date ,
activating mutations in the egf receptor kinase domain are the only known predictors of response , but these mutations have not been fully explored in ovarian tumors .
the monoclonal antibodies against the egf receptor ligand binding domain have some pharmacologic advantages and may perhaps lead to better clinical outcomes compared to the tkis .
cetuximab is the prototype mab and has been administered alone or in combination with carboplatin + / paclitaxel .
a gynecologic oncology group ( gog ) phase ii trial of cetuximab and carboplatin in platinum sensitive recurrent ovarian cancer showed a 35% response rate ( partial and complete responses ) in patients with tumors displaying egf receptor overexpression documented by immunohistochemistry ( ihc ) . of note , 93% of patients
although it is tempting to conclude that egf receptor immunohistochemical analysis of formalin fixed , paraffin embedded tissue is of predictive value for response rate , this has been neither quantified nor validated . a phase ii trial of emd 72000 ( matuzumab ) given at 800 mg iv weekly enrolled 37 women with heavily pretreated platinum resistant recurrent ovarian cancer .
egf receptor status was not evaluated for entry criteria or for correlation to clinical response and there were no objective responses in this group when matuzumab was used as monotherapy .
the authors concluded that matuzumab monotherapy was not effective for this heavily pretreated group of women .
panitumumab is a fully humanized egfr mab under active investigation , particularly in lung and colorectal cancer .
it is expected to elicit fewer hypersensitivity reactions than the chimeric human / mouse cetuximab , but to date , there is little direct clinical trial emphasis in ovarian cancer .
dual inhibition of erbb receptor family members is an interesting approach for targeted therapy as much of the signaling is generated by heterodimers , particularly heterodimers of egf receptor and erbb2 .
lapatinib is an oral small molecule tyrosine kinase inhibitor that reversibly inhibits both erbb1 and erbb2 .
it is well tolerated alone and in combination with chemotherapy as determined by phase i trials [ 4 , 47 ] .
our group recently completed a phase i / ii trial of weekly metronomic carboplatin and paclitaxel in combination with lapatinib ( 1250 mg daily ) in 25 evaluable patients with recurrent ovarian cancer .
interval evaluation showed a 50% response rate ( complete and partial response ) with the expected gastrointestinal and hematologic toxicities .
canertinib ( ci-1033 ) is a newer oral dual tki which inhibits autophosphorylation of all erbb receptors including a highly tumorigenic , constitutively active mutant form of the egf receptor ( egfrviii ) .
this agent showed no significant activity as a single agent in a phase ii study in patients with recurrent ovarian cancer .
pertuzumab is the prototype of this inhibitor class and prevents erbb2/her2 dimerization with the egf receptor , erbb3/her3 , and erbb4/her4 leading to inhibition of map kinase and pi3 kinase signaling .
a phase ii trial was conducted by gordon et al . that included 123 patients with recurrent ovarian cancer ( the majority platinum resistant ) .
two different dosing strategies of pertuzumab as a single agent demonstrated an overall response rate of 4.3% and a mean response duration of 18.6 weeks .
only 28 patients had biopsy material accessible for evaluation of phosphorylated her2 ( pher2 ) status by elisa .
of this group only 8 patients had pher2 + tissues with one patient in this group experiencing a partial response .
the 20 other tumors did not show pher2 expression and there were no treatment responses in this group .
this suggests that pher2 rather than her2 overexpression may be a viable biomarker for response although validation studies are desperately needed .
two ongoing randomized phase ii trials in relapsed ovarian cancer are evaluating pertuzumab versus placebo in combination with gemcitabine or carboplatin [ 54 , 55 ] . in these trials treatments
were tolerated , but clinical response endpoints have not yet been reached . in an early analysis of the data ,
low erbb3/her3 mrna levels as measured in 122 of the 130 patient archival tumor tissues appeared to predict clinical benefit in the cohort receiving gemcitabine + pertuzumab versus the gemcitabine + placebo group . final analyses of both pertuzamab trials are pending .
additional monoclonal antibodies developed to inhibit egf receptor family members are listed in table 2 and studies to test the toxicity and efficacy of these agents in ovarian cancer are needed .
in addition to therapies directed against the egf receptor as discussed previously , this receptor has been used to deliver imaging agents or therapeutics to tumors . to target the egf receptor on tumor cells , egf receptor ligands or anti - egf
egf receptor ligands such as mouse egf can be conjugated through its n - terminus without affecting receptor binding ability .
in contrast , human egf has two additional amino groups due to internal lysines , and their conjugation can interfere with receptor binding .
for that reason mouse egf rather than human egf is usually employed for egf receptor targeting .
such peptides have been identified either through screening of a virtual peptide library , or through screening phage display libraries for peptides that specifically bind to the egf receptor , including lysine - deficient egf variants .
egf receptor - targeting moieties are conjugated with imaging or therapeutic agents such as radionuclides , cancer chemotherapeutic agents , toxins , rnase , or photosenstizers .
in addition , delivery of oligonucleotides or expression vectors to either suppress or express certain genes in egf receptor - positive cells through the use of viral or nonviral delivery systems has been reported .
recently more complex systems have been designed that employ various nanocarriers as targeted delivery systems .
the simplest form of an egf receptor - targeting complex is radiolabelled - egf , tki inhibitor , anti - egf receptor mab , or engineered anti - egf receptor fragments , which can be used for in vivo imaging or for therapeutic purposes [ 59 , 60 ] .
the targeted radionuclide delivery serves as a cytotoxic agent by itself and has been employed in boron neutron capture therapy [ 61 , 62 ] , although optimal therapeutic effects may not be achieved with stand alone boron therapy .
radionuclides as imaging agents can be used to evaluate whether tumors are egf receptor positive and thus likely to respond to egf receptor - targeted therapies , or monitor response to therapy .
imaging techniques used to detect egf receptor - expressing tumors in small animals include positron emission tomography ( pet ) , magnetic resonance ( mr ) , and single photon emission computed tomography ( spect ) [ 59 , 60 , 64 ] .
these techniques involve positron emitting radionuclides ( such as c , f , among others ) , beta emitters ( such as technetium ( tc ) and lutetium ( lu ) ) , gamma emitters ( such as iodide ( i ) and indium ( in ) ) , and alpha emitters ( such as astatine ( at ) and bismuth ( bi , bi ) ) [ 59 , 60 , 64 , 65 ] .
numerous preclinical studies indicate that tumor targeting can be achieved through the egf receptor ; however , most of these studies did not include ovarian tumor models .
in addition to radionuclides , cancer chemotherapeutic agents such as cisplatin , doxorubicin [ 67 , 68 ] , carminomycin , and tyrosine kinase inhibitors [ 70 , 71 ] have been delivered to egf receptor - positive cells through conjugation to egf or to anti - egf receptor mab either directly or through a polymer linker .
numerous toxin conjugates that inhibit specific molecular targets within the cell have been delivered to egf receptor - positive cells including pseudomonas exotoxin ( pe ) , amanitin , gelonin , and ricin chain a [ 7578 ] .
furthermore , rnases targeted to the egf receptor were cytotoxic to cancer cells [ 7983 ] and photosensitizers used for photodynamic therapy have been successfully targeted to egf receptor - positive cells [ 8487 ] .
phase i clinical trials for tp-38 which is a fusion of a mutated pe and the egf receptor ligand transforming growth alpha demonstrate that it is well tolerated with promising clinical response in patients with recurring malignant brain tumors .
the main challenges to expanding use of these toxin conjugates in clinical trials include reducing their immunogenicity by shielding the toxin portion of the complex , and the need to improve delivery to solid tumors .
egf receptor targeted approaches have been used for viral and nonviral gene delivery to cells . as an example of viral systems , avidin - adenovirus ( adv ) that expresses gfp
was functionalized with egf , and gfp expression was enhanced in egf receptor - overexpressing cells compared to cells that moderately express egf receptor or relative to naked or peg - adv .
egf or anti - egf receptor mab was conjugated to cationic polymers such as poly - l - lysine ( pll ) [ 9095 ] or polyethyleneimine ( pei ) [ 96102 ] that are positively charged and thus interact with negatively charged oligonucleotides or expression vectors .
these systems efficiently transfected tumor cells in a receptor - dependent fashion . a number of strategies to improve egf receptor - specific gene transfer or specificity include peg or poly
other modifications that enhance egf receptor gene transfer include incorporation of melittin , a membrane active peptide , or incorporation of peg to reduce albumin - caused aggregation and protect the complexes from serum proteins .
new generations of nanocarriers are under intense investigation as they offer advantages over administering a drug alone or in a simple conjugated targeting moiety .
nanocarriers have numerous benefits including their ability to deliver hydrophobic drugs , increased drug loading , the potential to load multiple drugs or imaging agents , and the ability to functionalize nanocarriers with multiple molecules .
moreover , because of their size these nanocarriers can passively target tumors through the enhanced retention effect caused by large gaps between vascular endothelial cells tissue and defective lymphatic drainage in tumor tissue .
in addition to passive targeting , active targeting of cancer tissue can be achieved using nanocarriers functionalized with a targeting moiety such as an egf receptor ligand or an anti - egf receptor mab .
several nanocarriers have been employed as delivery vehicles for drugs or imaging agents to target egf receptor - positive cancers including liposomes [ 107112 ] , gelatin nanoparticles [ 113 , 114 ] , gold , dendrimers , and carbon nanotubes .
these nanocarriers specifically bound to and were internalized by egf receptor - expressing cancer cells in vitro [ 109 , 115 , 116 , 118 ] , or preferentially accumulated at tumor sites in vivo [ 107 , 109 , 113 ] .
we successfully targeted carbon nanotubes functionalized with egf and a peg - fluorescein conjugate to ovarian tumor cells .
specific egf receptor targeting and cellular uptake was achieved by coating the nanotubes with pl - peg2000 .
furthermore , we find that these vehicles were trafficked to lysosomes , consistent with the fate of ligand - activated egf receptor ( zeineldin , unpublished data ) .
lysosomes provide an acidic environment that is conducive to release of drugs attached to the delivery vehicle through acid - labile linkers .
this property may allow for the design of therapeutics that will release drugs intracellularly following egf receptor targeted internalization .
in addition , nanocarriers are being developed as efficient drug delivery systems to improve the cellular uptake of certain therapeutic agents such as inhibitory rna or to enhance the therapeutic efficacy of drugs .
a pioneering example of a targeted nanocarrier that just completed phase i trials is calaa-01 .
calaa-01 is a stabilized cyclodextrin - containing polymer that delivers inhibitory rna through transferrin targeting ( calando pharmaceuticals : http://www.insertt.com ) .
it is expected that nanotechnology will lead to innovative platforms for targeted drug delivery in future therapeutics .
there is abundant evidence that egf receptor activation drives cellular processes linked to ovarian tumor development , tumor cell survival , and metastasis .
however , the overall clinical impact of targeting the egf receptor and its dimers in ovarian cancer , either by monoclonal antibodies or inhibition of the tyrosine kinase domain , has been modest in unselected women with advanced or recurrent ovarian cancer .
although the egf receptor is a genetically validated target for non - small - cell lung cancer , therapeutic egf receptor inhibition results in significant tumor regression in only 1020% of patients . one key goal in applying these agents to ovarian and other cancers will be to identify patients most likely to benefit from targeted therapies and to validate biomarkers of response [ 2 , 4 ] .
this type of preselection is standard in breast cancer , for example , where the estrogen receptor status of a tumor plays a major role in therapeutic decision - making strategy . clearly , a better understanding of in vivo efficacy , improved predictive biomarkers of response , and an understanding of the molecular escape pathways for egf receptor antagonists is needed in ovarian cancer . given concurrent activation of signaling pathways and pathway crosstalk in tumor cells , inhibition of multiple pathways has been proposed as a strategy to improve the impact of targeted therapeutics .
accordingly , the latest approach in clinical trials is to combine the egf receptor antagonists with inhibitors of other related or downstream signaling pathways .
phase i clinical trials in solid tumors have been presented recently at the 2009 american society of clinical oncology ( asco ) meeting demonstrating this strategy ( table 3 ) .
agents such as the mtor inhibitor everolimus and vascular endothelial growth factor receptor inhibitor bevicizumab have been combined with panitumumab , and cetuximab has been combined with the bcr / abl and src tyrosine kinase inhibitor .
the impact on biologic endpoints in vivo will be critical to assess the mechanisms of action of these combined therapies .
ongoing research continues to identify new and more effective inhibitors of egf receptor activity , and novel approaches to target antitumor therapies via the egf receptor .
exploiting the egf receptor to target and deliver drugs or imaging agents to tumor cells shows promise in preclinical models and an egf receptor targeted toxin is in clinical trials for glioblastoma .
there is resurgence of interest in this strategy based on new generations of nanocarriers with improved drug delivery characteristics and the potential to deliver multiple drugs to tumor cells .
although application of egf receptor antagonists and egf receptor targeted therapies to ovarian cancer treatment lags behind that of certain other tumors such as lung and colorectal cancers , lessons learned in using these agents in other diseases are likely to benefit ovarian cancer patients in the future . | ovarian carcinoma is the leading cause of death from gynecologic malignancy in the us .
factors such as the molecular heterogeneity of ovarian tumors and frequent diagnosis at advanced stages hamper effective disease treatment .
there is growing emphasis on the identification and development of targeted therapies to disrupt molecular pathways in cancer .
the epidermal growth factor ( egf ) receptor is one such protein target with potential utility in the management of ovarian cancer .
this paper will discuss contributions of egf receptor activation to ovarian cancer pathogenesis and the status of egf receptor inhibitors and egf receptor targeted therapies in ovarian cancer treatment . |
considering the speed and the size of data as well as the challenges to be dealt by organizations , it appears increasingly vital to have some criteria available to decide about the success and to plan based on advantages and disadvantages .
the development of a seriously competitive atmosphere among organizations and their never - ending attempts toward the promotion of quality of services and products as well as satisfaction of customer expectations and demands have made the organizations to look for a comprehensive , reliable , and flexible method to measure their performance to obtain accurate and comprehensive information on their status , position , and performance in the society and to avoid further mistakes in the future while taking their past advantages and disadvantages into consideration . in this manner
the introduction of a performance measurement system into any organization has great significance and the lack of such a system at various dimensions of the organization , including the measurement of utilization of facilities and resources , purposes , strategies , etc . ,
therefore , to become aware of the acceptability and quality of its performance , especially in dynamic and complex spaces , every organization is in a serious need for a measurement system .
however , no serious attempt has been made so far to measure the performance of libraries .
as the ideal of all libraries is to increasingly improve their performance quality and to promote their excellence , it is primarily necessary to study the current status of management performance in these libraries to measure their distance from the ideal situation of excellence
. then , through the recognition of advantages and disadvantages , they would be able to make required decisions for taking measures toward promotion and excellence .
as a process for judging about the efficiency of the predetermined plans , evaluation needs to use specific instruments and models .
various models have been suggested for the evaluation of organizational performance and each one has its own specific features .
taking into consideration the significance of the implementation of a universal management as a universal solution to the organizational efficiency improvement , the model mostly focused by the specialists of management for organizational performance measurement is organizational excellence model , among other models .
organizational excellence model helps the directors as a universal instrument with a comprehensive attitude to all aspects of organizations in order to assist them in a better and more definite recognition of the organization .
they enable an organization not only to measure the level of its successes in implementation of the programs in different periods and times , but also to identify their advantages and disadvantages and to include some projects in their performance indicator , regardless of the sector that the organization belongs to or the size it may enjoy in economic scales .
it is worth noting to add that the organizational promotion is not a theoretical conception , rather it is the acquisition and presentation of tangible and observable results that are based on evidence , sustainability , and persistence .
they are formed based on the elements and values of total quality management . among these models , deming model , malcolm baldrige performance excellence , and efqm
excellence models are three most popular organizational excellence models with the highest level of applications for organizational performance measurement [ figure 1 ] .
an outline of efqm organizational excellence model 2010 as iranian national quality award ( inqa ) has been taken from the efqm excellence model , it can be predicted that the models used in the iranian libraries will be so much similar to the efqm model in the future .
five criteria of this model are related to the enablers and explain the components of an organization and the quality of their interactions ; and other four criteria form the results obtained from the performance of an organization .
the arrow of innovation and learning helps the improvement of enablers and this , in turn , leads to the improvement of the results .
the criteria of this model are mentioned below : leadership , strategy , people , partnership and resources , processes , products and services , customer results , people results , society results , and key results .
richness of libraries affects the support for the planning process ( reduction in costs , time , and creation of peace of mind ) .
library resources play a significant role in qualitative and quantitative promotion , determining the model , and introduction of new ideas .
in contrast , inefficiency of quantitative methods and increasing demand for justification of efficiency against costs and inputs have made the managers and authorities of libraries and information centers to resort to qualitative assessment .
the excellence model is used as a strong instrument for the measurement of establishment rate in different organizations . using this model ,
organizations assess their success in implementing improvement plans in several levels , on the one hand , and then they compare their performance with other organizations , especially with the best ones . as an inclusive instrument with a comprehensive view to all aspects of organizations , the efqm excellence model assists the managers in a better and more accurate recognition of their organization . considering the fact that management is considered as the center of gravity for all organizations ,
the analysis of management performance can provide us with an understanding of the current situation of management and it would enable us to compare the situation with a favorable one .
this performance measurement for the libraries in esfahan university of medical sciences will give an insight into the library managers regarding the current situation as well as the measures that should be taken in the future in order to enable them to plan for a desirable management and to improve the quality if their services , as a result .
therefore , we will measure the performance of the libraries in esfahan university of medical sciences , using the efqm excellence model in nine dimensions : leadership , strategy , people , partnerships and resources , processes , products and services , customer results , people results , society results , and key results .
then , we will determine its advantages and disadvantages and try to improve the performance and excellence of the libraries in esfahan university of medical sciences .
( 2005 ) presented an article on excellence and quality in andalusia university library system focused on this issue that the national plan for quality measurement and its approval by the regional and academic authorities since 1996 led to an expanded recognition of quality measurement in university libraries by the spanish academic society who benefit a lot from the challenges and opportunities provided as a result of quality measurement and quality assurance activities . to them
, the reason behind the utilization of the efqm excellence model was the increasing and remarkable influence it had on many service - providing and nonprofit units in europe .
( 2005 ) carried out a research on efqm and libraries : an organizational challenge for improving the provided services .
these researchers used the efqm excellence model to measure the service quality in the specialized libraries of national cancer institute in eastern italy . in their analysis ,
the lowest score was obtained by the criterion of customer results ( 74 out of 200 ) and the highest score belonged to the criterion of leadership ( 67 out of 100 ) .
the findings indicate that spain is one of the pioneers for the application of efqm excellence model in the libraries . in their research on excellence in libraries : a systematic and integrated approach , herget and hierl ( 2007 ) studied the application of efqm excellence model in the systematic and integrated evaluation process of libraries and they referred to the existing radar logic structure in this model as its advantage for the stability and persistence of the libraries .
they implemented a plan in estuary university in switzerland according to this model , which can be successfully used to analyze the exiting situation and to identify the library 's advantages and disadvantages .
the purpose of this study is to suggest an integrated concept for management that is in consistency with the context and future demands of the libraries .
the method used for this research is the content analysis for suggesting a convenient model for libraries that would improve the library management performance .
the findings of this research show that the suggested model results in the establishments of an integrated foundation for benchmarking and optimization of the libraries in the future .
in his article titled factors influencing self - assessment in a changing library environment : case study
, borbely ( 2010 ) explained the results of two consecutive self - assessment periods carried out in the debrecen library in 2007 and 2008 .
the method of this study was based on a former study carried out by the researcher , which resulted in the creation of an efqm - based instrument for library self - assessment .
the factors influencing the organizations were evaluated and analyzed based on the efqm model : leadership , strategy , people , partnerships and resources , and processes .
the findings of this research reveal that the results of the second self - assessment were normally more positive than the results of the first self - assessment .
for instance , the empowering of human resources had a constantly higher rate in 2007 than in 2008 .
the purpose of this study is to investigate an interesting dilemma , which resulted in the modification of senior management in the organizational bases .
mohammadesmaeil , ebrahimi , and ghavidel ( 2011 ) reported a study titled comparative study of application of the efqm excellence model in central libraries of governmental and medical sciences universities of tabriz ( iran ) .
the statistical society of this research included 22 managers and authorities in the central libraries of university of tabriz and tabriz university of medical sciences .
the findings of this study indicate that the score of quality management for the central library of tabriz university of tabriz was 446.07 out of 1000 .
akhishk and farajpahlou ( 2010 ) discussed the quality measurement in their study entitled the analysis of efqm excellence model and it management quality measurement in the library of jundishapur university of medical sciences .
this was a survey - based study and its statistical population included all it managers and personnel in the library of jundishapur university of medical sciences who consisted of 30 people .
a workshop was held for each group on the way of answering the questionnaire prior to the implementation of the questionnaire .
the findings show that the library of jundishapur university of medical sciences is at an average level , with the score of 441.4208 out of 1000 of which 203.304 is estimated out of 500 for the enablers indices and 238.3904 out of 500 for the result indices .
the results reveal that the management of jundishapur university of medical sciences in ahvaz and the senior decision - makers at this university have to pay more attention to the it quality issues , situation of the personnel , and customer satisfaction and to try to improve them .
esfandiarimoghadam , zarei , and familrohani ( 2011 ) carried out a study entitled performance measurement for all archives and libraries of the irib based on efqm excellence model .
the statistical population includes all managers , authorities , and personnel at the general department of archives and libraries of irib and the sample includes all statistical population .
forty out of 50 managers , authorities , and experts from the general department participated in this research .
the findings reveal that the general department obtained the lowest level of excellence with the score of 295.83 and the society results ( 56.7% ) have the highest score and customer results ( 6.67 ) have the lowest one .
( 2005 ) presented an article on excellence and quality in andalusia university library system focused on this issue that the national plan for quality measurement and its approval by the regional and academic authorities since 1996 led to an expanded recognition of quality measurement in university libraries by the spanish academic society who benefit a lot from the challenges and opportunities provided as a result of quality measurement and quality assurance activities . to them
, the reason behind the utilization of the efqm excellence model was the increasing and remarkable influence it had on many service - providing and nonprofit units in europe .
( 2005 ) carried out a research on efqm and libraries : an organizational challenge for improving the provided services .
these researchers used the efqm excellence model to measure the service quality in the specialized libraries of national cancer institute in eastern italy . in their analysis ,
the lowest score was obtained by the criterion of customer results ( 74 out of 200 ) and the highest score belonged to the criterion of leadership ( 67 out of 100 ) .
the findings indicate that spain is one of the pioneers for the application of efqm excellence model in the libraries . in their research on excellence in libraries : a systematic and integrated approach , herget and hierl ( 2007 ) studied the application of efqm excellence model in the systematic and integrated evaluation process of libraries and they referred to the existing radar logic structure in this model as its advantage for the stability and persistence of the libraries .
they implemented a plan in estuary university in switzerland according to this model , which can be successfully used to analyze the exiting situation and to identify the library 's advantages and disadvantages .
the purpose of this study is to suggest an integrated concept for management that is in consistency with the context and future demands of the libraries .
the method used for this research is the content analysis for suggesting a convenient model for libraries that would improve the library management performance .
the findings of this research show that the suggested model results in the establishments of an integrated foundation for benchmarking and optimization of the libraries in the future . in his article titled factors influencing self - assessment in a changing library environment : case study , borbely ( 2010 ) explained the results of two consecutive self - assessment periods carried out in the debrecen library in 2007 and 2008 .
the method of this study was based on a former study carried out by the researcher , which resulted in the creation of an efqm - based instrument for library self - assessment .
the factors influencing the organizations were evaluated and analyzed based on the efqm model : leadership , strategy , people , partnerships and resources , and processes .
the findings of this research reveal that the results of the second self - assessment were normally more positive than the results of the first self - assessment .
for instance , the empowering of human resources had a constantly higher rate in 2007 than in 2008 .
the purpose of this study is to investigate an interesting dilemma , which resulted in the modification of senior management in the organizational bases .
mohammadesmaeil , ebrahimi , and ghavidel ( 2011 ) reported a study titled comparative study of application of the efqm excellence model in central libraries of governmental and medical sciences universities of tabriz ( iran ) .
the statistical society of this research included 22 managers and authorities in the central libraries of university of tabriz and tabriz university of medical sciences .
the findings of this study indicate that the score of quality management for the central library of tabriz university of tabriz was 446.07 out of 1000 .
akhishk and farajpahlou ( 2010 ) discussed the quality measurement in their study entitled the analysis of efqm excellence model and it management quality measurement in the library of jundishapur university of medical sciences .
this was a survey - based study and its statistical population included all it managers and personnel in the library of jundishapur university of medical sciences who consisted of 30 people .
a workshop was held for each group on the way of answering the questionnaire prior to the implementation of the questionnaire .
the findings show that the library of jundishapur university of medical sciences is at an average level , with the score of 441.4208 out of 1000 of which 203.304 is estimated out of 500 for the enablers indices and 238.3904 out of 500 for the result indices .
the results reveal that the management of jundishapur university of medical sciences in ahvaz and the senior decision - makers at this university have to pay more attention to the it quality issues , situation of the personnel , and customer satisfaction and to try to improve them .
esfandiarimoghadam , zarei , and familrohani ( 2011 ) carried out a study entitled performance measurement for all archives and libraries of the irib based on efqm excellence model .
the statistical population includes all managers , authorities , and personnel at the general department of archives and libraries of irib and the sample includes all statistical population .
forty out of 50 managers , authorities , and experts from the general department participated in this research .
the findings reveal that the general department obtained the lowest level of excellence with the score of 295.83 and the society results ( 56.7% ) have the highest score and customer results ( 6.67 ) have the lowest one .
this descriptive study with a survey - based approach is carried out as a cross - sectional study in 2011 .
the statistical population for this research includes the librarians and managers of hospital , faculty , and central libraries including 12 hospital libraries in esfahan ( libraries of alzahra , imam mousa kazem , amin , seyed shohada , imam hossein , shahid beheshti , shahid chamran , eisa - ibn - maryam , farabi , nour , hazrat aliasghar , and feiz hospitals ) , 7 school libraries ( libraries of the faculties of medical library and information sciences , health and nutrition , medicine , pharmacy , dentistry , nursery and midwifery , and rehabilitation ) and a central library with a total staff number of 70 ( directors and librarians ) who were sampled by the census .
the data collection instrument was a standard functional assessment questionnaire based on the efqm excellence model .
the validity of the instrument was measured by the specialists in the field of management and library science . to measure the reliability of the questionnaire , cronbach 's alpha coefficient was used and its value was 0.93 .
the percentage and frequency distribution table were used at the descriptive statistics level and t - test , anova , and spearman 's rank correlation coefficient were used for the inferential statistics level .
the performance measurement was carried out in all school , hospital , and central libraries covered by esfahan university of medical sciences .
sixty - one out of 70 personnel filled in the questionnaires and the data were analyzed after studying and analyzing the completed questionnaires .
figure 2 illustrates the mean score for the points of view of the subjects with regard to nine dimensions in general . according to figure 2 ,
leadership has the highest score ( 65.3% ) and the people and society results have the lowest score ( 55.1% ) .
mean score for the point of view of the subjects in nine dimensions of the efqm model table 1 shows the mean score for the points of view of the subjects based on their sex and their positions .
the points of view of the female subjects with regard to the society results shows a significant difference with that of the male subjects .
generally , it can be claimed that the female subjects have a more positive point of view , as compared with the male subjects .
mean score of the points of view of the subjects about the nine dimensions of efqm based on sex and their position table 2 shows the mean score for the points of view of the subjects based on their field of study and libraries that they worked there . as it can be observed from table 2 ,
the mean score for different dimensions of the efqm model is not significant for the people studied in library science and those studied in other fields of study ( p > 0.05 ) .
as it can be observed from table 2 , the mean score of different dimensions of efqm is significant for the managers and librarians ( p > 0.05 ) .
mean score of the points of view of the subjects about the nine dimensions of efqm based on their field of study and library ( place of service ) according to table 2 , the dimensions of leadership , strategy , processes and products , services , and customer results have a significant relationship with the place of service and the people who work in central library have more positive points of view .
table 3 shows the spearman correlation coefficient for the score of the points of view of the subjects about the nine dimensions of efqm based on their educational level and work records .
the educational level is only directly related with leadership and the work record is directly related with the dimensions of partnerships and resources , processes , products and services , customer result , people result , society result , and key result and it has no significant relation with other dimensions .
spearman correlation coefficient for the score of the points of view of the subjects about the nine dimensions of efqm based on their educational level and work records the mean score of the efqm model criteria is illustrated in table 4 .
based on table 4 , the mean of performance measurement of enabler 's criteria was 290 out of 500 and in results criteria was 290.95 out of 500 .
generally , the mean score for this model is 586.95 out of 1000 in this study .
the performance measurement was carried out in all school , hospital , and central libraries covered by esfahan university of medical sciences .
sixty - one out of 70 personnel filled in the questionnaires and the data were analyzed after studying and analyzing the completed questionnaires .
figure 2 illustrates the mean score for the points of view of the subjects with regard to nine dimensions in general . according to figure 2 , leadership has the highest score ( 65.3% ) and the people and society results have the lowest score ( 55.1% ) .
mean score for the point of view of the subjects in nine dimensions of the efqm model table 1 shows the mean score for the points of view of the subjects based on their sex and their positions .
the points of view of the female subjects with regard to the society results shows a significant difference with that of the male subjects .
generally , it can be claimed that the female subjects have a more positive point of view , as compared with the male subjects .
mean score of the points of view of the subjects about the nine dimensions of efqm based on sex and their position table 2 shows the mean score for the points of view of the subjects based on their field of study and libraries that they worked there . as it can be observed from table 2 ,
the mean score for different dimensions of the efqm model is not significant for the people studied in library science and those studied in other fields of study ( p > 0.05 ) .
as it can be observed from table 2 , the mean score of different dimensions of efqm is significant for the managers and librarians ( p > 0.05 ) .
mean score of the points of view of the subjects about the nine dimensions of efqm based on their field of study and library ( place of service ) according to table 2 , the dimensions of leadership , strategy , processes and products , services , and customer results have a significant relationship with the place of service and the people who work in central library have more positive points of view .
table 3 shows the spearman correlation coefficient for the score of the points of view of the subjects about the nine dimensions of efqm based on their educational level and work records .
the educational level is only directly related with leadership and the work record is directly related with the dimensions of partnerships and resources , processes , products and services , customer result , people result , society result , and key result and it has no significant relation with other dimensions . spearman correlation coefficient for the score of the points of view of the subjects about the nine dimensions of efqm based on their educational level and work records the mean score of the efqm model criteria is illustrated in table 4 .
based on table 4 , the mean of performance measurement of enabler 's criteria was 290 out of 500 and in results criteria was 290.95 out of 500 . generally , the mean score for this model is 586.95 out of 1000 in this study .
the results of this study reveal that the score of the criteria of enablers and results is generally at a poor situation [ table 4 ] . among all these dimensions , the leadership has the highest score ( 65.3% ) and the dimensions of people and society results obtain the lowest score ( 55.1% ) .
the low score of the society score generally indicates that the libraries of esfahan university of medical sciences are at a poor position in fulfilling the expectations of the society . the low score of the people result shows that the library managers have totally had a poor performance in planning and in their attempt to improve the quality of performance among the personnel , to identify and to expand knowledge and qualifications of the personnel and staff of the library based on the context and objectives of the library , to involve them in library affairs , and to hold a mutual communication with these individuals .
the data show that the cause behind the poor management of the personnel in university libraries is the inconvenient attention paid to appreciation and encouragement of the personnel which undoubtedly results from the fact that the attempts and achievements of these people are not properly recognized and no record is kept for them .
the findings of this research was poor and the lower scores were obtained , as compared with those reported by akhishk ( 2007 ) , farajpahlou and akhishk ( 2009 ) , farajpahlou , mehralizadeh , and akhishk ( 2009 ) , akhishk and farajpahlou ( 2010 ) , and esfandiarimoghadam , zarei , and familrohani ( 2011 ) .
the scores lower than the ones obtained in other similar studies is indicative of the fact that the quality management in these libraries is at a poor situation and needs further reconsiderations and improvement of quality management .
hopefully , some measures have been taken during the last years which include holding several conferences and courses .
these activities have influenced the promotion of performance measurement concepts and the application of new evaluation methods in nonprofit organizations including libraries .
it should be noted that through this study and the training of and consultation , we were successful in ensuring the libraries that the purpose of performance measurement is to help the libraries themselves and we created incentives for providing accurate and precise information .
the results of the investigation can be helpful for identifying the advantages and disadvantages and the improvement areas .
as it was mentioned earlier , the main purpose of performance measurement based on the efqm excellence model is to primarily recognize the current situation of the organization and to identify its advantages and disadvantages . in the next step
, the attempt should be made to strengthen and support the advantages and to alleviate the deficiencies and to improve the situation of the organization .
one of the significant aspects of this analysis is to identify the advantages and disadvantages of the libraries under investigation .
these can be used as a guide and assistance for managerial decision - making and policymaking .
advantages and disadvantages of the libraries covered by esfahan university of medical sciences have been elicited as follows based on the results of performance measurement of these libraries . | introduction : performance measurement is inevitable for university libraries . hence , planning and establishing a constant and up - to - date measurement system is required for the libraries , especially the university libraries .
the primary studies and analyses reveal that the efqm excellence model has been efficient , and the administrative reform program has focused on the implementation of this model .
therefore , on the basis of these facts as well as the need for a measurement system , the researchers measured the performance of libraries in schools and hospitals supported by isfahan university of medical sciences , using the efqm organizational excellence model.materials and methods : this descriptive research study was carried out by a cross - sectional survey method in 2011 .
this research study included librarians and library directors of isfahan university of medical sciences ( 70 people ) .
the validity of the instrument was measured by the specialists in the field of management and library science . to measure the reliability of the questionnaire , the cronbach 's alpha coefficient value was measured ( 0.93 ) .
the t - test , anova , and spearman 's rank correlation coefficient were used for measurements .
the data were analyzed by spss.results:data analysis revealed that the mean score of the performance measurement for the libraries under study and between nine dimensions the highest score was 65.3% for leadership dimension and the lowest scores were 55.1% for people and 55.1% for society results.conclusion:in general , using the ninth efqm model the average level of all dimensions , which is in good agreement with normal values , was assessed .
however , compared to other results , the criterion people and society results were poor .
it is recommended by forming the expert committee on criterion people and society results by individuals concerned with the various conferences and training courses to improve the aspects . |
the first case was reported in the english medical literature by larsen and solomon in 1978 .
implantation of a pregnancy within the fibrous scar of a previous cesarean section is being reported more frequently , and the incidence is now higher than that of cervical ectopic pregnancies .
there has been a substantial increase in published cases of csps in the medical literature since its first reporting . while this may reflect a true
increase in incidence given the rise in cesarean section rates over the years , it may also be attributed to improved detection of this condition , with routine use of transvaginal ultrasound ( tvus ) .
early detection of a csp is critical , as delayed diagnosis and management of this potentially life - threatening condition may result in complications , such as uterine rupture and hemorrhage with serious maternal morbidity and potential hysterectomy .
diagnosis of such a condition should be made based on a high index of suspicion from clinical and imaging investigations combined with the patient 's history and clinical manifestations .
improvements in ultrasound and magnetic resonance ( mr ) technology may enable earlier detection of a csp , thus facilitating prompt intervention to avoid potential complications . to our knowledge , there is no standardized approach to treat this condition at present ; however , various treatment options are available and should be tailored to meet the needs of each individual case .
further characterization of a csp using magnetic resonance imaging ( mri ) after initial ultrasonography may provide additional information useful for directing patient management and therapy ( e.g. conservative versus surgical management ) .
we describe a case of an ectopic pregnancy implanted in the fibrous scar of a previous cesarean section , which led to vaginal bleeding .
the patient was initially managed conservatively with systemic intramuscular methotrexate and bilateral uterine artery embolization , but ultimately required hysterectomy .
a 36-year - old woman ( gravida 5 , para 2 ) at approximately 8 weeks and 6 days gestation presented to her obstetrician complaining of heavy vaginal bleeding that had started 3 days prior . at the time of presentation
she did report a medical history significant for beta thalassemia minor and a past surgical history of two prior cesarean sections for macrosomia .
serum beta - human chorionic gonadotropin ( beta - hcg ) level at the time of presentation measured 199,760 miu / ml . a subsequent pelvic ultrasound showed a retroflexed gravid uterus with a single viable pregnancy in the lower uterine segment abutting the urinary bladder [ figure 1a d ] .
two- and 3-dimensional multiplanar views on the tvus demonstrated a normal uterine fundus superior to the gestational sac .
fetal measurements were consistent with an estimated gestational age of 8 weeks and 5 days .
fetal cardiac activity measured 159 beats per minute ( bpm ) . given the position of the gestation in the lower uterine segment and the history of two prior cesarean sections , the findings on initial ultrasound examination raised the suspicion of a cesarean scar ectopic pregnancy with possible isthmic - cervical involvement [ figure 1a d ] .
in addition , the proximity of the gestation to the urinary bladder raised the possibility of urinary bladder involvement ; however , ultrasound examination could not exclude this possibility with certainty .
the patient was referred to the hospital for admission , further evaluation with mri , and medical management with the possibility of surgical intervention .
( a ) sagittal and ( b ) transverse images from a pelvic ultrasound show a gravid retroflexed uterus measuring 12.4 6.5 7.1 cm corresponding to a volume of 299.3 cc . ( c ,
d ) additional transabdominal images show a single intrauterine gestation with positive fetal cardiac activity measuring 159 beats per minute and a crown - rump length measuring 2.1 cm corresponding to an estimated gestational age of 8 weeks and 5 days mri confirmed an intrauterine pregnancy bulging through the myometrium of the lower uterine segment with resultant mass effect on the superior right parasagittal aspect of the urinary bladder [ figure 2a d ] without direct invasion of the urinary bladder wall .
the developing placenta was positioned inferiorly in this region with little or no surrounding myometrium .
the findings on mri were diagnostic of an ectopic pregnancy in the lower uterine segment , within the known cesarean scar , and confirmed the absence of urinary bladder involvement .
( a ) sagittal t1-weighted fat - saturated contrast - enhanced image of the uterus just off midline demonstrates an intrauterine gestational sac distending the endometrial cavity .
the sac produces an outward bulge in the lower uterine segment at the site of cesarean section scar ( white arrowheads ) and is intimately related to the urinary bladder roof . enhancing trophoblastic tissue / placenta ( red arrow ) is present at the bulging site .
( b ) sagittal t2-weighted image of the uterus again shows the outward bulging gestational sac through the cesarean scar . marked thinning of the myometrium
the central portion of the bulging gestation sac appears to be covered only by thin hypointense serosa ( yellow arrowheads ) .
of note on this image is the preserved urinary bladder wall with a separating hyperintense fat plane ( white arrows ) .
( c ) coronal t2-weighted and ( d ) coronal t2-weighted fat - saturated images demonstrate the preserved hypointense urinary bladder wall at the level where the gestational sac bulging through the scar sits atop the urinary bladder .
note also the marked thinning of the overlying myometrium ( black and white arrowheads ) upon admission , conservative management was initiated .
the patient underwent a successful bilateral uterine artery embolization via a right common femoral artery approach .
the right uterine artery was embolized using five vials of 500 - 700 m microspheres and stasis of blood flow was achieved .
the left uterine artery was embolized using four vials of 500 - 700 m microspheres and an additional vial of 700 - 900 m microspheres with near stasis of flow .
the patient also received systemic intramuscular methotrexate therapy ( 80 mg administered via the left gluteus muscle ) .
quantitative serum beta - hcg levels began to decrease , though fetal cardiac activity remained present and measured in the range of 160 bpm .
a second dose of 80 mg intramuscular methotrexate was administered 3 days later with serum beta - hcg levels continuing to decline ; however , follow - up serial ultrasound examinations continued to demonstrate fetal cardiac activity in the 160 bpm range .
the patient was deemed to have failed initial conservative therapy and was given the option of an intra - amniotic injection of potassium chloride with methotrexate or a hysterectomy .
the patient ultimately decided on surgical management and underwent a total abdominal hysterectomy with extensive lysis of adhesions .
intraoperative findings revealed the urinary bladder to be densely adherent to the lower uterine segment and the unruptured pregnancy , primarily on the right as demonstrated on the mri .
pathologic evaluation of the hysterectomy specimen revealed a gravid leiomyomatous uterus with decidua in the uterine corpus as well as serosal adhesions to adipose tissue and skeletal muscle at the prior cesarean section scar .
a portion of spongy , placental tissue measuring 5.0 4.0 2.7 cm was attached to the anterior lower uterine segment with extension to the upper endocervical canal .
an embryo with four limb buds and ambiguous genitalia was present within the gestational sac [ figure 3a c ] .
( a , b ) photographs of the gross specimen post - hysterectomy show a gestational sac with a single embryo ( dashed yellow arrow ) measuring 2.4 cm in length attached by a 2.6 cm long umbilical cord ( not shown ) .
( c ) microscopic evaluation of the sample from the scarred portion of the lower uterine segment in the implantation site shows myometrium ranging from 0.1 cm to 1.8 cm in thickness ( black arrowheads ) and chorionic villi with trophoblasts ( black arrows ) invading into 1 mm of fibroadipose tissue , suggesting impending rupture .
once considered an extremely rare entity , cesarean scar pregnancies are becoming a more common occurrence with the increase in elective cesarean procedures and improvement in detection of csps with tvus .
while the cause and pathophysiology are still not well understood , to our knowledge , many theories regarding the mechanism of a csp have been proposed .
it has been hypothesized that poor vascularity in the anterior lower uterine segment impairs healing after cesarean procedures in some women , rendering this area vulnerable to formation of small dehiscent tracts or defects into which a trophoblast can implant .
women who undergo multiple cesarean sections develop scars with larger surface areas , which may in turn increase the risk of a scar implantation .
, : ( i ) implantation of the trophoblast on a prior cesarean scar with growth toward the uterine cavity and ( ii ) implantation deep in the scar defect with progression toward the bladder and abdominal cavity .
the former type may progress to a viable pregnancy with significant risk of hemorrhage , while the latter type is at greater risk of rupture .
diagnosis of a cesarean scar pregnancy requires a high index of clinical suspicion , as up to 40% of patients may be asymptomatic .
symptoms of severe acute abdominal pain with heavy vaginal bleeding should raise concerns for impending rupture while hemodynamic instability indicates rupture of a csp .
ultrasound is the first - line imaging modality for evaluation of a potential csp , with the majority of csps diagnosed on the basis of tvus .
transvaginal ultrasound has a reported sensitivity of 84.6% , and has become the imaging examination of choice for diagnosis .
sonographic criteria initially proposed by vial et al . , have been adopted by others to aid in the diagnosis of a csp : ( i ) a trophoblast located between the bladder and anterior uterine wall at the presumed site of the cesarean section scar , ( ii ) a gestational sac which is ovoid and regular in shape , as opposed to distorted and collapsed as can be seen in miscarriages , and ( iii ) a thin or discontinuous myometrium between the gestational sac and urinary bladder wall on sagittal images of the uterus through the amniotic sac .
for example , vascularity of the sac on color doppler interrogation can aid in distinguishing a csp from the avascular sac of an aborting pregnancy .
alternatively , a negative sliding organ sign when gentle pressure is applied to a sac seen at the level of the internal orifice of the uterus using the endovaginal probe can help differentiate a csp from a spontaneous abortion in progress .
findings of high velocity ( peak velocity > 20 cm / sec ) and low impedance ( pulsatility index < 1 ) waveforms on pulsed doppler have been described with scar implantation . since transvaginal ultrasound combined with color and pulsed doppler evaluation is relatively reliable in diagnosing csps , most authors suggest limiting the use of mri to cases in which tvus findings are equivocal , or when the obstetrician requires additional information in preparation for surgery .
one drawback to evaluation of csps with mri is the long acquisition time ; thus , patients need be hemodynamically stable . despite this limitation , mri has been used previously in a small number of patients as an adjunct to ultrasound evaluation . though not essential for diagnosis
, mri can have an impact on patient management in rare or complicated forms of ectopic pregnancy , as with our patient . the decision to institute medical therapy versus
sagittal and axial t1- and t2-weighted sequences can depict the gestational sac implanted in the anterior lower uterine segment and more clearly define involvement of adjacent organs ( e.g. urinary bladder ) .
in addition , the multiplanar imaging capability of mr may be helpful in orienting the surgeon , should operative management be required .
the ultrasound findings in our patient were highly suspicious of a cesarean scar pregnancy ; however , the low position of the gestational sac and its proximity to the urinary bladder presented a problem in light of the patient 's desire for future fertility .
while the ultrasound images did not clearly demonstrate urinary bladder involvement , the mr images were more conclusive and showed the intrauterine pregnancy bulging through the myometrium of the lower uterine segment , immediately adjacent to and exerting mass effect on the superior aspect of the urinary bladder without invasion of the bladder wall .
though the patient ultimately required a hysterectomy , an attempt was made to preserve fertility according to the patient 's wishes . in the case of our patient , additional evaluation with mri played a role in the decision to institute conservative medical management and may have also helped with surgical planning .
while there is no standardized approach for the treatment of this condition to our knowledge , the general consensus is that cesarean scar pregnancies should not be managed expectantly due to the risk of uterine rupture and hemorrhage .
conservative medical management includes systemic or local administration of methotrexate , local injection of embryocides ( e.g. potassium chloride , hyperosmolar glucose , or crystalline trichosanthin ) into the gestational sac , or a combination of both .
the likelihood of treatment success with methotrexate is greatest when the serum beta - hcg level is less than 5,000 miu / ml , during the first 6 weeks of pregnancy , or when the embryo exhibits no cardiac activity . of note ,
our patient had an admission serum beta - hcg level of 199,760 miu / ml and the embryo did demonstrate cardiac activity .
if conservative management is instituted , close follow - up of the pregnancy with tvus is warranted .
while serial tvus with color doppler evaluation has been shown to correlate well with serum beta - hcg levels , it has been suggested that the high - velocity , low - impedance flow associated with the gestation may not change despite intervention until the serum beta - hcg levels return to normal .
patients who demonstrate such flow characteristics on follow - up tvus despite progressively declining serum beta - hcg levels on follow - up may be at increased risk of catastrophic uterine rupture and hemorrhage , as was the case in our patient .
surgical options include combined medical treatment with surgical sac aspiration , uterine curettage , hysteroscopic evacuation , laparoscopic removal , primary open hysterotomy , or hysterectomy .
the patient 's clinical symptoms and desire for future fertility , age and size of the gestation , and the clinician 's experience dictates which treatment option is most appropriate .
patients who choose conservative medical management should be counseled regarding the possibility of requiring definitive surgical treatment in the event of failed medical therapy .
in addition , it has been suggested that the risk of spontaneous uterine rupture is 17 times more likely in women with a prior cesarean section compared to those with an unscarred uterus .
this statistic , combined with the increased risk of uterine rupture after implantation of a pregnancy in the thin cesarean scar , should be taken into consideration when contemplating conservative versus surgical management .
an alternative to the more traditional treatment options presented is combined medical management with uterine artery embolization ( uae ) , which was the therapy initially instituted in our patient .
uae is minimally invasive , and may be an attractive option should patients present with vaginal bleeding yet maintain a desire to preserve fertility .
one study of 66 patients found uae combined with local methotrexate administration to be more effective in treating csps compared to traditional methods and had increased success rate , fewer complications , and a reduced risk for hysterectomy . in our patient , uae was combined with systemic methotrexate administration rather than local injection into the gestational sac .
it has been suggested that absorption of systemic methotrexate by the conceptus may be limited due to poor vascularization of the fibrous cesarean scar .
this may have been a contributing factor in the failure of conservative therapy in our patient , who ultimately required treatment with hysterectomy .
diagnosis of a cesarean scar pregnancy requires a high index of clinical suspicion , as up to 40% of patients may be asymptomatic .
symptoms of severe acute abdominal pain with heavy vaginal bleeding should raise concerns for impending rupture while hemodynamic instability indicates rupture of a csp .
ultrasound is the first - line imaging modality for evaluation of a potential csp , with the majority of csps diagnosed on the basis of tvus .
transvaginal ultrasound has a reported sensitivity of 84.6% , and has become the imaging examination of choice for diagnosis .
, have been adopted by others to aid in the diagnosis of a csp : ( i ) a trophoblast located between the bladder and anterior uterine wall at the presumed site of the cesarean section scar , ( ii ) a gestational sac which is ovoid and regular in shape , as opposed to distorted and collapsed as can be seen in miscarriages , and ( iii ) a thin or discontinuous myometrium between the gestational sac and urinary bladder wall on sagittal images of the uterus through the amniotic sac .
for example , vascularity of the sac on color doppler interrogation can aid in distinguishing a csp from the avascular sac of an aborting pregnancy .
alternatively , a negative sliding organ sign when gentle pressure is applied to a sac seen at the level of the internal orifice of the uterus using the endovaginal probe can help differentiate a csp from a spontaneous abortion in progress .
findings of high velocity ( peak velocity > 20 cm / sec ) and low impedance ( pulsatility index < 1 ) waveforms on pulsed doppler have been described with scar implantation . since transvaginal ultrasound combined with color and pulsed doppler evaluation is relatively reliable in diagnosing csps , most authors suggest limiting the use of mri to cases in which tvus findings are equivocal , or when the obstetrician requires additional information in preparation for surgery .
one drawback to evaluation of csps with mri is the long acquisition time ; thus , patients need be hemodynamically stable . despite this limitation , mri has been used previously in a small number of patients as an adjunct to ultrasound evaluation . though not essential for diagnosis
, mri can have an impact on patient management in rare or complicated forms of ectopic pregnancy , as with our patient . the decision to institute medical therapy versus
sagittal and axial t1- and t2-weighted sequences can depict the gestational sac implanted in the anterior lower uterine segment and more clearly define involvement of adjacent organs ( e.g. urinary bladder ) .
in addition , the multiplanar imaging capability of mr may be helpful in orienting the surgeon , should operative management be required .
the ultrasound findings in our patient were highly suspicious of a cesarean scar pregnancy ; however , the low position of the gestational sac and its proximity to the urinary bladder presented a problem in light of the patient 's desire for future fertility .
while the ultrasound images did not clearly demonstrate urinary bladder involvement , the mr images were more conclusive and showed the intrauterine pregnancy bulging through the myometrium of the lower uterine segment , immediately adjacent to and exerting mass effect on the superior aspect of the urinary bladder without invasion of the bladder wall .
though the patient ultimately required a hysterectomy , an attempt was made to preserve fertility according to the patient 's wishes . in the case of our patient ,
additional evaluation with mri played a role in the decision to institute conservative medical management and may have also helped with surgical planning .
while there is no standardized approach for the treatment of this condition to our knowledge , the general consensus is that cesarean scar pregnancies should not be managed expectantly due to the risk of uterine rupture and hemorrhage .
conservative medical management includes systemic or local administration of methotrexate , local injection of embryocides ( e.g. potassium chloride , hyperosmolar glucose , or crystalline trichosanthin ) into the gestational sac , or a combination of both .
the likelihood of treatment success with methotrexate is greatest when the serum beta - hcg level is less than 5,000 miu / ml , during the first 6 weeks of pregnancy , or when the embryo exhibits no cardiac activity . of note ,
our patient had an admission serum beta - hcg level of 199,760 miu / ml and the embryo did demonstrate cardiac activity .
if conservative management is instituted , close follow - up of the pregnancy with tvus is warranted .
while serial tvus with color doppler evaluation has been shown to correlate well with serum beta - hcg levels , it has been suggested that the high - velocity , low - impedance flow associated with the gestation may not change despite intervention until the serum beta - hcg levels return to normal .
patients who demonstrate such flow characteristics on follow - up tvus despite progressively declining serum beta - hcg levels on follow - up may be at increased risk of catastrophic uterine rupture and hemorrhage , as was the case in our patient .
surgical options include combined medical treatment with surgical sac aspiration , uterine curettage , hysteroscopic evacuation , laparoscopic removal , primary open hysterotomy , or hysterectomy .
the patient 's clinical symptoms and desire for future fertility , age and size of the gestation , and the clinician 's experience dictates which treatment option is most appropriate .
patients who choose conservative medical management should be counseled regarding the possibility of requiring definitive surgical treatment in the event of failed medical therapy .
in addition , it has been suggested that the risk of spontaneous uterine rupture is 17 times more likely in women with a prior cesarean section compared to those with an unscarred uterus .
this statistic , combined with the increased risk of uterine rupture after implantation of a pregnancy in the thin cesarean scar , should be taken into consideration when contemplating conservative versus surgical management .
an alternative to the more traditional treatment options presented is combined medical management with uterine artery embolization ( uae ) , which was the therapy initially instituted in our patient .
uae is minimally invasive , and may be an attractive option should patients present with vaginal bleeding yet maintain a desire to preserve fertility .
one study of 66 patients found uae combined with local methotrexate administration to be more effective in treating csps compared to traditional methods and had increased success rate , fewer complications , and a reduced risk for hysterectomy . in our patient ,
uae was combined with systemic methotrexate administration rather than local injection into the gestational sac .
it has been suggested that absorption of systemic methotrexate by the conceptus may be limited due to poor vascularization of the fibrous cesarean scar .
this may have been a contributing factor in the failure of conservative therapy in our patient , who ultimately required treatment with hysterectomy .
cesarean scar ectopic pregnancy remains a relatively rare entity ; however , this potentially life - threatening condition is being reported more often as greater numbers of elective cesarean procedures are performed worldwide .
tvus remains the imaging modality of choice for diagnosis , though mri may play a greater role in evaluating csps .
the superior soft tissue characterization and anatomical information provided by mri allows patients and clinicians to consider conservative management as initial therapy , especially with the increasing availability of minimally invasive uae .
combined with conservative medical therapy , minimally invasive uae makes preserving fertility in the face of a csp more achievable .
while there is no standardized approach at present , intervention must be instituted early to minimize complications and maternal morbidity regardless of which therapeutic option is decided upon .
treatment must be tailored to each individual case and requires careful discussion between the patient and clinician . in the end , the decision should be made based on the patient 's clinical presentation , age of the gestation , desire for future fertility , and clinician 's experience in managing this condition . | cesarean scar pregnancies ( csps ) are a relatively rare form of ectopic pregnancy in which the embryo is implanted within the fibrous scar of a previous cesarean section . a greater number of cases of csps are currently being reported as the rates of cesarean section are increasing globally and as detection of scar pregnancy has improved with use of transvaginal ultrasound ( tvus ) with color doppler imaging .
delayed diagnosis and management of this potentially life - threatening condition may result in complications , predominantly uterine rupture and hemorrhage with significant potential maternal morbidity .
diagnosis of a cesarean scar pregnancy ( csp ) requires a high index of clinical suspicion , as up to 40% of patients may be asymptomatic .
tvus has a reported sensitivity of 84.6% and has become the imaging examination of choice for diagnosis of a csp . magnetic resonance imaging ( mri )
has been used in a small number of patients as an adjunct to tvus . in the present report ,
mri is highlighted as a problem - solving tool capable of more precisely identifying the relationship of a csp to adjacent structures , thereby providing additional information critical to directing appropriate patient management and therapy . |
dental caries , more commonly known as cavities or tooth decay , is one of the most prevalent clinical conditions in the world .
theworld health organization estimates that 60 - 90% of school children and nearly 100% of adults have or have had caries , with indications that global incidence rates are increasing .
while it is assumed that industrialized nations have better oral hygiene due to implementation of public health measures ( e.g. water fluoridation ) and improved living conditions , some developed nations have caries incidence rates greater than those of resource - limited settings .
tooth decay occurs when oral bacteria metabolize sugars and starches found in food and produce acids that erode the enamel of the tooth .
caries are strongly correlated with the presence of dental plaque which is produced by the same antagonizing bacteria , . with time
, the enamel layer is compromised and the acids attack the inner dentin and eventually the soft pulp , causing pain , sensitivity , stains , and halitosis .
dentists treat caries by manually removing the compromised tooth material and filling the remaining void . to help locate and
determine the depth of the decay , dental professionals commonly rely on radiographs in conjunction with white light visual examination .
the most popular radiographic views are bitewing , periapical , and occlusal views . because traditional radiographs capture a two - dimensional projection of all superimposed material between the film and source
the orientation and severity of the feature directly affects its radiographic visibility ; as such , some incipient caries , early demineralization , and cracks will not appear on the x - rays .
caries adjacent to and within direct line - of - sight of opaque amalgam fillings , such as instances of secondary caries , may also not be visible .
furthermore , modern radiographs generate concern among patients who seek to reduce their exposure to ionizing radiation .
it is not uncommon for patients to refuse radiographs despite the wishes of dental professionals .
the scattering properties of enamel and dentin in the nir range were first characterized by fried et al . in 1995 .
depending on the wavelength , most nir light can be transmitted across healthy enamel with marginal scattering ; 1310 nm light represents an optimal imaging wavelength as it strikes a balance between enamel and water attenuation , .
however , dental caries and demineralization scatter transiting nir light and appear as dark areas .
the depth of caries and other features can be further interrogated when comparing the dark and translucent areas at different viewing angles , illumination modes , and nir wavelengths ; fig .
stains , which absorb light and hamper assessment of the underlying material , do not manifest at nir wavelengths of 1200 nm and greater . at 1310 nm
, mild fluorosis and other shallow defects may be discernable depending on their severity , geometry , and method of imaging ; hirasuna et al .
suggest multimodal nir imaging as a potential means for differentiating between mild fluorosis and deeper caries .
taken altogether , nir dental imaging has produced condition - dependent applications that challenge or exceed two - dimensional radiographs without the use of ionizing radiation , and work has been done to compare the diagnostic capabilities of both technologies , , .
( a ) reflectance imaging has light directed at the tooth , with reflected rays collected by a camera positioned in a desired orientation ; a beamsplitter is commonly employed to redirect the illuminated surface for imaging .
( b ) transillumination imaging focuses on capturing the source light after it has transited the tooth .
emitters of low coherence , such as slds and leds , are usually placed adjacent to the tooth to maximize the input of light ; lasers can be placed further away .
transillumination most commonly has the tooth positioned between the source and camera , although this orientation is not mandatory . if the camera is angled to view the occlusal surface and is orthogonal to light source , the tooth is undergoing occlusal transillumination imaging ( c ) .
( a ) reflectance imaging has light directed at the tooth , with reflected rays collected by a camera positioned in a desired orientation ; a beamsplitter is commonly employed to redirect the illuminated surface for imaging .
( b ) transillumination imaging focuses on capturing the source light after it has transited the tooth .
emitters of low coherence , such as slds and leds , are usually placed adjacent to the tooth to maximize the input of light ; lasers can be placed further away .
transillumination most commonly has the tooth positioned between the source and camera , although this orientation is not mandatory .
if the camera is angled to view the occlusal surface and is orthogonal to light source , the tooth is undergoing occlusal transillumination imaging ( c ) . while much of the research into nir dental imaging occurs around 1310 nm ,
neighboring nir wavelengths have also demonstrated potential application depending on the illumination mode and target feature , and it is not uncommon for researchers to image the tooth across the nir range and into the short - wavelength infrared range , , . as current
ccd and cmos technologies have sensitivities that extend into the nir range , , some nir imaging is realizable with many commercial cameras .
ingaas infrared cameras , as used in some of the previously cited work , are capable of capturing in the nir and short - wavelength range , typically operating onwavelengths of 0.9 - 1.7 \documentclass[12pt]{minimal }
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\end{document } with enhanced cooling . however , ingaas cameras are comparatively more expensive that ccd and cmos cameras , pose numerous challenges for use in vivo , and typically have restricted access . while not as spectrally powerful as an ingaas camera , and despite the increased scattering properties of enamel below 1300 nm , ccd and cmos technologies remain viable options for nir dental imaging closer to the visible spectrum . a small selection of market devices utilizing nir light are available for purchase , such as the dexis carivu ( dexis , llc , hatfield , pa ) and drr dental s vistacam ix ( drr dental , bietigheim - bissingen , germany ) .
the dexis carivu uses a ccd sensor and nir light maximized at 788 nm for fixed - angle occlusal surface transillumination imaging .
the dexis carivu has two light sources that enter the buccal and lingual side of a target tooth for transillumination , and its camera is oriented directly towards the occlusal surface .
the vistacam ix by drr dental is a software - assisted handpiece incorporating a variety of illumination modes , including a proxi
when the proxi head is attached to the vistacam ix handpiece , two nir emitters are positioned on either side of a cmos sensor . despite the range of technology available to the clinician , challenges in detecting secondary and intraproximal caries in both nir imaging and two - dimensional radiographs
furthermore , there have been no studies which utilize a ccd or cmos sensor in non - occlusal imaging schemes . to test the capability of an nir intraoral imager in both reflectance and transillumination modes , as well as make the device angle independent for flexibility in imaging occluded clinical features
the user then has improved control for manipulating the frame and angle of capture , the amount of illumination , and the manner in which the tooth is illuminated .
a study population of 10 individuals was recruited to test the nir system on a variety of teeth , ranging from healthy , restored , and those under suspicion of requiring excavation .
our results showthat the nir systemwas capable of detecting clinical conditions usually imaged via x - rays as well as new dental lesions currently lacking diagnostic imaging .
the first wand contained a usb cmos camera board with its infrared filter removed , inserted into a custom 3d - printed wand housing .
reduction of the housing profile was critical for granting the camera access to as much of the mouth as possible .
an 850 nm led was embedded into a second wand , to be used as the source illumination . wands were sealed with a hard epoxy resin . during operation , the nir system was connected to a computer and a generic webcam application launched ; live video feed was displayed onscreen . while using the nir system ,
the dental professional held a wand in each hand , allowing for positional flexibility of the camera , as well as the proximity of the illumination source to the tooth .
the massachusetts institute of technology s institutional review board , the committee on the use of humans as experimental subjects reviewed and approved the clinical protocol ( 1603518840 ) .
subjects aged 18 - 70 were invited at hampden dental care in lakewood , colorado to participate in the study via a phone call or email one week prior to visiting the clinic site for routine dental prophylaxes . during the actual visit , subjects were explained the details of the study and provided the opportunity to ask questions , refuse participation , or provide written consent .
after written consent was obtained , overhead lights were turned off while a dentist imaged sites of interest with both the nir system and a white light intraoral camera .
the nir system was used to capture approximal and occlusal views in both reflectance and transillumination modes .
the carivu was selected as a reference dental imaging technology as it was the most easily obtainable and widely used nir market device in north america .
periapical / bitewing radiographs from a kodak rvg 6100 digital radiography system ( carestream dental , atlanta , ga ) were recovered from patient histories and used for x - ray comparison .
10 individuals with a variety of fillings , caries , and other dental conditions were imaged .
2a shows a white light image of tooth 9 from a 56-year - old female .
2b , blue outlines ) appear smooth and homogenous in texture in comparison to the genuine tooth material .
reduced translucency located throughout the tooth is indicative of areas of demineralization . the large demarcated patch of demineralization ( fig
. 2b , yellow outline ) was dental attrition compounded from the patient s overbite ; repeated impact from the incisal edge of the matching lower tooth eroded the protective enamel .
numerous vertical craze lines are easily visible and appear to be miniscule sites of demineralization ; it is probable that their location renders them easily cleaned during brushing , mitigating progression of caries .
a horizontal fracture along the center of the tooth spans the distance between the two restorations ( fig . 2b , cyan outline ) .
caries appear as regions darker and more clearly defined than demineralization ; the secondary caries ( fig .
2d ) ; for instance , the horizontal fracture detectable in fig . 2b , but not in fig . 2d .
images are of tooth 9 in a 56-year - old female ; views in ( a)-(c ) are taken from the lingual side with the distal direction leftwards .
the nir system s transillumination image exhibits areas of reduced luminescence indicative of demineralization or caries .
select areas of interest are highlighted ; two proximal restorations ( blue ) displaying leakage and secondary caries ( red ) .
a large area of demineralization ( yellow ) denotes a region where the incisal edge of tooth 24 on the mandible has caused enamel attrition .
( c ) periapical radiograph section with both proximal restorations visible and darkening from corresponding secondary caries .
( d ) in the dexis carivu occlusal view , teeth lacking a larger occlusal surface , such as this incisor , are more difficult to image
images are of tooth 9 in a 56-year - old female ; views in ( a)-(c ) are taken from the lingual side with the distal direction leftwards .
the nir system s transillumination image exhibits areas of reduced luminescence indicative of demineralization or caries .
select areas of interest are highlighted ; two proximal restorations ( blue ) displaying leakage and secondary caries ( red ) .
a large area of demineralization ( yellow ) denotes a region where the incisal edge of tooth 24 on the mandible has caused enamel attrition .
( c ) periapical radiograph section with both proximal restorations visible and darkening from corresponding secondary caries .
( d ) in the dexis carivu occlusal view , teeth lacking a larger occlusal surface , such as this incisor , are more difficult to image .
3 compares images of a tooth with an amalgam filling extending over the occlusal surface , captured by multiple image systems .
however , using the nir system to scan along the margin of the filling revealed a hidden secondary caries that was not readily apparent in the white light ( fig .
the dentist confirmed the presence of the secondary caries under the amalgam post - extraction . evaluating the integrity of a filling is crucial for determining when
it warrants replacement , so any method that can bypass optically - opaque features and resolve secondary caries is of interest .
( c ) amalgam appears opaque in the two - dimensional radiograph and blocks evaluation of superimposed tooth material .
( d ) scanning of the amalgam margin with the nir system shows secondary caries along the bottom of the amalgam circled in red , as seen in the radiograph in ( c ) .
midway down the side of the filling , circled in blue , was another secondary caries spot undetected by the other imaging methods .
no indication of this caries is present in the white light image in ( b ) .
( a ) was taken in vivo , and ( b)-(d ) were taken after extraction .
( c ) amalgam appears opaque in the two - dimensional radiograph and blocks evaluation of superimposed tooth material .
( d ) scanning of the amalgam margin with the nir system shows secondary caries along the bottom of the amalgam circled in red , as seen in the radiograph in ( c ) .
midway down the side of the filling , circled in blue , was another secondary caries spot undetected by the other imaging methods .
no indication of this caries is present in the white light image in ( b ) . ( a ) was taken in vivo , and ( b)-(d ) were taken after extraction .
the images captured by the nir system revealed clinical conditions , including caries , demineralization , fractures , cracks , and craze lines .
the detail afforded by nir images offers the possibility of preemptive assessment of conditions prior to their visibility on a radiograph , such as incipient caries .
differentiation of deeper caries from surface artifacts such as stains was bypassed via altering the camera view and illumination , which could be observed in real - time ; however , the most extreme surface anomalies within any subject were marginal , and further testing would be required to see how capable the nir system would be at circumventing more extensive surface features .
the nir system can be deployed as quickly as any conventional intraoral camera and without requiring the patient to leave the examination chair .
as the nir system and the carivu could only image approximately one tooth at a time , radiographs remained a faster method for surveying multiple teeth at once .
the surface of the tooth opposite the camera , as well as larger teeth , were more difficult to image with the nir system .
radiographs could also provide information deeper into the tooth volume , on the root , and on the surrounding bone .
however , the use of non - ionizing radiation was a major incentive for the patients to be imaged with either the carivu or the nir system . despite this , the fixed angle of the carivu presented unique shortcomings primarily due to its fixed plane of imaging .
other reasons that inhibited satisfactory imaging with the carivu included tooth geometry , the carivu head not properly fitting a certain tooth , and the size and/or location of the target caries .
some of these factors contributed to the imaging difficulties present in the tooth featured in fig .
since the illumination and camera are separate wand entities in the nir system , a view or set of views could be established to overcome tooth geometry limitations . on the other hand
, a single - point light source did not transilluminate the tooth as uniformly as the carivu during occlusal imaging .
table 1nir dental imaging system comparisoncapability2d radiographicdexis carivunir systemmultiple view anglesyes - requires new x - ray administration for each viewno ( occlusal view only)yesincipient carious lesionslimited ( due to sensitivity)limited ( due to view)yesfine feature visualization ( early conditions , cracks , craze lines)nolimited ( due to view)yescapable of bypassing opaque amalgamlimited ( due to amalgam orientation and view)limited ( due to view)yesavoids ionizing radiationnoyesyes the single - point wand configuration was selected after preclinical prototypes of various fixed - angle geometries were used to probe extracted teeth .
leds of varying wavelength from the 780 - 1550 nm range were also tested on extracted teeth .
preliminary findings suggested that leds of 850 nm wavelength worked best with our selected cmos camera board for lesion contrast .
further testing revealed that a bandpass filter in front of the camera aperture was not necessary if the room was sufficiently dark ; thus , room lights were turned off during data collection , and an optical filter omitted from the camera probe to further minimize profiling . at the 850 nm wavelength , transillumination imaging was far more diagnostically functional than reflectance imaging .
attempts at using the nir system for pure reflectance imaging did not take advantage of the tooth material s optical channeling .
consequently , reflectance imaging light did not penetrate deep into the tooth to reveal subsurface conditions . while it is possible that led intensity
could have been increased or exchanged for a laser source , the led transillumination proved to be much more practical .
the intensity output of the selected leds was more than enough to optically saturate the tooth during transillumination .
furthermore , laser excitation harbors increased safety concerns in living patients ; even invisible , the amplified light from a coherent nir source can permanently damage the eye .
the cost to build the nir system was modest ( less than $ 100 ) and mostly attributed to the usb webcam , which was approximately $ 80 ; the emitter itself was a few dollars .
previous work from our group has constructed similarly - themed solutions for retinas , ear , and skin imaging . with the advent of rapid prototyping and computer vision algorithms , construction and use of cost - effective oral imaging technologies
in a study of 10 subjects with varying dental conditions , we test a custom nir system capable of illuminating at 850 nm and imaging with a standard cmos usb camera . the decentralization of camera sensor and illumination source into multiple wands was essential for navigating the non - uniform topology of the mouth , and is highly recommended as a nir dental imaging configuration .
the nir system was capable of displaying subsurface caries , including those around fillings , demineralization , fractures , and cracks , all without the use of ionizing radiation .
the superb detail of the images , primarily in comparison to conventional radiographs , potentially grants the clinician greater diagnostic power .
furthermore , the inexpensive cost of the components required to produce such high quality images adds a new dimension that is in contrast to conventional imaging approaches . | 2-d radiographs , while commonly used for evaluating sub - surface hard structures of teeth , have low sensitivity for early caries lesions , particularly those on tooth occlusal surfaces .
radiographs are also frequently refused by patients over safety concerns .
translucency of teeth in the near - infrared ( nir ) range offers a non - ionizing and safe approach to detect dental caries .
we report the construction of an nir ( 850 nm ) led imaging system , comprised of an nir source and an intraoral camera for rapid dental evaluations .
the nir system was used to image teeth of ten consenting human subjects and successfully detected secondary , amalgam occluded and early caries lesions without supplementary image processing .
the camera - wand system was also capable of revealing demineralized areas , deep and superficial cracks , and other clinical features of teeth usually visualized by x - rays .
the nir system s clinical utility , simplistic design , low cost , and user friendliness make it an effective dental caries screening technology in conjunction or in place of radiographs . |
periodontal diseases include a sizeable group of pathological alterations of the periodontal tissue . the commonest forms of the disease are gingivitis and periodontitis , and these figure among the most widespread human infectious diseases [ 1 , 2 ] .
gingivitis is very common and is characterized by reversible inflammation in the marginal periodontal tissues while periodontitis is an inflammation - based infection of the support structure of the teeth with progressive destruction of them that can result in dental loss .
although several factors including systemic pathologies such as diabetes and immunosuppression or environmental factors such as smoking are capable of contributing to the severity of periodontal diseases , bacterial infection is considered to be the leading cause . as far as adult chronic periodontitis is concerned , the role of a bacterial consortium of the dental plaque , mainly composed of gram - negative strict anaerobes has been demonstrated [ 57 ] .
several virulence factors including fimbriae , hydrolases , hemolysins , hemagglutinins , and lipopolysaccharides have been detected in this microorganism and confer bacterial coaggregation capability , adhesion to epithelial cells , invasion of epithelial cells , and induction of inflammatory lympho- , chemo- , and cytokines [ 5 , 8 ] .
it is generally accepted that lowering the oral bacterial biomass is an effective method for curing and/or preventing bacterial oral pathologies .
the manual dexterity of many individuals , however , is not efficient enough for them to obtain good oral hygiene , and therefore several methods have been proposed in order to assist such people .
the simpler the methods are the more diffuse and effective they prove . in this context ,
products for regular daily oral hygiene such as mouthwashes and toothpastes which contain compounds endowed with antimicrobial and antiplaque activities are considered excellent candidates .
contrary to the commonly held opinion that food has a negative impact on oral health , research conducted over the past three decades has shown that several foods contain a number of components endowed with antibacterial and antiplaque activity [ 9 , 10 ] .
very recently , it has been shown that regular consumption of foods enriched with these active molecules is associated with modification of the oral microbial community in the direction of less periodontopathogenic microbiota .
it therefore seems reasonable to encourage the consumption of such foods and/or to incorporate the active compound(s ) in cosmetic products for daily oral hygiene .
previous studies have shown antimicrobial and antiplaque activities in low - molecular - mass ( lmm ) fractions of extracts from both an edible mushroom ( lentinus edodes , very popular in japan and called shiitake ) or from italian red chicory ( early treviso red chicory , an anthocyanic cultivar of cichorium intybus ) [ 12 , 13 ] . in this work
we evaluate the mode of antimicrobial action of fractions of these two natural extracts on p. intermedia .
basingstoke , uk ) broth to which 5 g / ml hemin and 1 g / ml vitamin k ( sigma - aldrich co. , st .
louis , mo , usa ) were added ( bhi+hk ) or in blood agar ( ba , oxoid ) plates .
cultures in both liquid and solid media were incubated at 37c in an anaerobic chamber ( whitley dg 250 anaerobic workstation , don whitley scientific , shipley , uk ) with an atmosphere composed of 85% nitrogen , 10% hydrogen , and 5% co2 . in some experiments growth of p. intermedia in a biofilm structure
was allowed on ceramic hydroxyapatite ( ha ) discs ( clarkson chromatography products inc . , south
ha discs ( 2.5 mm diameter ) were coated with sterile human saliva collected from a pool of donors .
each disc was then incubated in 5 ml of bhi+hk medium spread with a p. intermedia culture in a well of a 6-well tissue culture plate ( 35 mm diameter , flat bottom , sarstedt , verona , italy ) at 37c in the anaerobic atmosphere . every day ( for a total of five days )
culture supernatants were removed by gentile aspiration and replaced with fresh preconditioned medium . in order to analyse the effects of the test compound , on day 4 suitable dilutions of lmm fractions of either mushroom or chicory
extract were added to the growth medium following incubation for an additional 15 hours . at the end of each incubation , biofilms were washed three times with sterile distilled water and then fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ph 7.2 for sem analysis .
aqueous extracts of both shiitake mushroom and red chicory and separation by ultrafiltration of the lmm ( < 5,000 daltons ) fraction were performed as described elsewhere .
lmm fractions were lyophilised and stored up to 3 months at 80c . immediately before use , a sample was rehydrated with sterile distilled water to obtain a 10x solution and kept at 4c for no longer than a week .
the 1x concentration of the lmm fraction after reconstitution represents the original concentration in the food .
optical density ( o.d . ) was measured at 640 nm wavelength with a beckman mod .
total bacteria were counted as cell particles with a coulter counter mod zbi ( coulter scientific ) equipped with a 30 m capillary .
viable cells were determined as colony forming units ( cfu ) per ml of culture .
suitable dilutions in sterile saline solution of the untreated and treated cultures were plated in ba plates and incubated at 37c for 48 hours in the anaerobic atmosphere .
0.2 unit ) were placed in the wells of a microtitre plate together with 5 ci of [ h ] thymidine ( spec
> 10 ci / mmol ) or [ h ] uridine ( spec . act .
20 ci / mmol ) or [ h ] leucine ( spec . act .
50 ci / mmol , perkinelmer life and analytical science , boston , ma , usa ) . in some wells
microtiter plates were incubated at 37c in the anaerobic atmosphere for 60 min . at the end of this time period
the culture was overlaid on a millipore gf / c glass fibre paper soaked in advance with 10% trichloroacetic acid ( tca ) and plunged in a cold 10% tca solution .
filters were then washed three times with cold tca and , finally , twice with acetone .
glutaraldehyde - fixed samples from both planktonic and biofilm cultures were washed three times with phosphate buffer followed by three rinses in distilled water .
specimens were then serially dehydrated in ethanol and subjected to critical - point drying with co2 , mounted on metal stubs and sputter - coated with gold . each specimen was viewed with an esem feg xl30 electron microscope ( fei - philips ) at magnifications of 5,000 to 40,000 .
different concentrations of the lmm fractions of both mushroom and chicory extracts ranging from 0.25 to 1x were tested in growing p. intermedia .
figure 1 shows the effects of these concentrations on increases in optical density , cell particle number , and cell viability in p. intermedia .
as far as the lmm fraction of mushroom extract is concerned , the concentration of 0.5x was the minimum dose capable of inhibiting cell division , as evaluated by cell particle counts . at this concentration a bacteriostatic effect was observed , as well as a slight increase in o.d . in the course of the experiment .
higher concentrations ( e.g. , 1x ) had a partial bactericidal effect with a 50% decrease in viable cells .
similar results were obtained when lmm fraction of chicory extract was tested : again the minimal dose capable of inhibiting cell division was 0.5x , but , as opposed to the same concentration of mushroom extract , this concentration reduced cell viability by 50% .
dna , rna and protein synthesis of p. intermedia were evaluated in the presence of the active concentrations reported above .
table 1 summarises the results : a strong inhibitory effect on dna synthesis was observed during treatment with 1x and 0.5x concentrations of both mushroom and chicory extracts with a residual synthesis lower than 10% of the untreated control .
rna synthesis , albeit to a lesser extent compared with dna synthesis , was reduced to roughly 25% and 50% of the control in the presence of 1x and 0.5x , respectively , no matter whether mushroom or chicory was tested .
protein synthesis was inhibited by 50% in the presence of 0.5x while 1x induced greater inhibition ( 70% ) .
bacteria treated as above were also collected for morphological analysis by both optical and scanning electron microscopy ( sem ) .
preliminary observation by optical microscopy showed the presence of elongated cells as a result of the treatment .
thus , we resorted to sem analysis in order to precisely evaluate cell size and distribution .
figure 2 shows the appearance of p. intermedia during treatment with mushroom or chicory extracts .
distinctly elongated cells and several filaments with interrupted septa were seen after a three - hour treatment while untreated control cells prevalently presented the typical shape of short rods / cocco - bacilli .
table 2 shows the mean cell lengths with additional parameters of the treated cells in comparison with the untreated ones .
however , it is worthy of note that elongation was also observed in cells treated with a subinhibitory concentration ( 0.25x ) .
furthermore , analysis of the size distribution of untreated control and treated p. intermedia cells confirmed cell elongation following treatment ( figure 3 ) . in a further set of experiments we evaluated the effects of the extracts on p. intermedia biofilm architecture .
figure 4 shows that untreated control biofilm appeared as a compact structure with bacteria close to one another , while in treated biofilms , both with mushroom and with chicory ( the latter not shown in the figure ) , several gaps were observable .
a substantial number of in vitro studies have shown that components of vegetal food exert intrinsic antibacterial , antiadhesive and antiplaque activity [ 9 , 10 ] .
the main family of substances endowed with such activity is that of the polyphenolic substances .
these properties have been mainly detected against oral bacteria , although microorganisms responsible for infections of other human sites ( e.g. , lower urinary tract ) may interact with them [ 14 , 15 ] .
these observations suggest that a diet rich in polyphenols may be useful for preventing the development of oral microbial pathologies , especially those known as plaque - dependent pathologies , such as caries and gingivitis / periodontitis .
hence , the main repercussion may be the development of so - called functional foods , that is , foods enriched with healthy substances . at the same time
although a large number of studies have been conducted to evaluate the efficacy of these compounds in terms of antimicrobial , antiadhesive , and antiplaque activity , very little is known about their mode of antibacterial action .
the main goal of this research was precisely to try to elucidate this aspect . throughout the study we used p. intermedia , one of the microorganisms included in a polymicrobial complex ( mainly composed of strict anaerobe bacteria ) which is involved in the etiology of adult chronic periodontitis , a pathology present worldwide and currently considered ( together with dental caries ) the commonest infectious disease .
we evaluated the mechanism of antimicrobial action of lmm ( < 5,000 da ) fractions of aqueous extracts from both mushroom ( shiitake ) and red chicory [ 12 , 13 ] .
this concentration of mushroom extract acted bacteriostatically while the same concentration of chicory extract was slightly bactericidal after a three - hour treatment . at this concentration dna synthesis
the persistence of protein synthesis over time allows a cell mass increase , as indicated by the increase in o.d .
in fact , nonseptate long rods and filaments were observed after treatment in comparison with untreated controls .
these data suggest that the main target of action of both mushroom and chicory extracts is dna synthesis . as a result of this , inhibition of septum formation occurs , but , as expected for rod - shaped bacteria , elongation still occurs .
morphological changes are compatible with the observation that protein synthesis persists ( about 50% of the untreated control ) and o.d .
this mode of action is reminiscent of that described for antibiotics belonging to the quinolone family ( e.g. , ciprofloxacin ) .
. a similar mode of action has been described for -lactam antibiotics ( e.g. , penicillins and cephalosporins ) . in this case
septum inhibition via blockade of the penicillin binding protein involved in septum synthesis is the main target of action , and , in this example too , filamentation occurs after septum inhibition .
thus , a mode of action comparable with that of antibiotics may be supposed for these natural extracts .
this statement is further supported by the observation that sub - mics display morphogenetic effects such as those induced by the mic and higher doses , as previously demonstrated for both -lactams and quinolones [ 16 , 18 ] .
thus , this preliminary report indicates that a specific mode of action exists and the identification of specific target(s ) is needed .
this result suggests that these extracts could be used to advantage for daily oral hygiene in formulations of cosmetic products such as mouthwashes and toothpastes .
the possibility of developing cosmetic products in which the active compound or compounds are natural molecules supports the tendency of consumers to prefer natural compounds to those obtained by chemical synthesis .
furthermore , foods enriched with these active compounds may be of great interest particularly in developing countries where severer economic conditions militate against the use of commercially available products for regular oral hygiene . | contrary to the common assumption that food has a negative impact on oral health , research has shown that several foods contain a number of components with antibacterial and antiplaque activity .
these natural compounds may be useful for improving daily oral hygiene . in this study
we evaluate the mode of antimicrobial action of fractions of mushroom and red chicory extracts on prevotella intermedia , a periodontopathogenic bacterium .
the minimal inhibitory concentration corresponded to 0.5x compared to the natural food concentration for both extracts .
this concentration resulted in a bacteriostatic effect in mushroom extract and in a slightly bactericidal effect in chicory extract .
cell mass continued to increase even after division stopped . as regards macromolecular synthesis ,
dna was almost totally inhibited upon addition of either mushroom or chicory extract , and rna to a lesser extent , while protein synthesis continued .
cell elongation occurred after septum inhibition as documented by scanning electron microscopy and cell measurement .
the morphogenetic effects are reminiscent of the mode of action of antibiotics such as quinolones or -lactams .
the discovery of an antibiotic - like mode of action suggests that these extracts can be advantageously employed for daily oral hygiene in formulations of cosmetic products such as mouthwashes and toothpastes . |
transcranial direct current stimulation ( tdcs ) is a non - invasive form of brain stimulation that is thought to provide a constant low level of electrical current to the brain .
approximately 1000 scientific studies have been published in peer - reviewed journals in the last decade , many of which suggest the beneficial effects of tdcs in both clinical populations , for treating a variety of conditions and psychiatric disorders , and in healthy individuals , for enhancing everything from creative problem solving to the acquisition of motor skills . because a tdcs device is relatively easy to make ( and cheap to acquire ) , there has arisen a movement wherein individuals stimulate their own brains with tdcs outside of research or medical settings for self - improvement purposes .
the movement has been colloquially referred to as do - it - yourself ( diy ) tdcs as it began with individuals constructing tdcs devices themselves . since
today many individuals who identify with the diy tdcs movement purchase ready - made , direct - to - consumer ( dtc ) devices , the border between diy and dtc has become muddled . in this paper ,
i refer to those who use tdcs devices outside of professional research and medical settings as home users. thus , there currently exists two groups researchers and home users who utilize a single technology ( sometimes even the exact same device ) in very different ways . whereas researchers apply tdcs to subjects within the controlled realm of the laboratory , home users apply tdcs on themselves , mostly in private settings , for cognitive enhancement or self - treatment .
unsurprisingly , the use of tdcs outside of research settings has not been well received by scientists , many of whom believe that home users may ruin it for the entire academic field .
researchers have publicly voiced their concerns : a 2013 nature editorial stated that to try to boost cognitive performance in this way might be a very bad idea indeed. one tdcs researcher published a letter in nature titled transcranial devices are not playthings writing : unorthodox technologies and applications must not be allowed to distort the long - term validation of tdcs. in addition , scientists have urged caution regarding the uncontrolled use of tdcs : although no serious adverse events have been reported among the 10,000 subjects studied to date , at least one study has found that tdcs can impair cognitive function in some individuals .
currently , tdcs is not approved in the united states by the food and drug administration ( fda ) as a medical treatment for any indication .
researchers ( but not the general public ) may obtain tdcs devices for investigational use from either soterix or neuroconn , the two us companies whose devices have an investigational device exemption from the fda .
however , as the soterix and neuroconn models cost thousands of dollars , some researchers have opted to repurpose cheaper iontophoresis devices ( current - providing machines used to treat various conditions , such as excessive sweating ) for tdcs use . by contrast , consumer tdcs devices which are not regulated as medical or investigational devices are available to the public ; there are currently at least a half dozen devices on the market ranging in price from $ 49 to $ 299 .
several scientists and neuroethicists have argued that there is a need for additional regulation to cover consumer tdcs devices .
one paper proposed extending medical device regulation in europe to include not just tdcs devices but also consumer electroencephalography ( eeg ) devices , which passively record electrical brainwaves and display them to users .
( i focus here only on non - invasive electrical brain stimulation devices , because as has been previously pointed out , eeg devices in themselves are measuring tools , akin to heart - rate monitors . )
some scholars have written extensively about the moral and ethical considerations related to non - invasive brain stimulation .
others have argued that there is a need for greater engagement with the diy brain stimulation community .
collectively , existing scholarship has contributed important normative and ethical perspectives on the regulation of such devices .
missing from this literature , however , are accounts that consider the practicalities of the law and how it applies to existing and foreseeable consumer brain stimulation devices . for example , as i will show in part i below , an examination of the current tdcs consumer device market reveals that the recommended extension of medical device regulation in europe would apply to only a small sliver of devices , rendering it largely ineffective .
furthermore , many proposals neglect to consider the practical differences between regulation and regulatory enforcement .
though an ideal model of the law envisions all regulations as being consistently and equally enforced , a more realistic view takes into account the resource - constrained nature of government bodies , who must formally , and sometimes informally , prioritize regulatory enforcement and often do so in unclear or unsystematic ways .
thus , before calling for additional regulation or concluding that there is a regulatory gap , it must first be determined that the problem is the lack of regulation , not the lack of enforcement .
for example , in the united states , the fda makes its determination of whether a product is a medical device based on the intended use of the device as stated by the manufacturer not based on the mechanism of action of the device itself .
one recent paper co - authored by tdcs experts failed to take into account the intricacies of the statute , stating that it would be logical to include tdcs devices as medical devices according to the fda , regardless of whether indicated for medical treatments , diagnostic purposes , wellness aids , entertainment devices , or any other purpose
. however , legal authority carves up jurisdiction much more finely than this logic suggests . within current statutory definitions , products marketed for
entertainment or wellness purposes would not fall under the scope of the fda ( as long as they make no medical - related claims about modifying the structure or function of the body , as will be discussed in part ii below ) , whereas a device intended for medical treatment or diagnosis would indeed be regulated as a medical device .
this paper contributes to the literature on the regulation of consumer brain stimulation devices in the usa by providing a fact - based analysis of the consumer tdcs market and relevant laws and regulations . in the first section ,
i present a short history of the diy tdcs movement and the subsequent emergence of dtc devices . in the second and third sections ,
i outline the basics of fda medical device regulation and discuss how the definition of a medical device which focuses on the intended use of the device rather than its mechanism of action is of paramount importance for discussions of consumer tdcs device regulation .
i then discuss how both the fda and the courts have understood the fda 's jurisdiction over medical devices in cases where the meaning of
intended use has been challenged . in the fourth section , i analyse the only instance of tdcs regulatory action to date , in which the california department of public health ( cdph ) forced a firm to recall several hundred consumer tdcs devices .
although there exists a common perception that the fda has not been involved with the regulation of consumer tdcs devices , the california case demonstrates that the cdph 's actions were instigated by an fda engineer .
finally , i discuss the multiple us authorities , other than the fda , that can regulate consumer brain stimulation devices . in sum
, this paper dispels the notion of a regulatory gap with regard to consumer non - invasive brain stimulation ( in the united states ) .
level of regulation seem to have assumed that additional regulation is the de facto appropriate response to the existence of consumer tdcs devices .
i argue , however , that there already exists a comprehensive regulatory framework for both consumer and medical tdcs devices in the united states .
thus , rather than calling for additional regulation , i suggest the need for a practical approach to consumer brain stimulation devices , one that outlines relevant issues of concern and considers multiple ways of addressing them .
the earliest mentions of diy brain stimulation date back to 2007 , in an online forum posting on longecity.org and in an article the phoenix , which described how one individual tried to treat his depression using a tdcs device he constructed by modifying a radio shack electronics learning lab .
home use of tdcs seems to have remained isolated until mid-2011 , when a yahoo group and a reddit forum ( called a
subreddit ) dedicated to diy tdcs were formed . by early 2012 , there were a number of blogs and sites dedicated exclusively to the topic , indicating that individuals were utilizing tdcs for both self - treatment and cognitive enhancement purposes .
the appearance of the movement corresponds to the increase in popularity of tdcs in scientific journals : in 2011 , there were over 130 peer - reviewed articles about tdcs , more than double that of the previous year .
the following year , 2012 , saw the greatest quantitative increase in mentions of tdcs in the popular press .
in addition , diy tdcs shares characteristics with related movements that were well established by 2010 , such as diy biology and quantified self .
finally , and perhaps most importantly , although there are other methods of stimulating the brain with electricity or magnetism , tdcs is unique in that the stimulation device is both non - invasive ( ie no surgical implantation is required ) and relatively inexpensive to acquire or create . during the early days of the diy tdcs
, most individuals built their devices from scratch , with the help of diagrams posted online and electronics assistance from other diyers . at its core
, a tdcs device contains a current - providing component ( such as a 9 v battery ) , wires that plug into the current source , and electrodes that interface between the wire and the skin .
when both electrodes are connected to the scalp , the electrical circuit closes , and current is thought to flow through the brain .
this simple construction battery , wires , and electrodes forms the essence of a tdcs device . compared to other techniques that stimulate the brain with electricity , such as electroconvulsive therapy ( ect ) , the level of current used in tdcs is relatively low : most tdcs studies use 0.52 ma , whereas ect utilizes 500900 ma .
the notion of a ready - to - wear consumer tdcs headset first hit the media in the spring of 2012 , when two undergraduates from the university of michigan , matt sornson and nick woodhams , built a prototype of a tdcs device called the goflow ( the world 's first tdcs kit ) and promised to sell it for $ 99 .
various press outlets picked up on the story , enthusiastically describing the initiative with headlines such as buy a diy brain supercharger for $ 100 and transcranial direct current stimulation works , and you can try it at home. two months later , in may 2012 , the company announced that they were being delayed due to fda concerns , and in early 2013 the co - founders made the decision to abandon plans for the headset .
rumors swirled online that the goflow team ran into some problems with the fda .
but sornson had not been contacted by the fda ; rather , he and woodhams abandoned the goflow for personal reasons , though potential future complications with the fda were also a factor in their decision .
the company quietly sold their mailing list and domain name to a firm building a consumer tdcs device called the foc.us , and in june 2013 announced that they would not be moving forward with the project . in the year between
sornson 's and woodham 's start up and shut - down announcements , a number of other consumer tdcs devices appeared on the market : hong kong - based trans cranial technologies began selling a $ 379 device , and at least three other websites offered more affordable tdcs device kits. the kits varied in both price and level of sophistication , but usually consisted of a 9 v battery enclosure ( or a snap connector that the battery attached to ) , wires , electrodes , and a headband to facilitate electrode placement .
they were seemingly geared to those who had knowledge of tdcs but lacked the necessary soldering skills to build their own device from scratch .
some home users , like scientists , began purchasing and repurposing iontophoresis devices , which legally require a prescription but in practice are widely available online .
the foc.us device , which was released in the summer of 2013 , was arguably the first true dtc tdcs device
. with its sleek , ready - to - wear headset design , it looked more like google glass than a cobbled - together diy device .
the company 's website , advertising campaign ( featuring photos of an attractive woman wearing the device ) , and promised smartphone integration made it clear that the product was a step up from the kits sold by small - scale vendors . though the foc.us device was ostensibly marketed to gamers , its release thrust the diy tdcs movement into the spotlight and brought the debate over the regulation of cognitive enhancement devices to public attention .
since 2013 , thousands of foc.us devices have been sold and the company has released a second generation of products ; it currently has a headset specially designed for exercise .
new consumer tdcs devices are constantly appearing on the market , most often manufactured in small runs by individuals interested in , or involved with , the diy tdcs movement .
though the prospect of regulation looms large for many of these manufacturers , the only legal action to date has come at a state level , from the cdph , which effectively halted sales from tdcsdevicekit.com in may 2013 ( the case is discussed in detail in part iv below ) . in 2014
, two silicon valley start - ups announced that they were entering the consumer brain stimulation device market .
one company , halo neuroscience , issued a press release in may 2014 , noting that it had received $ 1.5 million in venture capital funding and was developing wearable technology that boosts brain function. the company 's board includes well - known names such as reed hundt , former chairman of the federal communications commission ( fcc ) . in october 2014 , a company named thync announced that it had raised $ 13 million in venture capital funding . in june 2015
, the company released its device ( $ 299 ) , which is controlled via smartphone and provides a form of non - invasive brain stimulation for mood alteration purposes ( either a
the company has reportedly tested thousands of subjects , both on its own and in collaboration with tdcs researchers , and has posted some of its results online . given that both halo neuroscience and thync are well funded and highly connected , consumer non - invasive electrical brain stimulation is certain to hit the mainstream public in the near future .
thus , what began as diy brain stimulation is likely to be superseded by or at least dwarfed by dtc brain stimulation . at present
qualitative research has shown that while some individuals from the diy tdcs movement build their own devices , others acquire a wide range of devices , from device
kits ( which require assembly ) and iontophoresis devices ( which require repurposing ) to the foc.us headset . given the variation in existing consumer tdcs devices , the consumer non - invasive brain stimulation market does not lend itself easily to regulation . indeed , the proposed extension of medical device regulation in europe would apply only to true dtc headsets ; it would not encompass self - built devices and iontophoresis devices , and it is unclear whether it would cover
kits. furthermore , if the devices from thync and halo neuroscience are sold at competitive price points , it is possible that the market may take care of itself .
that is , absent a material price difference , consumers will opt for the safest and most effective product .
as companies such as thync have already been in dialog with the fda , it is unclear how additional regulation which could take years to implement might fit into the overall picture . prior to 1976 , medical devices were not required to secure fda approval before being marketed .
but after a series of tragedies related to the implantation of pacemakers and intra - uterine devices , congress passed the medical device amendments in 1976 , which set up a new regulatory scheme for devices , classifying them based on risk level .
class i devices are low - risk devices , such as band - aids and examination gloves , which are subject to
general controls ( eg registration of facilities , device labeling , compliance with good manufacturing practices ) .
class ii devices are moderate risk devices , such as surgical drapes and breast pump kits , and are subject to additional special controls that vary by product ( eg performance standards , special labeling , and post - market surveillance ) . to date , most devices that provide a low level of electrical stimulation to the body for medical purposes , such as transcutaneous electrical nerve stimulation ( tens ) devices and powered muscle stimulation devices , are considered class ii devices .
the only class iii non - invasive electrical stimulation devices are cranial electrotherapy stimulation ( ces ) devices and some iontophoresis devices , though in 2014 the fda indicated that it planned to recategorize both as class ii . prior to being marketed , a new class iii device must submit a premarket application ( pma ) demonstrating safety and efficacy for a specific indication .
the pma process , which is similar to the new drug approval process , is often a multi - year , multi - million dollar endeavor .
thus , most medical device manufacturers take a faster and cheaper path to market , by filing a 510(k ) application to demonstrate
nearly 99 per cent of new medical devices were cleared by the fda through the 510(k ) process .
note , however , that most new class iii devices ( which require pmas ) can not be cleared via the 510(k ) process .
while the level of risk determines the regulatory process for medical devices , a product must first meet the definition of a medical device to fall within the regulatory jurisdiction of the fda . according to section 201(h ) of the food , drug & cosmetic ( fd&c ) act ,
a medical device is :
an instrument , apparatus , implement , machine , contrivance , implant , in vitro reagent , or other similar or related article , including a component part , or accessory which is :
recognized in the official national formulary , or the united states pharmacopoeia , or any supplement to them , intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease , in man or other animals , orintended to affect the structure or any function of the body of man or other animals , and which does not achieve its primary intended purposes through chemical action within or on the body of man or other animals and which is not dependent upon being metabolized for the achievement of any of its primary intended purposes .
an instrument , apparatus , implement , machine , contrivance , implant , in vitro reagent , or other similar or related article , including a component part , or accessory which is :
recognized in the official national formulary , or the united states pharmacopoeia , or any supplement to them , intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease , in man or other animals , orintended to affect the structure or any function of the body of man or other animals , and which does not achieve its primary intended purposes through chemical action within or on the body of man or other animals and which is not dependent upon being metabolized for the achievement of any of its primary intended purposes .
recognized in the official national formulary , or the united states pharmacopoeia , or any supplement to them , intended for use in the diagnosis of disease or other conditions , or in the cure , mitigation , treatment , or prevention of disease , in man or other animals , or intended to affect the structure or any function of the body of man or other animals , and which does not achieve its primary intended purposes through chemical action within or on the body of man or other animals and which is not dependent upon being metabolized for the achievement of any of its primary intended purposes .
importantly , the definition of a medical device is not based on the mechanism of action of the device , but rather on its intended use : a product is a medical device if it is intended for use in diagnosis or treatment , or intended to affect the structure or function of the body .
intended use language dates all the way back to the federal food and drugs act of 1906 .
how does the fda establish intended use ? according to the code of federal regulations title 21 , part 801 , subpart a , section 801.4 ( meaning of intended uses ) , it focuses on the objective intent of the persons legally responsible for the labeling of devices as shown on both the product 's labels and advertising .
that is , the product is classified according to the manufacturer 's representation of it .
thus , to a large extent , manufacturers can maintain control over how their products are regulated .
indeed , this was the legislative intent : a 1935 senate report noted that the manufacturer of a laxative which is a medicated candy or chewing gum can bring his product within the definition of drug and escape that of food by representing the article fairly and unequivocally as a drug product. a product 's classification as a drug or device can have far - reaching consequences : as mentioned above , new drugs ( and many new devices , if they can not demonstrate substantial equivalence to a predicate device ) must undergo costly clinical trials to demonstrate safety and efficacy , and it often takes several years to obtain fda approval . in some industries , such as cosmetics , which have lenient regulatory requirements as compared to drugs , manufacturers take great pains to ensure that their advertising and product labels do not make disease or structure / function claims .
it is no exaggeration to say that a manufacturer 's fate may hinge on specific word choices . in the classic example , a cream that claims to reduce
wrinkles will be classified as a drug ( and subject to stringent regulatory requirements ) because the wording makes a specific structure / function claim , but a cream that claims to reduce the appearance of wrinkles will be classified as a cosmetic because the language makes a beautification claim , not a structure / function one .
given the importance of intended use claims for the classification of drugs and devices , it is worthwhile to examine how manufacturers of consumer tdcs devices have represented their products . as can be seen in table 1 , the overall intended use implied by most manufacturers ( based on their websites ) is related enhancement or optimization of brain function .
two manufacturers ( tct research limited and priormind , both based in hong kong ) make explicit disease claims about their products .
several other manufacturers mention , or link to , studies on the therapeutic use of tdcs , while others refrain from referring to any such research .
marketing language from the websites of consumer tdcs devices available for purchase as of june 2015 .
furthermore , it seems that at least some manufacturers have attempted to write around the provisions of the fda .
seven of the nine manufacturers display some form of health or medical - related disclaimer on their websites , noting either that their product is not a medical device or that it is not intended to cure , treat , or diagnose diseases . to date , at least two manufacturers of tdcs devices have used the term
kit in hopes that it would put distance between their product and the fda 's definition of a medical device . while products that make explicit or implied disease claims would be classified as medical devices , it is less clear whether those that mention , or link to , tdcs research could be considered medical devices .
for example , while a link to a study describing the benefits of tdcs for depression may be considered an implied medical claim , noting that scientists are studying the use of tdcs for various indications may not be .
each manufacturer 's claim and product would have to be reviewed in their own contexts .
although such individual reviews are outside the scope of this paper , the issue of implied therapeutic claims is considered again in part iv , in the discussion of the regulatory enforcement action taken by the cdph against a small - scale consumer tdcs manufacturer . the more complex question , however , is whether language such as power your mind and increase your attention span implies an intended structure / function claim .
the fda and courts have struggled to interpret the meaning of intended to affect the structure or any function of the body as a large number of products from high - heeled shoes to chairs to treadmills can be said to be intended to affect the body 's structure or function . in general , both the fda and courts have recognized that interpreting the statute literally would lead to a large number of consumer products coming under fda regulation .
circuit court noted that congress surely did not mean for there to be a broad reading of intended to affect the structure or function because otherwise a multitude of products anything that stimulates the sensescould be considered a medical device or drug .
instead , courts have leaned toward a narrow interpretation of the statute , in which a structure / function claim must have a medical or therapeutic connotation .
three cases in particular , related to seizures of wrinkle remover creams in the 1960s , have served to clarify this interpretation . in united states v. an article sudden change , an appeals court ruled that the question of whether a product is to intended to affect the structure or function of the body hinged on whether the claim may be said to constitute a representation that the product will affect the structure of the body in some medical or drug - type fashion. in united states v. an article of drug helene curtis magic secret , a district court also upheld that a
drug connotation was necessary for a claim to be considered a structure / function one .
.. line away , an appeals court ruled that the line away product was a drug , because its structure / function claims had therapeutic implications . though the latter three cases relate to drugs , the intended to affect
indeed , in a 2002 letter , fda chief counsel daniel troy stated that the food , drug , and cosmetic act regulates only those devices whose claims have medical or therapeutic connotations . in the letter , which can be said to reflect the fda 's most recent explication of its position on intended use and structure / function claims , the fda determined that an implantable radio - frequency identification chip intended for health applications
was considered a medical device , whereas the same chip intended for use only in security and personal identification applications was not .
one that clearly affects the structure or function of the body in some manner was considered a medical device by the fda only when its intended use was medically related .
brain optimization or cognitive enhancement claims be considered to have medical or therapeutic connotations ?
indeed , the issue of what sorts of optimization claims are permissible for consumer devices has become a pressing one in recent years , due to the increase in wearable technology devices and smartphone - based health applications . in at least some industries
wellness claims ( eg supports sleep ) as opposed to therapeutic ones ( eg reduces insomnia ) will place a product outside the definition of a medical device .
for example , at the 2014 neurogaming conference , a panel of neurotechnology investors emphasized the importance of distinguishing between wellness and therapy claims for avoiding fda regulation .
indeed , many consumer eeg devices which , as mentioned in the introduction , monitor rather than stimulate the brain 's activity seem to have strategically marketed their devices for improving mental fitness and optimizing brain performance. however , it should be noted that the lack of formal enforcement action on the part of the fda in the consumer neurotechnology arena ( ie with regard to both tdcs and eeg devices ) does not necessarily indicate that the fda shares this view . to address the regulatory status of health - related wearable technology devices ,
general wellness : policy for low risk devices. although guidances are non - binding , the fda has in recent years increasingly used them to reflect its latest thinking to industry .
the draft guidance which , it should be emphasized , is not the final version of the guidance , nor will it be binding or enforceable when it is finalized indicates that the fda does not intend to enforce device provisions for general wellness products presenting a low risk to safety .
the draft guidance defines a general wellness product in terms of intended use claims : a general wellness product is one that makes claims related to maintaining or encouraging a general state of health without references to diseases or conditions . among the examples of acceptable wellness claims
concentration , problem - solving , and relaxation and stress management. furthermore , the draft guidance indicates that even certain structure / function claims are permitted under the umbrella of wellness claims : examples of acceptable claims include those related to improving muscle size , toning the body , enhancing cardiac function , and improving sexual performance , among others .
thus , at first glance , the draft guidance seems to indicate that the fda does not intend to enforce regulations for tdcs devices marketed for wellness purposes .
however , the draft guidance also provides a second criterion : the product must be a low - risk device . according to the draft guidance , a product is not a low - risk device if
it involves an intervention or technology that may pose a risk to a user 's safety if device controls are not applied. the draft guidance also states that in determining whether a device is low risk , the manufacturer should consider whether a similar device is
though the guidance provides a number of examples of low - risk devices ( mobile apps , wearable heart - rate monitors ) and non - low - risk devices ( cosmetic implants , laser technology products ) , electrical brain stimulation devices are not mentioned .
on the one hand , tdcs has been consistently shown to have relatively mild side effects ( eg surface skin burns , headaches , and dizziness ) .
on the other hand , no serious or chronic adverse events have been reported in the literature .
the recent letter from the fda to thync regarding the classification of its device supports the notion that the fda does not view consumer neurotechnology devices that have intended uses related to wellness or recreation as medical devices .
prior to going to market , thync submitted a 513(g ) , requesting information from the fda regarding how it would classify its product . according to the company ,
the fda exempted the product from medical device requirements such as pre - market approval and clearance , as the product was intended for recreational purposes .
it should be noted , however , that the same decision might not hold true for manufacturers who do not limit their claims to recreational use .
furthermore , it is worth pointing out that the communication between the fda and thync has not been made public ; all information regarding the fda 's letter has come from the company itself .
still , one question remains : if the definition of a medical device is based on its intended use and neither on risk level nor mechanism of action
could a consumer device making only wellness or recreational claims ( and not disease or medical - related structure / function ones ) fall under the scope of fda regulation ?
furthermore , how can the draft guidance pose risk level as a criterion for regulation , if risk level only defines the regulatory process once a device is considered to be within the jurisdiction of the fda ?
answering these questions requires an examination of how courts have construed the fda 's statutory authority with regard to medical devices , which is the topic of the next section . when determining the intended use of a product , the fda has historically relied almost exclusively on manufacturers claims as represented on the product 's labeling and advertising .
for example , in two cases from the 1950s , district courts upheld the focus on marketing claims , finding that cigarettes making disease prevention or weight - loss claims came under the fda 's jurisdiction , whereas cigarettes that lacked such claims fell outside it .
( both of these cases came prior to the medical device amendments of 1976 , and the courts in each case considered the cigarettes to be drugs , not devices . ) indeed , today there are many
dual use products that are identical in technology but regulated differently based on intended use as represented by the manufacturer 's intent .
for example , exercise equipment marketed for medical purposes is regulated by the fda , but exercise equipment for recreational use is regulated by the consumer product safety commission ( cpsc ) .
other dual use products include bed rails , razors , and binoculars . though the fda has focused almost exclusively on marketing language to establish intended use , a close read of 21 c.f.r .
section 801.4 ( meaning of intended uses ) shows that the fda can consider a variety of factors : intent may be shown by the circumstances surrounding the distribution of the article or oral and written statements by the manufacturers and their representatives .
indeed , the courts have affirmed the fda 's extensive powers in this regard : in one case , the seventh circuit upheld the fda 's reliance on instruction booklets , financial arrangements , and individuals testimonies in determining the intended use of a product . in another , a district court agreed that the fda could rely upon the circumstances surrounding the distribution of the article to determine that a product was a drug , even in the absence of explicit labeling .
section 801.4 , if the fda can demonstrate that the article is , with the knowledge of such persons or their representatives , offered and used for a purpose for which it is neither labeled nor advertised , it can deem the device misbranded . with these broad criteria , the fda would likely be well within its authority to take action against many small - scale tdcs manufacturers .
even if a consumer tdcs device makes no explicit or implied medical - related claims , can the way that consumers actually use the product be sufficient to imply an intended use
? this question has previously been raised in cases regarding cigarette regulation . in the late 1970s ,
a citizen action group , action on smoking and health ( ash ) , attempted to compel the fda to regulate cigarettes , arguing that the way in which consumers actually used cigarettes demonstrated that the product 's
intended use was to affect the structure or function of the body. the fda refused to regulate , citing the absence of manufacturers health claims . in 1980 's action on smoking and health ( ash )
circuit ruled that while it may be possible to demonstrate intention by showing actual consumer use , the standard was high , as it had to be shown that consumers were using a product
ash did not establish , and arguably can not establish , the near - exclusivity of consumer use of cigarettes with the intent to affect the structure or any function of the body of man
however , even if ash had been able to demonstrate that consumers were indeed using cigarettes exclusively to affect the structure or function of the body , the court would have had to confront the question of whether the structure / function use had medical or therapeutic connotations .
exercise machines for recreational purposes , for example , are used by consumers nearly exclusively to affect the structure or function of the body , yet they are not regulated as medical devices . the question of whether the actual and foreseeable use of cigarettes could be sufficient to demonstrate an intended structure / function use was raised again in 1995 , when the fda , under commissioner david kessler , attempted to regulate tobacco .
the tobacco industry challenged the fda , and the case ultimately went to the supreme court . in food and drug administration v. brown & williamson ( 2000 ) , the court rejected the fda 's assertion of jurisdiction over cigarettes but did not address the question of actual and foreseeable use vs. intended use , and the matter has never been subsequently resolved in court .
however , it should be noted that in the 2002 letter from the fda chief counsel mentioned in the previous section , the fda took the position that [ f]oreseeability by the manufacturer does not suffice to establish intended use. thus , intended use has most often been determined with relation to marketing claims ( although the fda is fully within its power to consider a broad range of factors ) and actual or foreseeable use has not been sufficient to determine intended use . while the fda and courts have favored a narrow interpretation of intended use , recognizing that a literal reading of the structure / function definition would open up the door for a large number of consumer products to come under its jurisdiction , it is important to note that courts are often willing to give deference to the fda 's statutory interpretations .
for example , in united states v. an article of drug bacto - unidisk ( 1969 ) , the supreme court upheld the fda 's decision to classify an antibiotic sensitivity disc as a drug rather than a device , because doing so would subject the product to more stringent requirements .
the court reasoned that congress had not meant for the statute to be read narrowly : rather , the fd&c act is to be given a liberal construction consistent with the act 's overriding purpose to protect the public health. subsequent opinions , such as the dissent in brown & williamson , have quoted this line when reasoning in favor of a liberal interpretation of the fd&c act .
however , it is unlikely that a similar public health argument could be made with regard to consumer tdcs , as it would have to be demonstrated that tdcs posed a significant public health risk and according to one recent empirical study , the home use of tdcs does not seem to pose an imminent risk or danger to the public. importantly , when attempting to both understand and predict the actions of the fda , it should be emphasized that the agency does not act consistently over time ; instead there is variation across different leaderships and agency actions .
thus , it is impossible to draw a single coherent picture of how the fda ( and courts ) has acted .
for example , in at least one instance , the fda seemed to contradict its stance that structure / function claims must be medically related : in july 2000 , it sent a warning letter to a company marketing a battery - powered facial mask called rejuvenique , which applied electrical stimulation to the face .
in the warning letter which , it should be noted , is not final agency action
the fda wrote that regardless of the manufacturers claims , because the rejuvenique is intended to affect the structure or function of the body by providing electrical current to various facial muscles to repeatedly contract them , it is a device . the company 's lawyers responded with a 15-page letter , arguing that the product was not a medical device because the fda classifies devices based on intended use , not mechanism of action . evidently , however , the company decided it was in their best interest to work with the fda rather than litigating , because one year later the rejuvenique was cleared via the 510(k ) process .
the rejuvenique case is particularly informative , as it exemplifies the typical negotiations between government agency and manufacturer following a warning letter .
while the rejuvenique manufacturer could have challenged the fda in court , it chose the faster and cheaper option of working with the agency . indeed
, practical business considerations may be the best predictor of how a company will engage with the fda .
many companies that have a product of unclear regulatory status especially if they are investor backed or are looking for funding will open a dialog with the fda early in the development process , so as not to risk later regulatory action .
indeed , it is hard to imagine that venture capital - backed companies such as thync and halo neuroscience would have been able to raise millions of dollars without a solid regulatory plan .
( as noted in the previous section , thync was in contact with the fda prior to going to market ; press mentions of halo neuroscience have indicated that the company intends to work with the agency . ) by contrast , a small - scale tdcs consumer device manufacturer without the funds for legal counsel would probably attempt to place its device outside of the scope of fda regulation .
a manufacturer of this kind who received a warning letter or notice of violation from the fda could in theory litigate ( assuming that its product does not have an medical or disease - related intended use according to all the factors set out in 21 c.f.r .
section 801.4 ) , but in practice it would not likely be worth the time or money .
the section below illustrates what happened when the state of california acting on an email from an engineer at the fda sent a notice of violation to a small - scale consumer tdcs manufacturer .
thus far , the only instance of regulatory enforcement against a consumer tdcs device has come at a state level , from the cdph , which in may 2013 took action against a company called tdcs device kit , inc . , for violating california 's sherman food drug , and cosmetic law . according to california state law , medical devices that are not federally approved for market in the united states can be considered misbranded and/or adulterated under the sherman law .
furthermore , the sherman law requires anyone manufacturing a medical device in california to obtain a license from the cdph . according to the cdph report
, the investigation of tdcs device kit was initiated following receipt of an email and five - page analysis from a biomedical engineer at the fda 's center for devices and radiological health .
the company , which was based in california , had been selling a home tdcs device kit via its website ( www.tdcsdevicekit.com ) .
the fda engineer had concluded that the home tdcs device kit was a class iii medical device and would require 510(k ) clearance to be legally marketed in the usa , as the company 's website implied that the device could be used to treat a variety of disorders . although the company never explicitly claimed that its own device had medical benefits , its website did contain several paragraphs about tdcs , with sentences such as :
there have been therapeutic effects shown in clinical trials involving parkinson 's disease , tinnitus , fibromyalgia , and post - stroke motor deficits. in may 2013 , an investigator from the cdph contacted the president of tdcs device kit , richard o'rourke , who believed that his product did not require cdph clearance , since the product was a kit that was assembled by the user .
a few days later , the same investigator met o'rourke and inspected the manufacturing facility ; the company was issued a notice of violation that day for non - compliance with california 's sherman food , drug and cosmetic law for manufacturing medical devices without a license from the cdph .
two weeks later , the cdph issued tdcs device kit a second notice of violation , this time with 11 items related to selling and delivering misbranded , adulterated , and unapproved medical devices. according to the notice of violation , the device was unapproved by the fda ; it was adulterated in that it did not comply with good manufacturing practices and performance standards ; and it was misbranded in that the manufacturing establishment was not licensed by the fda or the cdph , and the label failed to bear adequate warnings , directions , and other information .
the company subsequently stopped selling the devices , and a sentence was added to its website , noting that the kits were
unavailable at this time. o'rourke , however , was reluctant to issue a recall , and evidently did not take action to meet with the fda or medical device regulatory counsel to negotiate a plan to secure approval for the device . therefore , the cdph moved forward , issuing a press release on june 28 , 2103 , titled
cdph warns consumers not to use tdcs home device kit. the next month , tdcs device kit sent out a recall email to over 200 customers . the investigation was officially closed in october 2013 , following a cdph determination that the firm 's recall efforts were adequate .
first , it remains an open question why the fda engineer conducted his or her own analysis , yet forwarded the issue to the cdph .
one possibility is that the fda engineer did not have evidence of the product being sold across state lines ; the fda 's jurisdiction lies only in interstate commerce , not in intrastate commerce .
another possibility is that it was simpler to hand the case off to the state and not engender complex federal regulatory processes . in response to a query regarding the frequency of fda referrals for medical device investigations , the cdph noted that such referrals occur
occasionally. second , all items in the 11-item notice of violation referred to the device as a prescription medical device ( emphasis added ) .
the fifth item on the notice of violation was based entirely on this characterization , stating that the device was misbranded because the label failed to bear the statement caution : federal law prohibits dispensing without a prescription or similar language .
however , there is no clinical use of tdcs that is fda approved , either for prescription or over - the - counter use . when queried about the use of the term
additional information was obtained , it was determined the product was an unapproved new medical device as opposed to a prescription medical device. indeed , the second , shorter cdph report does not mention the word
third , even though tdcs device kit never made a specific disease or structure / function claim for its product , the report states that treatment claims were
thus , if statements about the clinical effects of tdcs appear on a website that sells a consumer tdcs device , representatives from the fda and the state of california have inferred that the product is a medical device . furthermore , designing and marketing a consumer tdcs device as a kit made no meaningful difference .
thus , several manufacturers that currently sell consumer tdcs devices may be liable to receive notices of violation ( at the state or federal level ) for unapproved , adulterated , or misbranded medical devices .
as noted in part ii , several manufacturers link to , or mention , research regarding the therapeutic effects of tdcs .
fourth , according to the cdph report , the fda engineer 's five - page analysis concluded that the device was a class iii device that would require a 510(k ) clearance before marketing .
this implies that the fda engineer had characterized the device as substantially equivalent to an existing pre - amendment class iii device , which as mentioned in part ii , is a device that poses the highest risk of injury and illness .
the existing class iii device most similar to tdcs is a ces device , which according to the fda 's definition , applies electrical current to a patient 's head to treat insomnia , depression , or anxiety. the main difference between the two is that while tdcs uses direct current , ces uses alternating current .
the assumption of substantial equivalence to ces would explain the cdph report 's ( incorrect ) observation that tdcs is typically used to treat depression and other mood disorders. it would also explain the use of the term
prescription in the cdph 's violation notice , as ces is currently a prescription - only device .
the comparison of tdcs to ces is especially interesting in light of the ongoing battles about whether ces should be categorized as a class ii or class iii device .
as mentioned earlier , the vast majority of non - invasive electrical stimulation devices for medical purposes are categorized as class ii devices , and ces manufacturers have long attempted to have the ces device reclassified from a class iii to class ii . in recent years , however , the fda has tried unsuccessfully , and against heavy resistance , to increase the burden on ces manufacturers . in june 2014 , in response to a flood of public comments , the fda stated that it planned to reclassify the device as class ii , although it has yet to follow through with this reclassification .
it is likely that the particular ces troubles are a consequence of being grandfathered in under the 1976 medical device amendments and automatically categorized as a class iii device ; ces was therefore never required to submit a pma demonstrating safety and efficacy .
the lack of pma for ces also explains the fda engineer 's surprising determination that the home tdcs device kit was substantially equivalent to an existing class iii device and would require 510(k ) clearance , as most new class iii devices can not be cleared via the 510(k ) process .
the engineer likely had ces in mind , because the only instance where a new class iii device can be cleared via the 510(k ) process is if it can demonstrate substantial equivalence to a preamendment device that was automatically classified as class iii , such as ces . in sum , the cdph case shows that contrary to common perception , the fda has been involved in the regulation of consumer tdcs devices ; however , it should be emphasized that the fda engineer 's email and analysis does not represent the formal position of the fda . though there was initial uncertainty on the part of the cdph regarding the status of consumer tdcs devices , state and federal regulators still considered the product to be a medical device based on manufacturer 's statements about the therapeutic effects of tdcs .
finally , the only documented case to date of the fda 's involvement in tdcs indicates that at least one engineer in its offices has viewed tdcs for medical purposes as a class iii device . while this will likely be surprising for researchers who have assumed that tdcs for medical use would be a class ii device , the report should be taken with a grain of salt , as the fda engineer 's analysis is not representative of the agency 's formal position .
indeed , another device similar to tdcs one that provides electrical stimulation to the head for headache treatment was recently classified by the fda as a class ii device .
if the fda does not recognize consumer tdcs devices as medical devices ( or opts not to enforce existing regulations ) , such products would still be subject to a multitude of consumer product safety and advertising laws .
for example , the federal trade commission ( ftc ) has the authority to take relevant administrative action for unfair or deceptive business practices .
sales of hazardous products without adequate disclosuresand therefore issues related to the sale of consumer tdcs products could fall under its scope .
while the ftc regulates the advertising of consumer products as well as over - the - counter drugs and medical devices , the fda maintains regulatory authority over the advertising of restricted ( ie prescription ) drugs and medical devices , and for the labeling of all products under its jurisdiction .
thus , if the fda classifies consumer tdcs products as unrestricted medical devices , oversight of their advertising would fall under the ftc and labeling under the fda ; if such devices are considered consumer products , oversight of both advertising and labeling would fall under the broad brush of the ftc .
two recent fda guidances the wellness device draft guidance mentioned in part ii and a 2015 guidance regarding health - related mobile applications seem to shift a regulatory burden to the ftc by placing a large class of wellness products outside the scope of fda regulation .
indeed , the ftc recently filed a complaint against a company marketing a computer game that claimed to improve cognition in children .
it also has pursued action against various mobile medical health apps in recent years , such as ones that have claimed to treat acne and diagnose melanoma . with regard to safety ,
unreasonable risks of injury associated with consumer products , and regulates products such as table saws , cribs , and carbon monoxide detectors .
the cpsc has the authority to ban products , develop safety standards , and facilitate product recalls .
all medical devices ( and other products regulated by the fda ) fall outside the scope of the cpsc .
it should be noted , however , that as a matter of practice , some authorities may be reluctant to take enforcement action over a product that may fall under the primary jurisdiction of another agency .
for example , if a product has the appearance of a medical device , an agency such as the cpsc may be hesitant to assert jurisdiction .
another federal agency , the fcc , regulates the radio frequency output of various wireless technology devices , ensuring that they meet certain standards .
while most tdcs devices from small - scale vendors do not incorporate wireless technology , both the foc.us and the thync devices have fcc certification , as they can be controlled wirelessly from a smartphone .
the fcc and the fda in some cases have overlapping jurisdiction , as they both regulate radiation - emitting products .
for example , the fcc certifies cell phones and ensures that they meet certain radio frequency standards , whereas the fda is responsible for potential cell phone - related health issues .
in addition to federal regulations , individual states also have health , safety , and medical device laws . according to section 521 of the fd&c act ,
the fda 's medical device regulations preempt ( ie supersede ) state laws . though the overlap between federal and state regulations is complex , in general
, a state law can not interfere with fda regulations ( eg it can not set lesser criteria for what constitutes a medical device ) .
however , in some cases a state can apply additional rules or more stringent requirements .
for example , the first notice of violation issued to tdcs device kit was related to a state requirement ( lack of licensing from the cdph ) , not a federal one .
currently , all us - based consumer tdcs device manufacturers are selling their products on an interstate level , but if every part of a device were to be manufactured and sold entirely within a single state
which , of course , is highly unlikely the product would fall under state , and not fda , jurisdiction .
states also have their own consumer protection laws ( often for unfair or deceptive business practices ) that would be applicable in the realm of consumer tdcs . with regard to the importation of foreign tdcs devices into the usa ( such as those from the hong kong - based companies tct research limited and priormind , both of which make explicit medical claims ) , the fda imposes identical requirements on both foreign and domestic devices .
the fda works with the department of homeland security to inspect food , drugs , cosmetics , and medical devices , and the government has broad powers to detain or seize imports .
however , the fda recognizes that it is not practical to inspect every item imported into the usa , and the agency has therefore been permissive with small quantities of drugs or devices imported for personal use . according to the fda 's regulatory procedures manual , the agency exercises enforcement discretion even for items that are in clear violation of fda regulations .
however , the manual notes that although the fda may use discretion to allow admission of certain violative items , this should not be interpreted as a license to individuals to bring in such shipments. the fda is more likely to take enforcement action when the product presents a health risk , if it is being actively promoted to us customers , or if it is being shipped repeatedly and/or in large quantities . finally , the better business bureau ( bbb ) , a non - profit non - governmental organization , plays a major role in what it is referred to as industry self - regulation. the bbb interfaces between companies and consumers to settle complaints outside of court ; it has already helped one consumer with a complaint about the foc.us device .
the bbb also has a national advertising division , which monitors companies advertising claims and attempts to resolve matters through settlements .
thus , even if consumer tdcs devices are not regulated as medical devices by the fda , they would still be subject to numerous federal and state consumer product laws .
in sum , this paper has offered an in - depth , fact - based perspective on the regulation of consumer tdcs devices in the united states . rather than a
regulatory gap , there are multiple , distinct pathways by which consumer tdcs devices can be regulated in the united states .
an examination of the existing consumer device market illustrated the complex array of various tdcs devices and the shift from diy devices to dtc ones . a review of the statutory language of the fd&c act as well as related judicial and agency interpretations demonstrated the importance of considering the precise language of the law ( and the intended use claims made for each consumer tdcs device ) as well as how the courts have construed the fda 's authority . although it is unclear whether the fda or states will attempt to assert jurisdiction over small - scale consumer tdcs manufacturers , if regulatory action is initiated , such small - scale manufacturers may be able to challenge the fda in limited circumstances , though in practice this is unlikely to be a successful or worthwhile endeavor .
in addition , an analysis of the state regulatory action taken against tdcs device kit revealed one fda engineer 's interpretation that a consumer tdcs product was a class iii device ; it also provided an indication of the level of scrutiny that states and federal bodies might adopt in determining what constitutes an implied therapeutic use claim .
finally , i have shown that if the fda does not regulate consumer tdcs products as medical devices , such products would still be subject to a multitude of consumer safety and advertising regulations , although enforcement may not be vigorous .
for example , products such as the foc.us and tdcs device kits could be regulated as consumer devices , and therefore would be subject to the consumer safety and advertising laws outlined in the previous section .
foreign device manufacturers who ship consumer tdcs devices to the usa ( such as tct research limited and priormind ) might not encounter regulatory issues if they ship in limited quantities to individuals for personal use .
companies with greater resources , such as thync and halo neuroscience , may be more likely to work with the fda ; as mentioned above , according to thync 's press release , the fda exempted the company from obtaining approval or clearance for its device .
separate from consumer tdcs devices , medical and investigational devices have their own regulatory pathways .
neuroconn could continue to provide its tdcs device to clinicians and researchers for investigational purposes only , under the fda 's investigational device exemption .
scientists could continue to repurpose iontophoresis devices for use in research studies as long as such devices are deemed to be non - significant risk devices by their local institutional review board . with regard to treatment
, psychiatrists and other medical professionals could continue to repurpose iontophoresis devices for off - label use for either cognitive enhancement or treatment ; such a practice is legal in the united states . in the future
, a company could demonstrate the safety and efficacy of its device for a specific clinical use ( eg depression ) and see it approved or cleared as a prescription - only medical device .
thus , while all of the above - mentioned examples involve some form of tdcs , each is subject to different forms of oversight .
given the multitude of regulation covering various forms of tdcs devices , it is unclear how additional regulation might fit into the picture . indeed , as i mentioned at the outset , many scholars have conflated the lack of enforcement with the lack of regulation . furthermore ,
much of the existing literature has neglected to consider current legal frameworks and factors such as the practical feasibility of implementation ( ie the procedures , costs , and length of time required to modify regulation ) , the precise targets of regulation ( exactly which devices additional regulation would affect , and how ) , and possible social implications ( such as the possibility that home users might go further
indeed , modifying a regulatory framework to questionably encompass a small sliver of devices that have yet to cause any serious adverse effects seems both impractical and unrealistic . rather than focusing on the enforcement of existing regulation which would not affect the use of home - built devices , the thync device and iontophoresis devices
a more productive method might be to begin with a review of existing devices and the populations who use them , outlining the relevant issues of concern surrounding consumer non - invasive brain stimulation .
while there has been a large body of literature on the ethics of cognitive enhancement drugs ( eg inequality with regard to distribution , compelled use ) , the bulk of the conversation surrounding consumer tdcs devices has focused on issues of safety and risk . however , further clarification is necessary when using these terms , as even a device that complies with regulatory standards may not be
safe under certain usage practices ( eg long - term use of the thync device ) ; whereas a device that does not meet technical output standards may be safe when used in a specific manner ( eg a
in other words , who is using the device is just as important as what device they are using .
furthermore , it is crucial to differentiate between short - term safety issues ( eg side effects such as skin irritation and headaches ) and long - term unknowns ( eg the possibility of deleterious cognitive effects ) .
importantly , the review could focus on establishing guidelines that address usage practices , rather than devices themselves . such guidelines , which would be along the lines of the engagement approach that has been previously suggested in the literature , could target home users , the general public , and physicians who currently administer tdcs for off - label use .
guidelines could reflect best usage practices , as well as current unknowns in the literature , and leave it to the users to make informed decisions .
such an approach could be quickly implemented , flexible , and would reach a global audience , rather than being limited to a specific country .
indeed , qualitative studies have shown that tdcs home users look to the scientific community for guidance and would be open to guidelines from researchers .
though an engagement approach to the regulatory issues surrounding diy tdcs was first proposed two years ago , as of yet , no initiative has come to fruition .
first , as home users are perceived as fringe or unorthodox by much of the scientific community , there is likely a stigma attached to engaging with them directly .
second , given that tdcs is still very much in research and investigational stages , most scientists are probably unwilling to make proclamations about what is safe and what is not , and would likely not want to take steps that may be perceived as encouraging unsupervised use .
third , guidelines could lead to liability issues ( eg if a home user injured himself despite following the guidelines ) .
fourth , given the lack of consensus within the tdcs field itself ( eg with regard to optimal dosage and potential long - term deleterious effects ) , it may be difficult for researchers to agree on a single set of guidelines . given these obstacles , it may be beneficial for such guidelines to be developed under the umbrella of a reputable third party , such as the national academy of medicine ( formerly known as the institute of medicine ) or a neuroethics committee .
a third - party might alleviate some of the stigma of interacting directly with the home users , and protections could be put into place to shield individual researchers from liability issues . rather than reflecting a single consensus
, guidelines could summarize the latest research regarding the short- and long - term issues regarding tdcs , with a focus on minimizing side effects and encouraging safe usage practices .
importantly , the committee could be an avenue not just for one - way communication from researchers to home users and the general public , but a venue for representatives of various groups to interact .
qualitative and quantitative work should continue to track tdcs home use and its potential uptake by the general public , in order to provide ongoing information regarding the use of non - invasive brain stimulation outside of academic settings .
looking ahead , it is clear that electrical brain stimulation will soon become available to a larger audience than it ever has before .
if thync 's initial results are borne out and its device does have the same effect as a glass of wine or a cup of coffee then the technology could be profoundly transformative in ways that we can not yet grasp .
rather than adopting an alarmist approach to the new reality of consumer brain stimulation , we must navigate this unfamiliar terrain with practical , grounded assessments of social and regulatory issues . establishing a forum for
the continued interaction among manufacturers , home users , and regulators would represent an important first step in this direction . | several recent articles have called for the regulation of consumer transcranial direct current stimulation ( tdcs ) devices , which provide low levels of electrical current to the brain .
however , most of the discussion to - date has focused on ethical or normative considerations ; there has been a notable absence of scholarship regarding the actual legal framework in the united states .
this article aims to fill that gap by providing a pragmatic analysis of the consumer tdcs market and relevant laws and regulations . in the five main sections of this manuscript , i take into account ( a ) the history of the do - it - yourself tdcs movement and the subsequent emergence of direct - to - consumer devices ; ( b ) the statutory language of the federal food , drug and cosmetic act and how the definition of a medical device which focuses on the intended use of the device rather than its mechanism of action is of paramount importance for discussions of consumer tdcs device regulation ; ( c ) how both the food and drug administration ( fda ) and courts have understood the fda 's jurisdiction over medical devices in cases where the meaning of intended use has been challenged ; ( d ) an analysis of consumer tdcs regulatory enforcement action to - date ; and ( e ) the multiple us authorities , other than the fda , that can regulate consumer brain stimulation devices . taken together
, this paper demonstrates that rather than a
regulatory gap, there are multiple , distinct pathways by which consumer tdcs can be regulated in the united states . |
genomic medicine and personalized medicine have become buzz phrases that pervade all fields of medicine .
omics research ( chief of which are genomics , proteinomics , and epigenomics ) over the last few years have allowed us to dissect the molecular signatures and functional pathways that underlie disease initiation and progression and to identify molecular profiles that help the classification of tumor subtypes and determine their natural course , prognosis , and responsiveness to therapies .
genomic medicine implements the use of traditional genetic information , as well as modern pangenomic information , with the aim of individualizing risk assessment , prevention , diagnosis , and treatment of cancers and other diseases .
it is of note that personalizing medical treatment based on genetic information is not a revolution of the 21st century .
indeed , the use of genetic information , such as human leukocyte antigen - matching for solid organ transplantation or blood transfusion based on abo blood group antigens , has been standard of care for several decades . however , in recent years rapid technical advances have allowed us to perform high - throughput , high - density molecular analyses to describe the genomic , proteinomic , and epigenomic make - up of an individual at a reasonable cost .
hence , the so - called genomic revolution is more or less the logical evolution from years of bench - based research and bench - to - bedside translational medicine .
the challenge is to determine how the structural changes , both within the germline and soma , affect how genes globally interact within the germline , how they interact locally between the germline and tumor and , finally , how the environment and lifestyle choices can alter genes at a functional level in order to modulate thyroid cancer susceptibility , aggressiveness , and the likelihood of cure .
molecular testing for non - inherited genetic profiles ( i.e. , the somatic alteration of genes such as kit , braf , and egfr ) to tailor and optimize treatment approaches has become implemented in the routine armament for the diagnosis and treatment of various tumors ( i.e. , lung , colon , breast , gastrointestinal stromal , thyroid , etc . ) and will not be discussed in this review ( 1)(4 ) .
for instance , it will not be long before we can tailor our surgical approaches and surveillance of patients with papillary micro - carcinomas based on the braf ( v600e ) mutation status ( 5 ) .
on the other hand , human disease susceptibility can be seen as the result of either rare germline genetic variations of high penetrance or as common genomic variants of low penetrance .
the latter path on the road to practicing genomic medicine is using the technical advances of high - throughput , high - density analyses ( e.g. , whole - genome linked analysis ) to correlate common genetic variations ( e.g. , single nucleotide polymorphisms ) to common diseases ( 6 ) . in this way , a vast variety of allelic alterations have been identified that correlate with the risk of cancer , other common diseases ( e.g. , diabetes , hypertension , autism , obesity , etc . ) or even a patient 's ability to respond to severe sepsis ( 7)(10 ) .
however , there are some obstacles to clinical implementation that must not be underestimated and the impact on public health must be critically discussed ( 12)(14 ) . beyond ethical and legal issues , numerous studies have shown that susceptibility variants for common diseases only marginally contribute to the disease risk with odd ratios of around 1.5 ( 6 ) .
for instance , in coronary heart syndrome , other medical conditions and lifestyle factors , such as blood pressure , diabetes , or smoking , are better predictors of cardiovascular events than are genomic variants ( 15 ) . furthermore , easy access to affordable , high - volume sequencing / genotyping techniques ( i.e. , the ability to sequence thousands of genes or millions of single nucleotide polymorphisms ) resulted in a flood of studies reporting the association of one or another genetic variant to disease or disease outcome ( 6 ) .
for instance , to detect an association with an odds ratio of 1.5 ( i.e. , a patient with the variant is 1.5 times more likely to have the disease or outcome ) more than 1,000 people need to be tested when the suspected susceptibility variant occurs with a frequency of 0.2 ( 20% of the population ) .
furthermore , none of the technologies used today achieves 100% accuracy . even if we propose a test accuracy of 99%
to be more realistic , we should estimate it between 80% and 95% at least one of 100 patients would receive an erroneous result .
hence , there is the widely accepted opinion that clinical implementation should be scrutinized under the framework of analytic validity , clinical validity , clinical utility , and ethical , legal , and social implications .
the last of these implies that for each test there has to be a medical treatment available to prevent the outbreak or influence the course of the disorder in those patients who are identified as being at risk .
omics screening methods should help to establish which genetic changes , whether static ( structural genomic ) or dynamic ( expression profiling ) , predispose under which environmental conditions to disrupt the usual cellular balances of apoptosis , survival , and maintenance of differentiation . in the future , this will provide us with unique genetic signatures for the prediction of outcomes and choices of therapy or prevention , or , in short , personalized genomic healthcare .
furthermore , the field of pharmacogenomics allows us to anticipate what the future can hold .
for instance the hcp5 single nucleotide polymorphism serves as a predictor for a hypersensitivity to antiviral drug medications , and the cyp2c19 variant is associated with a diminished clopidogrel response ( 16),(17 ) .
hence , patients at risk of adverse events or with limited response to medication can be identified using virtually bed - side
this review will focus on the implementation of validated personalized healthcare practices and the utilization of evidence - based genetic tests for the individualized treatment of inherited endocrine , especially thyroid , cancers .
looking back in history , one finds that endocrine neoplasias and those involved in their treatment have pioneered the field of clinical cancer genetics and personalized medicine .
there is an array of heritable endocrine neoplasias for which genes that connote a high degree of penetrance have been identified and validated . in these situations ,
molecular diagnostic and predictive testing leads to tailored surveillance and clear therapeutic implications that , at best , allow for preventive surgery .
the inherited tumor syndrome multiple endocrine neoplasia type 2 ( men 2 ) can be considered the role model for the practice of genomic medicine ( 4),(18)(20 ) .
men 2 is an autosomal - dominant , transmitted tumor syndrome caused by germline mutation in the ret ( rearrangend during transfection ) proto - oncogene and is comprised of the key endocrine neoplasia components of pheochromocytoma , hyperparathyroidism , and , importantly , medullary thyroid carcinoma ( mtc ) as the life - limiting alteration ( 21),(22 ) . about 25% of all mtcs occur as part of the autosomal - dominant men 2 syndrome , hence all patients treated for mtc should be tested for this mutation regardless of age or known family history .
the presence of the ret mtc susceptibility gene enabled us to implement a powerful molecular diagnostic test to identify mutation carriers at premorbid stages ( i.e. , predictive testing ) ( table 1 ) .
in this situation , family members who are found to carry the family - specific mutation but who are as yet unaffected can be subjected to life - saving prophylactic surgery and surveillance ( 18 ) .
importantly , the delineation of a genotype - phenotype correlation allows us to tailor surgical techniques based on the exact amino acids that are altered ( 23 ) .
furthermore , family members not harboring family - specific disease - associated mutations can be definitively advised that they only have the same risk as the general population of developing the component neoplasias of men 2 , so could be excluded from lifelong clinical surveillance and prophylactic surgeries .
first , men 2a , the most common form , is characterized by the components mtc : primary hyperparathyroidism and pheochromocytoma .
because mtc can show a high penetrance at a young age , prophylactic surgery should be offered depending on the risk genotype before the age of 510 years .
men 2b is the most aggressive form caused by germline mutations in codon 918 ( 98% ) or codon 883 ( 2% ) and is associated with an age of onset for mtc in the first year of life .
therefore , genetic testing should be offered shortly after birth in order to identify those infants who require early preventive surgery .
of note , in men 2b mtc occurs together with pheochromocytoma ( 50% ) and marfanoid habitus ( 100% ) , intestinal ganglioneuromatosis , mucosal neuromas , and thickening of the corneal nerve ( all up to 90% ) , but not primary hyperparathyroidism .
familial mtc is often considered a separate entity giving rise to mtc as the sole clinical manifestation .
most likely , familial mtc is just the result of variability in penetrance , thus surveillance for hyperparathyroidism and pheochromocytoma should be offered to these patients as well .
importantly , for the most common exon 11 mutation ( ret c634 g ) , a penetrance of 21% by age 50 for hyperparathyroidism and 52% by age 30 and 83% by age 50 for pheochromocytoma have been reported .
however , it is not possible to predict precisely if and when an individual patient might suffer from parathyroid hyperplasia or pheochromocytoma ( 24 ) .
hence , all patients should undergo lifelong surveillance . furthermore , there are other clinical aspects that can not be entirely explained by the traditional mutations in ret . for instance
, within some men 2 families , the age of onset and severity of disease vary considerably among the affected family members despite carrying identical germline ret mutations .
for example , for the most common exon 11 mutation ( cys634trp ) the age of onset for mtc varies between families from 3 years to > 60 years . to what extent allelic variants play a role is a matter of continued investigation ( 25 ) .
for instance , the variant ivs1 - 126 g > t in a large family with the g533c mutation was associated with an early onset of disease ( 26 ) .
one might envision that the integration of classic mendelian cancer predisposition genes with genomic alterations of low penetrance might bring the practice of personalized genomic medicine to the next level ( 26),(27 ) .
heritable adrenal neoplasias can affect the medulla ( pheochromocytoma ) as well as the adrenal cortex .
diagnostic work - up , management , and surveillance of patients with hereditary diseases can differ significantly from their sporadic counterparts , so it is important to identify those patients .
for instance , in men 1 , adrenocortical lesions occur in as many as 40% of patients .
most manifest as non - functional , hyperplastic lesions and present an indolent course . although the incidence of adrenocortical carcinoma has been reported to be between 2% and 6% , there is no a consensus regarding the management of men 1-associated adrenocortical lesions . even though there is no genotype - phenotype correlation , it is a widely accepted protocol to surgically remove lesions exceeding a cutoff size of 3 cm in diameter .
a considerable proportion of pheochromocytomas are heritable and form part of tumor syndromes other than men 2 . in addition
, studies have shown that pheochromocytomas can precede the development of mtc , so knowledge about differential diagnosis is important .
pheochromocytomas can occur in the adrenal medulla but also extra - adrenally in the head - and - neck region as well as being abdominal and thoracic , where they are termed paraganglioma ( pgl ) .
it used to be thought that about 10% of all pheochromocytomas occur in a familial setting and are caused by germline genetic alterations .
today , there is growing evidence that about 2530% of all patients with pheochromocytoma and paraganglioma harbor a germline mutation .
six tumor syndromes , including men 2 , von hippel lindau disease , neurofibromatosis ( nf1 ) and pheochromocytoma - paraganglioma syndrome ( pgl1 , pgl2 and pgl3 ) , and their predisposing genes have been identified ( table 3 ) .
the predominant signs of neurofibromatosis type 1 include characteristic caf - au - lait spots , neurofibromas , and other lesions such as freckling of the skin .
although penetrance of pheochromocytoma in nf1 is low ( < 5% ) , malignant pheochromocytomas are not infrequent . in patients with nf1 and pheochromocytoma
there are no mutation hot spots , instead the alterations are spread all over the gene and include large deletions or rearrangements in about 10% of patients .
as outlined above , pheochromocytoma in men 2 shows a variable penetrance that can be as high as 50% .
most notably , mutations in codons 634 and 918 harbor a very high risk of developing this disease .
von hippel - lindau syndrome is an autosomal - dominant disorder that predisposes individuals to pheochromocytoma and other important neoplasias such as clear cell renal carcinomas , pancreatic islet cell tumors , and cystic disease of the kidney and pancreas .
interestingly , although the overall penetrance of pheochromocytoma ranges between 20% and 30% of patients with von hippel - lindau syndrome , in certain families the prevalence of pheochromocytoma can be more than 90% . as a rule
, all tumorous components of von hippel - lindau syndrome can be effectively treated if diagnosed in time .
three paraganglioma syndrome predisposition genes have been identified so far , namely , sdhb the predisposition gene for pgl4 , sdhc ( pgl3 ) , and sdhd ( pgl1 ) .
patients with pgl1 nearly always display benign but multiple adrenal pheochromocytomas and paragangliomas of the head and neck .
patients with pgl4 often display extra - adrenal , thoracic , or head - and - neck paragangliomas .
in contrast to pgl1 , they are frequently malignant and about one - third of patients develop metastases .
pgl3 is not associated with malignant pheochromocytoma / paraganglioma and patients display the characteristics of age , manifestation , and tumor number similar to those with sporadic hnps .
one has to be aware of the clinical symptoms and signs that allow for an operational division , which in turn is the cornerstone of prioritized genetic testing and personalized management of patients and their relatives ( 28 ) .
hereditary pheochromocytoma and paraganglioma syndromes should be recognized as early as possible in order to tailor medical management and follow - up . whereas the clinical and pathologic call of malignancy remains a diagnostic challenge and preventive therapy
can not be advised , it is key to conduct predictive testing of as - yet - unaffected relatives , so that mutation carriers can received a tailored screening and surveillance program ( 29 ) .
in contrast to heritable mtc , for which the genetic differential diagnosis is straightforward and results in only men 2 , heritable non - mtc ( nmtc ) is more complex ( 4),(20),(24),(30 ) . in this
setting we have to differentiate between nmtcs that are part of a heritable syndrome and those that occur non - syndromically as familial nmtc .
cowden syndrome , gardner syndrome ( familial adenomatous polyposis syndrome ) , and werner syndrome all of which are transmitted in an autosomal - dominant fashion ( 31),(32 ) .
patients with cowden syndrome present with characteristic multiple hamartomas and mucocutaneous lesions ( e.g. , oral papillomatous papules , trichilemmoma , and acral keratosis ) .
in addition , affected patients are at high risk of developing various benign and malignant neoplasias , especially of the breast ( up to 50% lifetime risk in affected females ) and thyroid gland .
while benign thyroid nodules are observed in up to 75% of patients , nmtc commonly presents as a follicular thyroid carcinoma and is associated with a 10-fold increased lifetime risk compared with the general population .
gardner syndrome is caused by germline mutations in the apc gene and is characterized by gastrointestinal adenomatous polyps and a high risk of developing gastrointestinal and other cancers .
it is estimated that the lifetime risk of developing nmtc ranges between 2% and 10% in those affected with familial adenomatous polyposis syndrome .
in contrast , the gastrointestinal manifestation will progress to colorectal cancer in 100% of affected patients and preventive surgery is advised for these patients .
interestingly , nmtc will present in this setting as a unique pathologic variant known as cribriform morular variant of papillary thyroid carcinoma ( 33 ) .
therefore , any patients with this diagnosis who are operated on for thyroid disease should be screened for familial adenomatous polyposis syndrome as well .
werner syndrome is a rare autosomal recessive disease that results in premature aging ( 34 ) . besides their elderly appearance , affected patients develop various age - related disorders such as diabetes , osteoporosis , heart disease , and other malignancies .
the incidence of thyroid cancer ( e.g. , papillary , follicular , and anaplastic thyroid cancer ) is about 20% . because of the characteristic physical appearance , genetic testing
molecular diagnosis and predictive testing for these syndromes are available and highly accurate , and the results change medical management with regard to prophylaxis and surveillance .
what can not be done currently is accurately predict who will develop thyroid cancer and what the age of maximal risk is .
however , the high incidence of malignant thyroid disease warrants regular thyroid screening and a low threshold for recommending thyroidectomy . it has been suggested that there is an association between non - mtc and two other familial cancer syndromes , carney complex ( prkar1a ) and pendred syndrome .
based on a small series of 15 kindreds with carney complex the prevalence of thyroid cancer is estimated 4% in affected family members ( 35 ) . for pendred syndrome the estimated 1% prevalence for thyroid cancer is deduced from a single family report in which two of the three affected family members developed follicular thyroid cancer ( 36 ) . whether these are true associations due to the germline alterations that are involved , or if additional modifying events participate , is a matter of debate .
however , patients with carney complex typically develop nodular thyroid disease in childhood ( about 60% ) and in pendred syndrome there is a high frequency of multinodular goiters and hypothyroidism . hence , while a preventive thyroidectomy can not be advised for any of these syndromes , a regular thyroid work - up should be part of the routine surveillance for patients identified as being at risk . for
non - syndromic familial nmtc ( fnmtc ) , epidemiologic data indicate a very high likelihood of familial aggregation and , hence , a strong genetic component ( 4),(37 ) .
indeed , it is estimated that about 10% of all cases of nmtc are hereditary , putting affected first - degree relatives at up to a 10-fold increased risk compared with the general population ( 38),(39 ) .
in fact , differentiated epithelial thyroid carcinomas have one of the highest familial risks among all cancer sites .
fnmtc is characterized by two or more first - degree relatives who are affected by thyroid carcinomas ( nmtc ) but lack the signs of other hereditary syndromes or exposure to other risk factors ( e.g. , radiation ) .
this operational definition is based on the assumption that the chance of clustering of three or more relatives with nmtc in one family by chance is less than 6% ( 40 ) .
in contrast , there is a > 60% likelihood that two affected first - degree relatives will harbor sporadic disease ( 40 ) .
the standardized incidence ratio is an index that estimates the familial risk of developing a malignancy based on epidemiologic data , and as a result may be viewed as a measure of cancer risk for the children of parents with a specific malignancy . for nmtc ,
one might argue that the familial aggregation might be the result of environmental factors shared by the family members because environmental factors have been implicated in thyroid carcinogenesis .
however , studies comparing cancer risk between spouses showed that the standardized incidence ratio does not exceed 1.4 .
therefore , for nmtc , it has been argued that heritability is most likely the main contributor to the high standardized incidence ratios and that familial aggregations of nmtc are most likely the result of germline mutations in susceptibility genes . despite the impressive standardized incidence ratio for fnmtc and tremendous efforts over the last 10 years to identify the cancer - associated gene ,
no susceptibility genes have been identified so far ( 4),(42 ) . nevertheless , at least five putative susceptibility loci have been identified .
the caveat remains that for each locus evidence both in favor and opposing association with fnmtc has been published ( 42 ) .
this can be explained by the fact that the operational definition for fnmtc varies between studies ( some already include families with two affected first - degree relatives ) and some include benign thyroid diseases as well .
of the five loci on 1q21 ( prn ) , 2q21 ( nmtc1 ) , 8p23.1-p22 ( ften ) , 14q31 ( mng1 ) , and 19p13.2 ( tco ) , only tco and nmtc1 have been replicated in independent family sets , but the susceptibly genes mapped to these loci have not been identified ( 42)(46 ) .
the nmtc1 locus has been replicated in 10 kindreds and linkage to fnmtc is proposed with a logarithm of the odds ( lod ) score of 2.85 ( where a lod score > 3 is accepted to confirm linkage ) is suggested ( 42 ) .
however , it has been suggested that nmtc1 has a more significant association with the follicular variant of papillary thyroid carcinoma . a linkage between the tco ( thyroid tumors with cell oxyphilia ) locus and nmtc has been identified and validated in four studies comprised of 21 families ( lod score of 3.01 ) ( 42),(43 ) . in contrast , studies with a total of 65 relatives failed to show a linkage , so tco might contribute only slightly to fnmtc and be associated with cell oxyphilia . the mng1 locus has only shown linkage ( lod score of 4.88 ) in one kindred with two family members who were affected by papillary thyroid carcinoma .
interestingly , 18 family members showed multinodular goiters ( 44 ) . given that six other studies that included > 100 kindreds failed to reproduce this association , mng1 might rather account for a familial form of benign nodular disease rather than fnmtc .
linkage of the prn locus ( lod score of 3.58 ) was identified in one large kindred with five cases of papillary thyroid carcinoma and two cases of papillary renal neoplasm ( 45 ) .
thus , the 1q21 locus might be associated with rare phenotypes of papillary thyroid carcinoma and papillary renal neoplasm .
recently , another association was identified in one kindred with five cases of thyroid cancer and 11 cases of benign nodular disease ( 47 ) .
the putative susceptibility locus was mapped to 8q23.1-p22 with a lod score of 4.41 . however , a follow - up study by the same group included six families but failed to validate the linkage ( 48 ) .
notably , there is ongoing debate as to whether or not fnmtc displays a more aggressive phenotype compared with its sporadic counterpart ( 49 ) . indeed
, several studies have found that fnmtc presents at a younger age and displays more aggressive features , such as multifocality , local and lymph node invasion , and diminished tumor - free and overall survival rates ( 50)(53 ) . in contrast , other studies have failed to show a difference between fnmtc and sporadic cancer ( 49),(54),(55 ) . a recent study that included 67 patients from 46 families with fnmtc and 375 controls was not able to identify a more aggressive phenotype ( 49),(54 ) .
still , there is a tendency for a lower age of onset ( 42 vs. 47 years ) in the fnmtc group compared with the control group .
others have proposed prophylactic thyroidectomy for fnmtc ( 42 ) . however , from a surgeon 's standpoint , the current data are too weak and inconsistent for such a general demand .
thus , in the setting of two or more family members with nmtc , a thyroid work up should be offered to all first - degree relatives and the presence of thyroid nodular disease should have a lower threshold for recommending thyroidectomy to these patients .
the inherited tumor syndrome multiple endocrine neoplasia type 2 ( men 2 ) can be considered the role model for the practice of genomic medicine ( 4),(18)(20 ) .
men 2 is an autosomal - dominant , transmitted tumor syndrome caused by germline mutation in the ret ( rearrangend during transfection ) proto - oncogene and is comprised of the key endocrine neoplasia components of pheochromocytoma , hyperparathyroidism , and , importantly , medullary thyroid carcinoma ( mtc ) as the life - limiting alteration ( 21),(22 ) .
about 25% of all mtcs occur as part of the autosomal - dominant men 2 syndrome , hence all patients treated for mtc should be tested for this mutation regardless of age or known family history .
the presence of the ret mtc susceptibility gene enabled us to implement a powerful molecular diagnostic test to identify mutation carriers at premorbid stages ( i.e. , predictive testing ) ( table 1 ) .
in this situation , family members who are found to carry the family - specific mutation but who are as yet unaffected can be subjected to life - saving prophylactic surgery and surveillance ( 18 ) .
importantly , the delineation of a genotype - phenotype correlation allows us to tailor surgical techniques based on the exact amino acids that are altered ( 23 ) .
furthermore , family members not harboring family - specific disease - associated mutations can be definitively advised that they only have the same risk as the general population of developing the component neoplasias of men 2 , so could be excluded from lifelong clinical surveillance and prophylactic surgeries .
first , men 2a , the most common form , is characterized by the components mtc : primary hyperparathyroidism and pheochromocytoma . because mtc can show a high penetrance at a young age , prophylactic surgery should be offered depending on the risk genotype before the age of 510 years .
men 2b is the most aggressive form caused by germline mutations in codon 918 ( 98% ) or codon 883 ( 2% ) and is associated with an age of onset for mtc in the first year of life .
therefore , genetic testing should be offered shortly after birth in order to identify those infants who require early preventive surgery . of note ,
in men 2b mtc occurs together with pheochromocytoma ( 50% ) and marfanoid habitus ( 100% ) , intestinal ganglioneuromatosis , mucosal neuromas , and thickening of the corneal nerve ( all up to 90% ) , but not primary hyperparathyroidism .
familial mtc is often considered a separate entity giving rise to mtc as the sole clinical manifestation .
most likely , familial mtc is just the result of variability in penetrance , thus surveillance for hyperparathyroidism and pheochromocytoma should be offered to these patients as well .
importantly , for the most common exon 11 mutation ( ret c634 g ) , a penetrance of 21% by age 50 for hyperparathyroidism and 52% by age 30 and 83% by age 50 for pheochromocytoma have been reported .
however , it is not possible to predict precisely if and when an individual patient might suffer from parathyroid hyperplasia or pheochromocytoma ( 24 ) .
hence , all patients should undergo lifelong surveillance . furthermore , there are other clinical aspects that can not be entirely explained by the traditional mutations in ret . for instance , within some men 2 families , the age of onset and severity of disease vary considerably among the affected family members despite carrying identical germline ret mutations .
for example , for the most common exon 11 mutation ( cys634trp ) the age of onset for mtc varies between families from 3 years to > 60 years . to what extent allelic variants
for instance , the variant ivs1 - 126 g > t in a large family with the g533c mutation was associated with an early onset of disease ( 26 ) .
one might envision that the integration of classic mendelian cancer predisposition genes with genomic alterations of low penetrance might bring the practice of personalized genomic medicine to the next level ( 26),(27 ) .
heritable adrenal neoplasias can affect the medulla ( pheochromocytoma ) as well as the adrenal cortex .
diagnostic work - up , management , and surveillance of patients with hereditary diseases can differ significantly from their sporadic counterparts , so it is important to identify those patients .
for instance , in men 1 , adrenocortical lesions occur in as many as 40% of patients .
most manifest as non - functional , hyperplastic lesions and present an indolent course . although the incidence of adrenocortical carcinoma has been reported to be between 2% and 6% , there is no a consensus regarding the management of men 1-associated adrenocortical lesions . even though there is no genotype - phenotype correlation , it is a widely accepted protocol to surgically remove lesions exceeding a cutoff size of 3 cm in diameter .
a considerable proportion of pheochromocytomas are heritable and form part of tumor syndromes other than men 2 . in addition , studies have shown that pheochromocytomas can precede the development of mtc , so knowledge about differential diagnosis is important .
pheochromocytomas can occur in the adrenal medulla but also extra - adrenally in the head - and - neck region as well as being abdominal and thoracic , where they are termed paraganglioma ( pgl ) .
it used to be thought that about 10% of all pheochromocytomas occur in a familial setting and are caused by germline genetic alterations .
today , there is growing evidence that about 2530% of all patients with pheochromocytoma and paraganglioma harbor a germline mutation .
six tumor syndromes , including men 2 , von hippel lindau disease , neurofibromatosis ( nf1 ) and pheochromocytoma - paraganglioma syndrome ( pgl1 , pgl2 and pgl3 ) , and their predisposing genes have been identified ( table 3 ) .
the predominant signs of neurofibromatosis type 1 include characteristic caf - au - lait spots , neurofibromas , and other lesions such as freckling of the skin .
although penetrance of pheochromocytoma in nf1 is low ( < 5% ) , malignant pheochromocytomas are not infrequent . in patients with nf1 and pheochromocytoma
there are no mutation hot spots , instead the alterations are spread all over the gene and include large deletions or rearrangements in about 10% of patients .
as outlined above , pheochromocytoma in men 2 shows a variable penetrance that can be as high as 50% .
most notably , mutations in codons 634 and 918 harbor a very high risk of developing this disease .
von hippel - lindau syndrome is an autosomal - dominant disorder that predisposes individuals to pheochromocytoma and other important neoplasias such as clear cell renal carcinomas , pancreatic islet cell tumors , and cystic disease of the kidney and pancreas .
interestingly , although the overall penetrance of pheochromocytoma ranges between 20% and 30% of patients with von hippel - lindau syndrome , in certain families the prevalence of pheochromocytoma can be more than 90% . as a rule
, all tumorous components of von hippel - lindau syndrome can be effectively treated if diagnosed in time .
three paraganglioma syndrome predisposition genes have been identified so far , namely , sdhb the predisposition gene for pgl4 , sdhc ( pgl3 ) , and sdhd ( pgl1 ) .
patients with pgl1 nearly always display benign but multiple adrenal pheochromocytomas and paragangliomas of the head and neck .
patients with pgl4 often display extra - adrenal , thoracic , or head - and - neck paragangliomas .
in contrast to pgl1 , they are frequently malignant and about one - third of patients develop metastases .
pgl3 is not associated with malignant pheochromocytoma / paraganglioma and patients display the characteristics of age , manifestation , and tumor number similar to those with sporadic hnps .
one has to be aware of the clinical symptoms and signs that allow for an operational division , which in turn is the cornerstone of prioritized genetic testing and personalized management of patients and their relatives ( 28 ) .
hereditary pheochromocytoma and paraganglioma syndromes should be recognized as early as possible in order to tailor medical management and follow - up . whereas the clinical and pathologic call of malignancy remains a diagnostic challenge and preventive therapy can not be advised , it is key to conduct predictive testing of as - yet - unaffected relatives , so that mutation carriers can received a tailored screening and surveillance program ( 29 ) .
in contrast to heritable mtc , for which the genetic differential diagnosis is straightforward and results in only men 2 , heritable non - mtc ( nmtc ) is more complex ( 4),(20),(24),(30 ) . in this
setting we have to differentiate between nmtcs that are part of a heritable syndrome and those that occur non - syndromically as familial nmtc .
cowden syndrome , gardner syndrome ( familial adenomatous polyposis syndrome ) , and werner syndrome all of which are transmitted in an autosomal - dominant fashion ( 31),(32 ) .
patients with cowden syndrome present with characteristic multiple hamartomas and mucocutaneous lesions ( e.g. , oral papillomatous papules , trichilemmoma , and acral keratosis ) .
in addition , affected patients are at high risk of developing various benign and malignant neoplasias , especially of the breast ( up to 50% lifetime risk in affected females ) and thyroid gland .
while benign thyroid nodules are observed in up to 75% of patients , nmtc commonly presents as a follicular thyroid carcinoma and is associated with a 10-fold increased lifetime risk compared with the general population .
gardner syndrome is caused by germline mutations in the apc gene and is characterized by gastrointestinal adenomatous polyps and a high risk of developing gastrointestinal and other cancers .
it is estimated that the lifetime risk of developing nmtc ranges between 2% and 10% in those affected with familial adenomatous polyposis syndrome .
in contrast , the gastrointestinal manifestation will progress to colorectal cancer in 100% of affected patients and preventive surgery is advised for these patients .
interestingly , nmtc will present in this setting as a unique pathologic variant known as cribriform morular variant of papillary thyroid carcinoma ( 33 ) .
therefore , any patients with this diagnosis who are operated on for thyroid disease should be screened for familial adenomatous polyposis syndrome as well .
werner syndrome is a rare autosomal recessive disease that results in premature aging ( 34 ) . besides their elderly appearance , affected patients develop various age - related disorders such as diabetes , osteoporosis , heart disease , and other malignancies .
the incidence of thyroid cancer ( e.g. , papillary , follicular , and anaplastic thyroid cancer ) is about 20% . because of the characteristic physical appearance , genetic testing
is commonly used to confirm the clinical diagnosis . molecular diagnosis and predictive testing for these syndromes are available and highly accurate , and the results change medical management with regard to prophylaxis and surveillance .
what can not be done currently is accurately predict who will develop thyroid cancer and what the age of maximal risk is .
however , the high incidence of malignant thyroid disease warrants regular thyroid screening and a low threshold for recommending thyroidectomy . it has been suggested that there is an association between non - mtc and two other familial cancer syndromes , carney complex ( prkar1a ) and pendred syndrome .
based on a small series of 15 kindreds with carney complex the prevalence of thyroid cancer is estimated 4% in affected family members ( 35 ) . for pendred syndrome
the estimated 1% prevalence for thyroid cancer is deduced from a single family report in which two of the three affected family members developed follicular thyroid cancer ( 36 ) . whether these are true associations due to the germline alterations that are involved , or if additional modifying events participate , is a matter of debate .
however , patients with carney complex typically develop nodular thyroid disease in childhood ( about 60% ) and in pendred syndrome there is a high frequency of multinodular goiters and hypothyroidism . hence , while a preventive thyroidectomy can not be advised for any of these syndromes , a regular thyroid work - up should be part of the routine surveillance for patients identified as being at risk .
for non - syndromic familial nmtc ( fnmtc ) , epidemiologic data indicate a very high likelihood of familial aggregation and , hence , a strong genetic component ( 4),(37 ) .
indeed , it is estimated that about 10% of all cases of nmtc are hereditary , putting affected first - degree relatives at up to a 10-fold increased risk compared with the general population ( 38),(39 ) .
in fact , differentiated epithelial thyroid carcinomas have one of the highest familial risks among all cancer sites .
fnmtc is characterized by two or more first - degree relatives who are affected by thyroid carcinomas ( nmtc ) but lack the signs of other hereditary syndromes or exposure to other risk factors ( e.g. , radiation ) .
this operational definition is based on the assumption that the chance of clustering of three or more relatives with nmtc in one family by chance is less than 6% ( 40 ) .
in contrast , there is a > 60% likelihood that two affected first - degree relatives will harbor sporadic disease ( 40 ) .
the standardized incidence ratio is an index that estimates the familial risk of developing a malignancy based on epidemiologic data , and as a result may be viewed as a measure of cancer risk for the children of parents with a specific malignancy .
for nmtc , the standardized incidence ratio exceeds an impressive value of 3.8 ( 38),(41 ) .
one might argue that the familial aggregation might be the result of environmental factors shared by the family members because environmental factors have been implicated in thyroid carcinogenesis .
however , studies comparing cancer risk between spouses showed that the standardized incidence ratio does not exceed 1.4 .
therefore , for nmtc , it has been argued that heritability is most likely the main contributor to the high standardized incidence ratios and that familial aggregations of nmtc are most likely the result of germline mutations in susceptibility genes . despite the impressive standardized incidence ratio for fnmtc and tremendous efforts over the last 10 years to identify the cancer - associated gene ,
no susceptibility genes have been identified so far ( 4),(42 ) . nevertheless , at least five putative susceptibility loci have been identified .
the caveat remains that for each locus evidence both in favor and opposing association with fnmtc has been published ( 42 ) .
this can be explained by the fact that the operational definition for fnmtc varies between studies ( some already include families with two affected first - degree relatives ) and some include benign thyroid diseases as well .
of the five loci on 1q21 ( prn ) , 2q21 ( nmtc1 ) , 8p23.1-p22 ( ften ) , 14q31 ( mng1 ) , and 19p13.2 ( tco ) , only tco and nmtc1 have been replicated in independent family sets , but the susceptibly genes mapped to these loci have not been identified ( 42)(46 ) .
the nmtc1 locus has been replicated in 10 kindreds and linkage to fnmtc is proposed with a logarithm of the odds ( lod ) score of 2.85 ( where a lod score > 3 is accepted to confirm linkage ) is suggested ( 42 ) .
however , it has been suggested that nmtc1 has a more significant association with the follicular variant of papillary thyroid carcinoma .
a linkage between the tco ( thyroid tumors with cell oxyphilia ) locus and nmtc has been identified and validated in four studies comprised of 21 families ( lod score of 3.01 ) ( 42),(43 ) .
in contrast , studies with a total of 65 relatives failed to show a linkage , so tco might contribute only slightly to fnmtc and be associated with cell oxyphilia .
the mng1 locus has only shown linkage ( lod score of 4.88 ) in one kindred with two family members who were affected by papillary thyroid carcinoma .
interestingly , 18 family members showed multinodular goiters ( 44 ) . given that six other studies that included > 100 kindreds failed to reproduce this association , mng1 might rather account for a familial form of benign nodular disease rather than fnmtc .
linkage of the prn locus ( lod score of 3.58 ) was identified in one large kindred with five cases of papillary thyroid carcinoma and two cases of papillary renal neoplasm ( 45 ) .
thus , the 1q21 locus might be associated with rare phenotypes of papillary thyroid carcinoma and papillary renal neoplasm . recently
, another association was identified in one kindred with five cases of thyroid cancer and 11 cases of benign nodular disease ( 47 ) .
however , a follow - up study by the same group included six families but failed to validate the linkage ( 48 ) .
notably , there is ongoing debate as to whether or not fnmtc displays a more aggressive phenotype compared with its sporadic counterpart ( 49 ) . indeed
, several studies have found that fnmtc presents at a younger age and displays more aggressive features , such as multifocality , local and lymph node invasion , and diminished tumor - free and overall survival rates ( 50)(53 ) .
in contrast , other studies have failed to show a difference between fnmtc and sporadic cancer ( 49),(54),(55 ) .
a recent study that included 67 patients from 46 families with fnmtc and 375 controls was not able to identify a more aggressive phenotype ( 49),(54 ) .
still , there is a tendency for a lower age of onset ( 42 vs. 47 years ) in the fnmtc group compared with the control group .
others have proposed prophylactic thyroidectomy for fnmtc ( 42 ) . however , from a surgeon 's standpoint , the current data are too weak and inconsistent for such a general demand .
thus , in the setting of two or more family members with nmtc , a thyroid work up should be offered to all first - degree relatives and the presence of thyroid nodular disease should have a lower threshold for recommending thyroidectomy to these patients .
over the recent years , interest in the field of personalized medicine has increased . in fact
, one has to wonder if personalized medicine is snowballing out of control when direct - to - consumer genomic testing is becoming increasingly available ( 56 ) . indeed , since 2007 the interested consumer is able to receive a whole - genome scan for less than us$500 , without ever having to see a doctor or receive medical advice .
this kind of self - administered genetic testing is problematic , as shown by bloss et al .
( 56 ) who found a correlation between test - related distress and increased lifetime risk .
various professional organizations , including the american medical association and the american college of medical genetics , state that such genetic testing should not be performed without the supervision of healthcare providers .
however , centers for genomic medicine , and even clinical cancer genetics , are far from being ubiquitously established even in high - level medical centers .
it is estimated that only 1% of patients that would benefit from this 21st century approach to medical treatment will be referred to professional genetic counseling ( 31 ) .
the implementation of state - of - the - art clinical genetic counseling within the constraints of budget and limited human resources is one of the important tasks that have to be tackled .
eventually , one has to keep in mind that , notwithstanding all of these technological advances , several tumor syndromes can be identified by rigorous clinical analysis ( 57 ) . especially regarding endocrine - related neoplasia
, the medical practitioner has to be aware of the phenomics , or phenotypic profiles , that comes along with the array of inheritable endocrine tumor syndromes .
it is of no surprise that several of the inherited tumor syndromes were first described and named long before genetic causes had been identified .
in fact , it should be paramount to the medical professional to use meticulous observation to personalize the decision for treatment options and , much more importantly , to prioritize medical genetic testing .
frank weber is deeply grateful to his mentor , charis eng , who always encouraged and inspired him | genomic and personalized medicine have become buzz phrases that pervade all fields of medicine .
rapid advances in -omics fields of research ( chief of which are genomics , proteinomics , and epigenomics ) over the last few years have allowed us to dissect the molecular signatures and functional pathways that underlie disease initiation and progression and to identify molecular profiles that help the classification of tumor subtypes and determine their natural course , prognosis , and responsiveness to therapies .
genomic medicine implements the use of traditional genetic information , as well as modern pangenomic information , with the aim of individualizing risk assessment , prevention , diagnosis , and treatment of cancers and other diseases .
it is of note that personalizing medical treatment based on genetic information is not the revolution of the 21st century .
indeed , the use of genetic information , such as human leukocyte antigen - matching for solid organ transplantation or blood transfusion based on abo blood group antigens , has been standard of care for several decades . however , in recent years rapid technical advances have allowed us to perform high - throughput , high - density molecular analyses to depict the genomic , proteinomic , and epigenomic make - up of an individual at a reasonable cost .
hence , the so - called genomic revolution is more or less the logical evolution from years of bench - based research and bench - to - bedside translational medicine . |
the associations between diabetes and cancer have been the subject of debate for over a century .
the length of this debate suggests , perhaps , that any such associations are relatively weak , and it is safe to say that most practicing diabetologists do not view cancer as a specific , diabetes - associated risk in day - to - day practice . that diabetes is not more clearly associated with cancer
is surprising , since diabetes may be viewed as a disease of increased intracellular oxidative stress , as well as accelerated biochemical aging caused by the accumulation of advanced glycation end - products ( ages ) on proteins and other macromolecules ( reviewed in ) .
an additional concern is the role of insulin , which , in various forms , is increasingly used in the management of type 2 diabetes , a condition that is now epidemic worldwide .
specifically , the ability of insulin to bind and activate the insulin - like growth factor-1 ( igf-1 ) receptor has raised concerns that it may promote the growth of nascent malignancies and even the establishment of new ones .
, there has been a recent surge in interest in the role of diabetes , insulin therapy , and specifically the effects of newer forms of insulin in promoting cancer .
recent rigorous epidemiologic studies have confirmed that type 2 diabetes does indeed confer increased risk for certain types of malignancies , in particular breast , colon , and pancreatic tumors .
many patients with type 2 diabetes eventually require insulin , and the long - acting insulin analogue , glargine , is one of the most commonly used agents .
several recent studies have demonstrated the potential mitogenic and anti - apoptotic effects of glargine on cultured cancer cells , and have implicated activation of the igf-1 pathway .
however , the clinical implications remain controversial , particularly when studies pertaining to untransformed cells did not support any mitogenic potential . in addition , according to some authors , glargine compared with human insulin does not seem to display increased mitogenic effects , despite its higher affinity for the igf-1 receptor ; rather , any type of insulin has the potential to promote growth in cancer cells , especially if administered at sufficiently high , supra - physiologic doses .
the question now is whether glargine is associated with increased cancer risk compared with other commonly used forms of insulin .
this question has come to the fore with the publication of several studies in diabetologia [ 12 - 15 ] , which will be reviewed briefly here .
the epidemiologic study that initiated the recent debate was performed in germany and suggested a specific cancer risk associated with glargine . based upon the mixed opinions of six reviewers , the editors of diabetologia , the journal to which it was submitted , commissioned three additional studies [ 13 - 15 ] , aiming to confirm or refute its findings . in the initial study , hemkens et al
. followed 127,031 german patients who were being treated with human insulin and human insulin analogues ( aspart , lispro , or glargine ) over a mean of 1.6 years between january 1998 and june 2005 .
those on two insulin analogues or a combination of human insulin and an analogue were excluded from the analysis .
patients with known or suspected malignant disease within 3 years prior to study start were also excluded .
the primary outcome was occurrence of a malignant neoplasm , and the secondary outcome was all - cause mortality . the gross malignancy incidence rates for human insulin , aspart , lispro , and glargine were 2.50 , 2.16 , 2.13 , and 2.14 per 100 patient - years , respectively , in the unadjusted analysis .
however , when the data were adjusted for daily insulin dose , glargine was found to be associated with a greater risk of malignancy , and the risk was incremental with higher glargine doses when compared with human insulin { adjusted hazard ratio [ hr ] of 1.09 for a daily dose of 10 iu [ 95% confidence interval ( ci ) 1.00 - 1.19 ] , 1.19 for a daily dose of 30 iu [ 95% ci 1.10 - 1.30 ] , and 1.31 for a daily dose of 50 iu [ 95% ci 1.20 - 1.42]}. aspart and lispro were not associated with an increased risk for cancer compared with human insulin .
in addition , regardless of the type of insulin , higher doses and longer duration of exposure increased the risk of malignancy .
finally , the mean insulin dose until occurrence of a neoplasm ( cumulative dose divided by time until an event ) was significantly lower for glargine ( 25.9 iu / day ) than for human insulin , aspart , and lispro ( 43.8 , 38.9 , and 36.2 iu / day , respectively ) . the major weaknesses of the study included the fact that patients were not allocated to treatment groups in a prospective manner and that insulin dose was calculated during follow - up from mean values , and not determined at baseline .
in addition , the exclusion of patients on combination insulin therapy may not reflect common practice , in which patients are typically on basal / bolus regimens requiring the use of different types of insulin .
finally , the duration of diabetes , as well as smoking status , body mass index ( bmi ) , and other potential confounding factors were not taken into account .
the three additional , commissioned studies were published in the same issue of diabetologia . in sweden , jonasson and
colleagues evaluated 114,842 patients with diabetes on insulin and noted that women on glargine alone , but not glargine combined with other types of insulin , had a higher risk of breast cancer than women on other types of insulin ( hr adjusted for age , smoking , bmi , and other confounding factors was 1.97 , 95% ci 1.29 - 3.00 ) .
however , this increased risk was not associated with an increased mortality rate ; instead , all - cause mortality was actually decreased in women on glargine alone versus women on other types of insulin ( relative risk [ rr ] 0.83 , 95% ci 0.71 - 0.96 ) .
the associations with other malignancies such as prostate and gastrointestinal tumors did not reach statistical significance .
no analysis taking into account insulin dose was possible in this study , and therefore the incremental risk conferred by higher doses as observed by hemkens et al .
the short duration of exposure and observation as well as the lack of data on how much insulin the patients were taking limit the strength of the conclusions . in a study in scotland , no increased risk of total or site - specific cancer associated with overall glargine use was found in a cohort of 12,852 patients followed for 4 years ; in particular , no increase in breast cancer risk ( hr 1.49 , 95% ci 0.79 - 2.83 ) was found .
however , exclusive use of glargine did seem to confer higher risk ( adjusted hr 3.39 , 95% ci 1.46 - 7.85 ) , but the number of cases was small ( six events ) .
in addition , glargine - only users tended to be older , were more likely to be women , and to have a higher bmi , reflecting a baseline imbalance .
finally , the restricted use of glargine in scotland may have contributed to a skewing of the results , since there were a limited number of patients taking this form of insulin . in a uk
studied 62,809 patients with diabetes on metformin , sulfonylurea monotherapy , a combination of both , or insulin therapy .
the latter were further divided into glargine - treated versus other human insulin - treated ( long - acting , biphasic , and biphasic analogue ) . the results indicated that any type of insulin therapy increased the risk of occurrence of solid tumors and that concomitant metformin therapy attenuated this effect .
indeed , the adjusted hrs relative to metformin monotherapy were 1.08 ( 95% ci 0.96 - 1.21 ) for the metformin / sulfonylurea combination , 1.36 ( 95% ci 1.19 - 1.54 ) for sulfonylurea alone , and 1.42 ( 95% ci 1.27 - 1.60 ) for insulin - based therapies .
concomitant metformin therapy decreased the risk of malignancy associated with insulin by 46% ( hr 0.54 , 95% ci 0.43 - 0.66 ) . when compared with glargine
, other human insulins displayed comparable rates , suggesting no added risk attributable to glargine ( adjusted hr 1.24 , 95% ci 0.90 - 1.17 for basal human insulin ; hr 0.88 , 95% ci 0.66 - 1.19 for biphasic human insulin ; and hr 1.02 , 95% ci 0.76 - 1.37 for analogue biphasic insulin ) .
malignancies that had the strongest association with insulin therapy compared with metformin were colorectal ( hr 1.69 , 95% ci 1.23 - 2.33 ) and pancreatic ( hr 4.63 , 95% ci 2.64 - 8.10 ) tumors .
breast and prostate cancer risks did not seem to be affected by treatment with insulin or insulin secretagogues , and a prior history of solid tumor strongly increased the overall risk ( hr 3.86 , 95% ci 3.46 - 4.31 ) . in a letter published in a subsequent issue of diabetologia , rosenstock et al .
, reported a study of glargine versus neutral protamine hagedorn ( nph ) insulin in type 2 diabetes patients randomly assigned to one insulin or the other for over 4 years . although malignancy risk was not a primary endpoint for the study , no increased cancer risk with glargine compared to nph was found ( rr 0.90 , 95% ci 0.64 - 1.26 ) . home and lagarenne , using the sanofi - aventis
pharmacovigilance database for all randomized company - sponsored clinical trials , published a systematic review of 31 trials comparing glargine to other agents ( nph for most studies ) .
no evidence for an increased risk of malignancy attributable to glargine was found ( rr 0.90 , 95% ci 0.60 - 1.36 ) .
all but one of these studies lasted less than 12 months ( on average , 6 months ) , so their major limitation was the short duration of exposure and follow - up .
indeed , the majority of reported cases of malignancy were derived from the study of rosenstock and colleagues , discussed above , in which follow - up exceeded 4 years .
finally , a meta - analysis comparing cancer risk associated with another new , long - acting insulin , insulin detemir , versus nph and glargine was performed .
it used data from randomized controlled trials sponsored by novo nordisk ( bagsvaerd , denmark ) and found comparable malignancy risks associated with detemir and glargine in the five trials that compared both drugs .
however , the number of events was very small ( n = 8 ; event rate per 100 exposure - years = 0.87 for detemir , and n = 8 ; event rate per 100 exposure - years = 1.27 for glargine ) .
the issues are highly emotive : cancer and diabetes eventually affect a large proportion of the world 's population .
in addition , there are enormous financial implications surrounding the controversy , and unfortunately , complex issues tend to be oversimplified and are often sensationalized .
either a diagnosis of cancer or the institution of insulin therapy for diabetes provokes great anxiety in many people .
criticism of the publication of the diabetologia papers came in the correspondence section of the lancet , where pocock and smeeth raised concerns regarding the quality of the statistical analyses and contended that these analytical flaws , especially in the german paper , had raised unsubstantiated anxieties .
they criticized the swedish study for its finding of an association of insulin glargine with breast cancer , arguing that the latter was not a
pre - defined site for analysis but rather a secondary finding ultimately not confirmed by the scottish study ( the latter did find an increased risk of breast cancer associated with the exclusive use of glargine , but only affecting very few , older patients ) .
further criticism came in an editorial by garg , hirsch , and skyler in diabetes technology and therapeutics , questioning the motivations behind the german study and raising concerns similar to those brought up by pocock and smeeth .
this paper also emphasized the lack of evidence of an increased overall cancer risk associated with glargine based on the swedish , scottish , and uk studies , concluding that glargine use should not be discontinued based on unsubstantiated allegations. the letter of pocock and smeeth elicited a prompt response from the editor of diabetologia .
the editor , while acknowledging the imperfections of the studies ( which he had already discussed in detail in an accompanying editorial ) , argued that the epidemiologic data can not and should not be dismissed , especially when considered in the context of animal and cell culture work , but rather should generate further research to define risk .
indeed , in his initial editorial on the subject , he had earlier stated that
the evidence presented in this set of papers is sufficient to establish that there is a case to answer , but is entirely insufficient to bring a verdict. at least on the latter conclusion , all parties agreed .
these thought - provoking studies , although inconclusive , do raise concerns about the long - term effects of diabetes itself , insulin treatment , and specifically , glargine , on cancer development .
it must be remembered that the greatly increased mortality among patients with diabetes has little to do with cancer , but instead is related to vascular complications .
the overall benefit - to - risk ratio of good glycemic control is likely to support extensive use of insulin .
all are agreed that the current evidence does not warrant any change in clinical practice , specifically with regard to the use of glargine .
further research will need to address the relationship between diabetes , insulin , insulin analogues and secretagogues , and cancer .
it will also explore potentially protective effects of metformin and perhaps the most tantalizing question : why is diabetes not more strongly associated with cancer ?
| diabetes , in particular type 2 , is associated with an increased incidence of cancer .
although the mortality attributable to cancer in type 2 diabetes is overshadowed by that due to cardiovascular disease , emerging data from epidemiologic studies suggest that insulin therapy may confer added risk for cancer , perhaps mediated by signaling through the igf-1 ( insulin - like growth factor-1 ) receptor .
co - administered metformin seems to mitigate the risk associated with insulin .
a recent series of publications in diabetologia addresses the possibility that glargine , the most widely used long - acting insulin analogue , may confer a greater risk than other insulin preparations , particularly for breast cancer .
this has led to a heated controversy . despite this
, there is a consensus that the currently available data are not conclusive and should not be the basis for any change in practice .
further studies and more thorough surveillance of cancer in diabetes are needed to address this important issue . |
caries activity usually causes tooth decay or cavities and can even lead to the loss of afflicted teeth , which is particularly harmful to children 's growth and development . the world health organization ( who ) reported that 6090% of school children experience caries globally .
preservation of the remaining vital portion of curiously exposed pulpal tissue in primary teeth , where the demand is to keep a functioning tooth in site , was one of the most frequent problems in pediatric dentistry . to solve this problem ,
pulpotomy is amputation of the coronal portion of the affected or infected dental pulp and treatment of the remaining vital radicular pulp tissue surface for preserving the vitality and function of all or part of the remaining radicular portion of the pulp .
furthermore , it is an accepted procedure for treating both primary and permanent teeth with carious pulp exposures .
these include formocresol , glutaraldehyde , ferric sulfate , collagen material , and mineral trioxide aggregate .
however , none of them have met the same degree of effectiveness and success rate as formocresol .
possible hazards of formocresol such as cytotoxicity , carcinogenicity , immunologic , biochemical , mutagenic , and teratogenic changes in the host have been reported .
the tissue changes have been found to occur in various internal organs , particularly in kidney and liver , and the quantity of circulating formocresol was found to increase with the number of teeth treated .
hence , increased utilization of indigenous plant medicines in developing countries became a policy of who in the 1970s . in this regard , a demand for natural medicament to replace formocresol as a pulp dressing material became imperative .
historically , garlic has been used around the world to treat many conditions , including hypertension and infections .
currently , garlic is used for reducing the cholesterol levels and cardiovascular risk , as well as for its antineoplastic and antimicrobial properties .
allium sativum is one of the most extensively researched medicinal plants and its antibacterial activity depends on allicin produced by the enzymatic activity of allinase ( a cysteine sulfoxide lyase ) .
allicin and other thiosulfinates are believed to be responsible for the range of therapeutic effects reported for garlic ; there is extensive literature on the antibacterial effects of fresh garlic extract .
garlic extract has been reported to inhibit growth of various gram - positive and gram - negative bacteria .
a. sativum extract has been known to have inhibitory activity on various pathogenic bacteria , viruses , and fungi .
the inhibitory activity of garlic extract on multidrugresistant ( mdr ) strains of streptococcus mutans isolated from human carious teeth has been reported .
interest in medicinal plants has burgeoned due to the increased efficacy of new plant - derived drugs and the growing interest in natural products . because of the concerns about the side effects of conventional medicine
, the use of natural products as an alternative to conventional treatment in healing and treatment of various diseases has been on the rise in the last few decades .
the aim of this study was to compare the clinical and radiographic effects of a. sativum oil with those of formocresol in mortal pulpotomy in primary teeth .
the aim of this study was to compare the clinical and radiographic effects of a. sativum oil with those of formocresol in mortal pulpotomy in primary teeth .
a total of 20 children with age ranging from 4 to 8 years were selected from the outpatient clinic of pediatric dentistry department , faculty of dental medicine , alazhar university .
every one of those children had a pair of nonvital primary molars ( upper or lower second primary molars ) .
caregiver 's approval was takenapproval of faculty of oral and dental medicine , al - azhar university , egypt ( under number 327/2013 ) was obtained .
caregiver 's approval was taken approval of faculty of oral and dental medicine , al - azhar university , egypt ( under number 327/2013 ) was obtained .
patient 's and parental cooperationabsence of any systemic disease which would contraindicate pulp therapyno previous history of antibiotic therapy for at least 2 weekspresence of clinical signs or symptoms suggesting a non - vital tooth , such as localized swelling , suppurating sinus , tenderness on percussion , or slight mobilitypossibility for establishing a final restoration of the tooth .
patient 's and parental cooperation absence of any systemic disease which would contraindicate pulp therapy no previous history of antibiotic therapy for at least 2 weeks presence of clinical signs or symptoms suggesting a non - vital tooth , such as localized swelling , suppurating sinus , tenderness on percussion , or slight mobility possibility for establishing a final restoration of the tooth .
cavity outline was established with a sterile , # 330 , high - speed , pear - shaped carbide bur with air / water spray .
caries was removed with a slow - speed , sterile , round , steel bur .
the roof of the pulp chamber was removed with a sterile , non - end cutting , slow - speed bur .
removal of the coronal pulp tissue was achieved with a sterile , low - speed carbide round bur no . 6 and/or sharp , large spoon excavator .
the pulp stumps of molars in the right side were dressed with a cotton pellet damped with a. sativum oil [ captin company ( cappharm ) registration no .
952/94 , cairo , egypt ] and in the left side with a cotton pellet damped with formocresol ( petrpolis - rj - industria brasileira , dentsply , latin america ) for 1 week . then , temporary filling was added .
after 1 week , the temporary filling and cotton pellet were removed and the pulp stumps of molars treated with a. sativum oil were dressed with zinc oxide a .
sativum oil paste , while those of teeth treated with formocresol were dressed with a thick paste prepared by mixing zinc oxide powder with one drop of eugenol .
then , a layer of intermediate restorative material zinc phosphate cement had been placed over the dressing materials .
after that , the molars were restored with suitable final restoration and then with stainless steel crowns .
the molars were clinically and radiographically evaluated after 6 months using standard clinical and radiographic criteria .
history of pain related to the treated molars
sensitivity to percussionteeth mobilitysigns of erythema , swelling , and the presence of fistulous tract in the surrounding gingival tissues and mucosa .
sensitivity to percussion signs of erythema , swelling , and the presence of fistulous tract in the surrounding gingival tissues and mucosa .
periapical radiographs were taken for all treated molars ( ultra - speed kodak periapical films , size 0 or 1 film ) .
all radiographs taken during the follow - up period were screened for their diagnostic quality prior to being included in radiographic evaluation .
acceptable radiographs should have non - distorted images of the treated molars and the osseous structures immediately adjacent to the roots .
radiographs that did not meet these criteria were excluded from the evaluation . during radiographic evaluation ,
the following were determined :
presence or absence ( or reduction in size ) of furcation or periapical radiolucencypresence or absence of pathologic internal or external root resorptionpresence or absence of widening in the periodontal membrane .
presence or absence ( or reduction in size ) of furcation or periapical radiolucency presence or absence of pathologic internal or external root resorption presence or absence of widening in the periodontal membrane .
statistical analysis was done between the clinical and radiographic effects of a. sativum oil and those of formocresol in non - vital pulpotomy in primary teeth using ttest and the level of significance was taken as p 0.05 .
caregiver 's approval was takenapproval of faculty of oral and dental medicine , al - azhar university , egypt ( under number 327/2013 ) was obtained .
caregiver 's approval was taken approval of faculty of oral and dental medicine , al - azhar university , egypt ( under number 327/2013 ) was obtained .
patient 's and parental cooperationabsence of any systemic disease which would contraindicate pulp therapyno previous history of antibiotic therapy for at least 2 weekspresence of clinical signs or symptoms suggesting a non - vital tooth , such as localized swelling , suppurating sinus , tenderness on percussion , or slight mobilitypossibility for establishing a final restoration of the tooth .
patient 's and parental cooperation absence of any systemic disease which would contraindicate pulp therapy no previous history of antibiotic therapy for at least 2 weeks presence of clinical signs or symptoms suggesting a non - vital tooth , such as localized swelling , suppurating sinus , tenderness on percussion , or slight mobility possibility for establishing a final restoration of the tooth .
cavity outline was established with a sterile , # 330 , high - speed , pear - shaped carbide bur with air / water spray .
caries was removed with a slow - speed , sterile , round , steel bur .
the roof of the pulp chamber was removed with a sterile , non - end cutting , slow - speed bur .
removal of the coronal pulp tissue was achieved with a sterile , low - speed carbide round bur no .
6 and/or sharp , large spoon excavator . the pulp stumps of molars in the right side were dressed with a cotton pellet damped with a. sativum oil [ captin company ( cappharm ) registration no .
952/94 , cairo , egypt ] and in the left side with a cotton pellet damped with formocresol ( petrpolis - rj - industria brasileira , dentsply , latin america ) for 1 week . then , temporary filling was added .
after 1 week , the temporary filling and cotton pellet were removed and the pulp stumps of molars treated with a. sativum oil were dressed with zinc oxide a .
sativum oil paste , while those of teeth treated with formocresol were dressed with a thick paste prepared by mixing zinc oxide powder with one drop of eugenol .
then , a layer of intermediate restorative material zinc phosphate cement had been placed over the dressing materials .
after that , the molars were restored with suitable final restoration and then with stainless steel crowns .
the molars were clinically and radiographically evaluated after 6 months using standard clinical and radiographic criteria .
history of pain related to the treated molars
sensitivity to percussionteeth mobilitysigns of erythema , swelling , and the presence of fistulous tract in the surrounding gingival tissues and mucosa .
sensitivity to percussion signs of erythema , swelling , and the presence of fistulous tract in the surrounding gingival tissues and mucosa .
periapical radiographs were taken for all treated molars ( ultra - speed kodak periapical films , size 0 or 1 film ) .
all radiographs taken during the follow - up period were screened for their diagnostic quality prior to being included in radiographic evaluation .
acceptable radiographs should have non - distorted images of the treated molars and the osseous structures immediately adjacent to the roots .
radiographs that did not meet these criteria were excluded from the evaluation . during radiographic evaluation ,
the following were determined :
presence or absence ( or reduction in size ) of furcation or periapical radiolucencypresence or absence of pathologic internal or external root resorptionpresence or absence of widening in the periodontal membrane .
presence or absence ( or reduction in size ) of furcation or periapical radiolucency presence or absence of pathologic internal or external root resorption presence or absence of widening in the periodontal membrane .
statistical analysis was done between the clinical and radiographic effects of a. sativum oil and those of formocresol in non - vital pulpotomy in primary teeth using ttest and the level of significance was taken as p 0.05 .
the clinical findings of the primary molars after 6 months of treating with a. sativum oil were as follows .
there was no tooth presenting with a draining sinus , gingival inflammation , and increased mobility .
the clinical findings of primary molars after 6 months of treatment with formocresol were as follows .
comparisons between the clinical findings of vital pulpotomy in primary molars treated with a. sativum oil and formocresol are presented in table 1 and figure 1 .
comparison between the effects of a. sativum oil and formocresol on the clinical findings of non - vital primary teeth after 6 months a histogram showing a comparison between the effects of a. sativum oil ( as ) and formocresol ( fc ) on the clinical findings at 6 months postoperatively statistically , these results revealed the differences between the clinical findings of non - vital pulpotomy in primary molars treated with either a. sativum oil or formocresol throughout the study interval .
the radiographic outcomes of primary molars after 6 months of treatment with a. sativum oil figure 2 . among the 20 non - vital pulpotomies , 8 had widening in the periodontal membrane space and 8 showed periapical radiolucency throughout the follow - up period .
( a ) preoperative radiograph ( b ) primary molar showed no changes at 6 months postoperatively the radiographic outcomes of teeth after 6 months of treatment with formocresolare given in figure 3 .
widening in the periodontal membrane space , periapical radiolucency was present in 12 cases . however , 8 showed furcation .
( a ) preoperative radiograph ( b ) primary molars showed no changes at 6 months postoperatively comparisons between the radiographic findings associated with the two medicaments in non - vital pulpotomy performed in primary molars are presented in table 2 and figure 4 . statistically , these results revealed significant difference between the radiographic findings of vital pulpotomy in primary molars with the two medicaments .
comparison between the effects of a. sativum oil and formocresol on the radiographic findings of non - vital primary teeth after 6 months a histogram showingcomparison between the effects of a. sativum oil and formocresol on the radiographic findings at 6 months postoperatively
the clinical findings of the primary molars after 6 months of treating with a. sativum oil were as follows .
there was no tooth presenting with a draining sinus , gingival inflammation , and increased mobility .
the clinical findings of primary molars after 6 months of treatment with formocresol were as follows .
comparisons between the clinical findings of vital pulpotomy in primary molars treated with a. sativum oil and formocresol are presented in table 1 and figure 1 .
comparison between the effects of a. sativum oil and formocresol on the clinical findings of non - vital primary teeth after 6 months a histogram showing a comparison between the effects of a. sativum oil ( as ) and formocresol ( fc ) on the clinical findings at 6 months postoperatively statistically , these results revealed the differences between the clinical findings of non - vital pulpotomy in primary molars treated with either a. sativum oil or formocresol throughout the study interval .
the radiographic outcomes of primary molars after 6 months of treatment with a. sativum oil figure 2 . among the 20 non - vital pulpotomies , 8 had widening in the periodontal membrane space and 8 showed periapical radiolucency throughout the follow - up period .
( a ) preoperative radiograph ( b ) primary molar showed no changes at 6 months postoperatively the radiographic outcomes of teeth after 6 months of treatment with formocresolare given in figure 3 .
widening in the periodontal membrane space , periapical radiolucency was present in 12 cases . however , 8 showed furcation .
( a ) preoperative radiograph ( b ) primary molars showed no changes at 6 months postoperatively comparisons between the radiographic findings associated with the two medicaments in non - vital pulpotomy performed in primary molars are presented in table 2 and figure 4 . statistically , these results revealed significant difference between the radiographic findings of vital pulpotomy in primary molars with the two medicaments .
comparison between the effects of a. sativum oil and formocresol on the radiographic findings of non - vital primary teeth after 6 months a histogram showingcomparison between the effects of a. sativum oil and formocresol on the radiographic findings at 6 months postoperatively
pulpotomy means removal of the coronal portion of the affected dental pulp and treatment of the remaining radicular pulp tissue .
furthermore , it is an accepted procedure for treating both primary and permanent teeth with carious pulp exposures .
pulpotomy has become the dominating pulp therapy for deciduous dentition because of the complicated anatomy of the root canals in the primary teeth , the proximity of the permanent tooth germ , and the difficulties in finding a root canal filling material compatible with physiological root resorption .
so , pulpotomy is indicated for teeth with deep carious lesion with no history swelling , presence of fistula , or tooth mobility ; primary teeth with vital pulp exposure as a result of carious process ; absence of radiographic evidence for internal or external root resorption or radiolucency in the furcation area ; no more than one - third of physiological root resorption ; and possibility for further tooth restoration .
pulpotomy is not suitable for teeth when there is perforation of the floor of pulp chamber , internal or external root resorption , and inability to isolate the tooth .
a number of studies on this alternative therapeutic system as well as its use for several purposes have been reported .
it serves as a therapeutic alternative , safer choice , or in some cases , the only effective treatment .
people in separate cultures and places are known to have used the same plants for similar medical problems .
nigella sativa oil ( ns ) extracted from black seed was used in pulpotomies for dogs .
specimens in ns groups showed mild to moderate vasodilatation , continuous odontoblastic layer , and few samples showed scattered inflammatory cell infiltration . in spite of
the large number of pharmacological studies carried out worldwide on a. sativa , scrutiny of the published articles showed that there is a need to investigate its application in dental practice .
therefore , this study was the first to find out the effect of a. sativa in treatment of pulpal affected primary teeth .
teeth included in the present study were non - vital primary molars in which the infected coronal pulp remained and , hence , microorganisms invaded the radicular pulp resulting in irreversible pulpitis and necrosis . at this stage ,
however , pulpectomy of primary molars is often impractical because of difficulty in obtaining adequate access to the root canals in small mouth openings of children , as well as the complexity of the root canals ( ribbon shaped , lateral branching , and ramifications ) .
therefore , non - vital ( mortal ) pulpotomy technique is often used and preferred by clinicians . in this study , a comparison between the clinical and radiographic outcomes of a. sativum oil and formocresol as pulpotomy medicaments in primary molars was successfully made .
since formocresol is still considered the standard in primary teeth pulp therapy , it was used in the present study as a control .
a systemic uptake of formocresol has been found from pulpotomized teeth ; lead totissue changes in various internal organs , particularly in kidney and liver , and in lymphatic and venous vascular flow due to uptake from the coronal pulp .
therefore , the local insult results in systemic distribution . the quantity of circulating formocresol was found to increase with the number of teeth treated .
considering these facts , the formaldehyde component of formocresol during a pulpotomy is insignificant . in this regard , a demand for medicament to replace formocresol as a pulp dressing material became imperative . despite the penetration of modern medicine
, traditional medicine continues to be a viable health alternative for the large underprivileged sections of the world .
medicinal plants constitute a promising source of several drugs . among the promising medicinal plants ,
a. sativa is considered to be an amazing herb with a rich historical and religious background . in spite of the fact that a. sativa oil was used for the first time in treatment of vital primary molars , the results were remarkable and promising , as none of the succeeded cases showed any signs or symptoms of morbidity during the entire follow - up period .
results of the present studyrevealed that the success rate of treatment with a. sativum oil was 95% , while the success rate of treatment with formocresol was 80% . indeed , these results confirm the success rate of formocresol in treatment of non - vital pulps of primary molars and it is in agreement with other studies ; however , the success rate of formocresol in the present study was lower than that suggested by others . rlling and thylstrup , in their study , reported 100% clinical success rate with formocresol .
the promising clinical outcome of vital pulpotomy performed with a. sativum oil may be explained based on the following reasons .
the oil was found to possess potent analgesic and anti - inflammatory properties and it is used in the folk medicine for treatment of toothache without any side effects .
ajoene inhibits prostaglandins through the suppression of cyclooxygenase ( cox ) enzyme in the inflamed area and inflammatory cytokines .
in addition , allicin and ajoene ( component of a. sativum ) appear to inhibit nitric oxide ( a pro inflammatory mediator ) which leading to anti inflammatory effect . in the present study ,
radiographic investigation revealed an improvement in both a. sativum oil and formocresol treated teeth at the end of the follow - up period .
if the recall periods had been continued longer , complete resolution of these changes would have occurred .
statistical analysis revealed significant difference between the effects of both medicaments on the clinical and radiographic outcomes .
a. sativum oil has potent antibacterial properties that enable it to combat intracanal microbes in the infected pulp of primary molarsas the usage of a. sativum oil in the pulp treatment of vital and non - vital primary molars was promising , this will encourage dentists to use it in the future as an alternative to formocresol .
a. sativum oil has potent antibacterial properties that enable it to combat intracanal microbes in the infected pulp of primary molars as the usage of a. sativum oil in the pulp treatment of vital and non - vital primary molars was promising , this will encourage dentists to use it in the future as an alternative to formocresol .
| objective : the objective of this study was to compare the clinical and radiographic effects of allium sativum oil and formocresol in nonvital pulpotomy in primary teeth.materials and methods : twenty children ranging in age from 4 to 8 years were included in the study . in every one of those children ,
pulpotomy was indicated for the primary molars .
pulpotomy procedure was performed and the radicular pulp tissue of one molar was capped with a. sativum oil in a cotton pellet while the other molar was capped with formocresol .
the teeth were evaluated clinically and radiographically before and after 6 months using standard clinical and radiographic criteria . statistically , these results revealed significant difference between the radiographic findings of nonvital pulpotomy in primary molars with the two medicaments .
statistical analysis was performed using independent t - test and paired t - test at the significance level of = 0.05.results:a .
sativum oil has potent antibacterial properties that enable it to combat intracanal microbes in the infected pulp of primary molars .
better results were obtained when a. sativum oil was used.conclusion:a .
sativum oil had more powerful effects than formocresol on the infected pulp of primary nonvital molars . |
obesity is a serious health problem , which may range from a simple complaint of disability to premature death . in many epidemiological studies , the body mass index(bmi 30 kg / m ) or percentage of body fat ( pbf > 25% in men and > 35% in women )
the pbf is measured by several methods , such as magnetic resonance imaging ( mri ) , bioelectrical impedance analysis ( bia ) , and x - ray .
the world health organization ( who ) , based on a 2004 study of 3,378 adults in southern iran , reported 21 to 25 kg / m as the range of the bmi and that 8% of women and 2.5% of men were obese without reporting the suitable cutoff points .
mirzazadeh et al . conducted a meta - analysis of published reports in iran , including proceedings of professional meetings , books , journal articles , and estimated the prevalence of obesity in men and women to be 12.9% and 26.2% , respectively . in some iranian articles , suitable bmi cutoff points for detecting obesity
for example , in one , it is reported to be 26.6 kg / m based on data from 206 elderly . in another study , data from 4,756 diabetic people were used to estimate the cutoff points as 24.8 kg / m and 26.3 kg / m for males and females , respectively . in the review of the literature , we identified iranian studies in which the bia had been used to measure body composition in samples ranging from 25 to 300 people .
some reported that the bmi was accurate in detecting obesity while others concluded that it was not an accurate indicator of obesity .
we also found non - iranian studies which supported the pros and cons of employing the bmi to detect obesity .
gallagher classified individuals based on the pbf for males and females in various age groups , namely , 18 , 19 , 20 - 39 , 40 - 59 , and > 59 years old .
a systematic review of the literature showed no published iranian studies using these age groups and no international studies focusing on either the accuracy of gallagher s classification or its comparison with the bmi .
the purpose of this study was twofold , ( i ) to examine the comparability of the bmi and gallagher s classification in diagnosing obesity , and ( ii ) to determine the suitable bmi cutoff points for detecting obesity in a large sample of iranians , using the bia method .
the significance of the study was that it compared two popular methods in detecting obesity , which had not already been done .
the permission to conduct the study and the ethics approval were obtained from the office of vice - chancellor for research and technology at hormozgan university of medical sciences ( # 6-hec-88 - 2 - 16 , 6 may 2009 ) .
the prevalence of obesity in the iranian adult population is reported as 21.50% , which we used to estimate the initial sample size at the 0.05 level of significance for testing non - directional hypotheses .
however , due to the duration of the investigation ( 2009 - 2014 ) , 23,300 individuals who visited a health and diet therapy center in bandar abbas , iran , and agreed to participate in the study were recruited .
those who were not at least 18 years old , had pacemakers , were pregnant , and were hospitalized at least 3 months prior to entering the study were excluded . in total , 20,163 adults met the inclusion criteria and their data were used in the study .
this device uses the frequency range between 50 khz and 250 khz , and utilizes the method of direct segmental multi - frequency bia . with a high level of accuracy ,
this method enabled us to measure various body composition indices ( i.e. , the bmi , waist to hip ratio , percentage of body fat , total body fat , proteins , minerals , soft lean mass , fat free mass , muscle quantity , lean body mass , total body water , total energy expenditure , basal metabolic rate , fat - trunk , and muscle - trunk ) .
the standard positions of outer and inner electrodes on the right hand and foot were employed for measuring the whole body impedance .
the device was explained to all research participants and trained technicians were in charge of all measurements .
the height ( cm ) was measured to the nearest 0.50 cm by a stadiometer . weight ( kg )
was divided by the squared height ( cm ) to measure the bmi , which was used to classify participants into four groups , ( i ) underweight ( under 18.50 ) , ( ii ) normal weight ( 18.50 to 24.99 ) , ( iii ) overweight ( 25.00 to 29.99 ) , and ( iv ) obese ( 30.00 and higher ) .
the participants were also classified into four groups ( underweight , normal weight , overweight , and obese ) using gallagher s classification and subdivided the sample by sex and age .
table 1 shows the classification of the participants based on the pbf using the gallagher s classification .
distribution of participants based on gallagher s classification pbf : percentage of body fat the who gold standards were used to determine the accuracy of the bmi and gallagher s classification in detecting obesity .
as noted earlier , pbf > 25% in men and > 35% in women indicate obesity . in the bmi and gallagher s classifications ,
all individuals in the subgroups of underweight , normal weight , and overweight were classified as non - obese and the rest were considered obese . for the purpose of the study , sensitivity , specificity ,
positive predictive value ( ppv ) and negative predictive value ( npv ) were computed using contingency tables .
receiver - operating characteristic ( roc ) curves were used to describe the diagnostic performance of screening test in terms of diagnostic accuracy or the ability to classify participants correctly into clinically relevant subgroups , as defined by a reference test .
version 22 of the statistical package for the social sciences ( spss ) was used for the purpose of data manipulation and analysis , which included contingency tables , linear regression , and hanely formula to compare the two roc curves .
the participants were selected from 14,270 ( 70.80% ) women and 5,893 ( 29.20% ) men aged between 18 and 85 years .
prevalence of obesity was estimated at 51% based on the gold standard 38.5% based on bmi and 27.4% by gallagher s classification .
based on the gold standard 49.9% and 54.4% of women and men respectively , were classified as obese .
results showed that the bmi was not suitable for detecting obesity among males . in this category ,
the following ranges were calculated : sensitivity ( 51.4% to 80% ) , specificity ( 89.4% to 100% ) , ppv ( 88.7% to 100% ) , and npv ( 53.2% to 93.9% ) . in the 20 - 39 , 40 - 59 , and 60 - 81 age groups , the bmi classified 34% , 45% , and 49% of male obese individuals as non - obese , respectively .
the following ranges were obtained : sensitivity ( 41.1% to 100% ) , specificity ( 99.8% to 100% ) , ppv ( 99.8% to 100% ) , and npv ( 51.2% to 100% ) . in the 18 and 19 age groups , sensitivity , specificity , npv , and ppv were 100% . in the 20 - 40 age group
in the 20 - 39 years age group , 9% , in 40 - 59 years age group , 46% , and in greater than 60 age groups , 59% of obese individuals were wrongly classified as non - obese .
thus , it was concluded that the gallagher s classification is better than the bmi for detecting obesity in men with the exception of those who are older than 59 years ( table 2 ) .
sensitivity , specificity , ppv , and npv based on the bmi and gallagher s criteria for overweight and obese men bmi : body mass index ; ppv : positive predict value ; npv : negative predictive value the number of women who were classified as obese by the bmi was 5,544 ( 38.9% ) .
the bmi classified 25.7% of the obese women as non - obese , which led to a conclusion that it is suitable for detecting obesity in females .
in this category , the following ranges were obtained : sensitivity ( 70.6% to 84.2% ) , specificity ( 92.8% to 98.3% ) , ppv ( 94.1% to 97.9% ) , and npv ( 56.1% to 93.5% ) .
in the five age groups , the bmi classified 16% , 23% , 23% , 27% and 29% of obese womens as non - obese , respectively . based on this indicator
the sensitivity ranged from 27.9% to 86.8% , and the npv s range was 35.2% to 94.7% .
sensitivity and npv decreased as age increased , which was consistent with the bmi s findings .
the range of decreases in sensitivity , based on the bmi and gallagher , were 13.6% and 58.9% , respectively .
beyond 19 years of age , based on the gallagher s and the bmi , 55 - 72% and 29 - 33% of the obese individuals were wrongly classified as overweight , respectively .
results showed that the bmi is better than gallagher in classifying the women ( table 3 ) .
sensitivity , specificity , ppv , and npv based on the bmi and gallagher s criteria for overweight and obese women bmi : body mass index ; ppv : positive predict value ; npv : negative predictive value gallagher s classification can correctly classify men and women who are less than 40 and 20 years old , respectively . in both classifications , an increase in age was associated with a decrease in sensitivity and the npv . in all participants , a suitable cutoff point for
bmi in detecting obesity was 27.70 kg / m ( sensitivity=88% , specificity=88.6% ) . among females , the suitable cutoff point was also found to be 27.7 kg / m ( sensitivity=92% , specificity=90% ) and among males , it was 27.3 kg / m ( sensitivity=84% , specificity=84% ) .
the comparison of the two roc curves , using the hanely formula , showed no statistically significant differences between these cutoff points ( figure 1 ) .
receiver operating characteristic for body mass index to detect obesity in all participants , women and men .
in this category , the following ranges were calculated : sensitivity ( 51.4% to 80% ) , specificity ( 89.4% to 100% ) , ppv ( 88.7% to 100% ) , and npv ( 53.2% to 93.9% ) . in the 20 - 39 , 40 - 59 , and 60 - 81 age groups , the bmi classified 34% , 45% , and 49% of male obese individuals as non - obese , respectively .
the following ranges were obtained : sensitivity ( 41.1% to 100% ) , specificity ( 99.8% to 100% ) , ppv ( 99.8% to 100% ) , and npv ( 51.2% to 100% ) . in the 18 and 19 age groups , sensitivity , specificity , npv , and ppv were 100% . in the 20 - 40 age group all ranges were between 90.8% and 100% . beyond 39 years of age , sensitivity and npv decreased as age increased .
in the 20 - 39 years age group , 9% , in 40 - 59 years age group , 46% , and in greater than 60 age groups , 59% of obese individuals were wrongly classified as non - obese .
thus , it was concluded that the gallagher s classification is better than the bmi for detecting obesity in men with the exception of those who are older than 59 years ( table 2 ) .
sensitivity , specificity , ppv , and npv based on the bmi and gallagher s criteria for overweight and obese men bmi : body mass index ; ppv : positive predict value ; npv : negative predictive value
the number of women who were classified as obese by the bmi was 5,544 ( 38.9% ) .
the bmi classified 25.7% of the obese women as non - obese , which led to a conclusion that it is suitable for detecting obesity in females .
in this category , the following ranges were obtained : sensitivity ( 70.6% to 84.2% ) , specificity ( 92.8% to 98.3% ) , ppv ( 94.1% to 97.9% ) , and npv ( 56.1% to 93.5% ) .
sensitivity and npv decreased as age increased . in the five age groups , the bmi classified 16% , 23% , 23% , 27% and 29% of obese womens as non - obese , respectively .
the sensitivity ranged from 27.9% to 86.8% , and the npv s range was 35.2% to 94.7% .
sensitivity and npv decreased as age increased , which was consistent with the bmi s findings .
the range of decreases in sensitivity , based on the bmi and gallagher , were 13.6% and 58.9% , respectively .
beyond 19 years of age , based on the gallagher s and the bmi , 55 - 72% and 29 - 33% of the obese individuals were wrongly classified as overweight , respectively .
results showed that the bmi is better than gallagher in classifying the women ( table 3 ) .
sensitivity , specificity , ppv , and npv based on the bmi and gallagher s criteria for overweight and obese women bmi : body mass index ; ppv : positive predict value ; npv : negative predictive value gallagher s classification can correctly classify men and women who are less than 40 and 20 years old , respectively . in both classifications , an increase in age was associated with a decrease in sensitivity and the npv .
in all participants , a suitable cutoff point for bmi in detecting obesity was 27.70 kg / m ( sensitivity=88% , specificity=88.6% ) . among females ,
the suitable cutoff point was also found to be 27.7 kg / m ( sensitivity=92% , specificity=90% ) and among males , it was 27.3 kg / m ( sensitivity=84% , specificity=84% ) .
the comparison of the two roc curves , using the hanely formula , showed no statistically significant differences between these cutoff points ( figure 1 ) .
receiver operating characteristic for body mass index to detect obesity in all participants , women and men .
the aim of the study was to examine the comparability of the bmi and gallagher s classifications in detecting obesity in a large sample of iranian adults .
a systematic review of the literature showed that the accuracy of gallagher s classification and its comparison with the bmi classification had not been adequately investigated .
results showed that gallagher s classification is better than the bmi in detecting obesity in men younger than 40 years old ; beyond this age , both methods wrongly classified nearly half of the obese males as non - obese .
the bmi was better than gallagher s classification in detecting obesity in females and an increase in age was associated with a decrease in the accuracy of both techniques , especially in gallagher s .
the bmi correctly classified 70.8% of the obese persons , suggesting that it is a moderate indicator of obesity , which is supported by other studies .
reported that the bmi accurately diagnosed excess adiposity in 84% of those participated in their study . in our study , the bmi and gallagher s criteria correctly identified 92.4% and 99% of males , and 96.5% and 100% of females as being non - obese , respectively . although the bmi is a good indicator for detecting non - obese individuals , but gallagher s classification is better than the bmi in classifying non - obese males and females . nearly 74.3% and 42% of females , and 63% and 79.4% of males were correctly classified as being obese by the gallagher s classification and the bmi , respectively .
neovius reported low sensitivity and high specificity for the bmi in both sexes with the average age of 15 years . in spite of age differences ,
the results of this study are similar to those reported by neovius . based on the bmi , among females and males 16%-29% and 20%-49% of obese individuals
these findings suggest that the bmi is a good indicator of obesity in females but not males .
accuracy of the bmi in both females and males increased after the age of 19 years .
coe et al . reported that the accuracy of the bmi decreased in females and no apparent trend was noted in males .
it contradicts the results of this study , which could be due to age , ethnicity , and sample size differences .
the correlations between the bmi and pbf were 0.86 and 0.91 in men and women , respectively ; similar to those reported by heyadari , et al .
thus , it was concluded that the bmi is a good predictor of the pbf but not adequate in diagnosing excess adiposity , especially in males as supported by romero et al . and contradicted in other studies .
the contradiction could be due to the selection of individuals , race , and sample size .
the bmi among males had 9.1% false positive and 32.4% false negative , which were similar to a study by jitnarin et al . , who studied firefighters between the ages of 20 and 62 years .
in short , there are errors in the identification of obese males with the bmi ; however , it is more suitable for females . in males under the age of 40 and females who are less than 20 years old , gallagher s classification is a better determinant of obesity than the bmi .
reported a paradox in the findings of elderly patients with coronary heart disease with gallagher s classification by reporting the highest mortality in the underweight and normal groups and the lowest mortality in the overweight group . in our study ,
more than 46% of the male obese and 62% obese females older than 39 years were wrongly classified as overweight by this indicator ; thus , we concluded that it is not a good indicator to distinguish between older overweight and obese individulas , which made schutter s paradox questionable .
a systematic comparison of all results showed that gallagher s classification is better in detecting obesity in men , especially among those who are less than 40 years old . on the other hand ,
the statement by rothman , the use of bmi as an indicator of obesity can introduce misclassification problems that may result in important bias in estimating the effects related to obesity is confirmed . in spite of the large sample
, it should be noted that the participants were recruited from southern iran and they do not represent the whole population of the country .
we recommend replication of the study in other regions of iran , which could enhance the generalizability of the results .
gallagher s classification is recommended for non - obese males and females and obese males .
additionally , for the iranian population , it is recommended to employ 27.7 kg / m as the cutoff point for detecting obesity among women and men , 27.7 kg / m for women , and 27.3 kg / m for men .
the results of the study would enlighten scientists to consider other methods in examining body conditions rather than the status quo . | background : the study was conducted to examine the comparability of the bmi and gallagher s classification in diagnosing obesity based on the cutoff points of the gold standards and to estimate suitable cutoff points for detecting obesity among iranians.methods:the cross - sectional study was comparative in nature .
the sample consisted of 20,163 adults . the bioelectrical impedance analysis ( bia )
was used to measure the variables of interest .
sensitivity , specificity , positive predictive power ( ppv ) , and negative predictive power ( npv ) were used to evaluate the comparability of the two classification methods in detecting obesity.results:the bmi wrongly classified 29% of the obese persons as overweight . in both classifications , as age increased ,
the accuracy of detecting obesity decreased .
the gallagher s classification is better than mbi in detecting obesity in men with the exception of those older than 59 years . in females ,
the bmi was better in determining sensitivity . in both classifications , either female or male ,
an increase in age was associated with a decrease in sensitivity and npv with the exception of the bmi for the 18 year olds .
gallagher can correctly classify males and females who are less than 40 and 19 years old , respectively.conclusion:gallaghers classification is recommended for non - obese in both sexes and in obese males younger than 40 years old .
the bmi is recommended for obese females .
the suitable cutoff points for the bmi to detect obesity are 27.70 kg / m2 for females and males , 27.70 kg / m2 for females , and 27.30 kg / m2 for males . |
leptomeningeal carcinomatosis ( lc ) , also called carcinomatous leptomeningitis , neoplastic ( lepto)meningitis , leptomeningeal metastasis , or more commonly leptomeningeal seeding , is the dissemination and growth of cancer cells within the leptomeningeal space ; it is one of the most serious complications that can occur in cancer patients1 ) .
lc is frequently found in patients with leukemia , breast cancer , lymphoma , and lung cancer2 ) .
gastric cancer ( gc ) is one of the most common causes of cancer related death in asian countries including korea .
kim , et al3 ) reported that the incidence of lc with gc was 0.06% of all cases of gastric cancer .
lc is a more frequent manifestation of advanced or metastatic carcinoma rather than associated with early disease .
we report a rare case with lc as the presenting symptom and review the medical literature .
a 49-year - old woman was admitted to our hospital with headache , dizziness , easy fatigability , and melena for 10 days .
her vital signs were as follows : a blood pressure of 130/75 mmhg , a pulse rate of 95 beats / min and a respiration rate of 21 breaths / min .
the mri of brain showed no abnormalities , but the esophagogastroduodenoscopy showed thickening of the gastric folds on the greater curvature of the stomach and on the posterior wall of the stomach compatible with a borrman type iv gastric cancer ( figure 1 ) .
the ct scan of abdomen and pelvis showed an advanced gastric cancer involving the posterior wall of the lower body of the stomach ; in addition , the angle and the antrum had extensive metastatic lymphadenopathy .
the patient complained of an intractable persistent headache as well as nausea and vomiting on admission day 6 . a lumbar puncture and analysis of the cerebrospinal fluid ( csf )
there were many mucin - secreting cells and signet ring cell types , which were strongly positive for mucicarmin and alcian blue in the csf ( figure 4 ) .
since the first report of leptomeningeal carcinomatosis ( lc ) in 1870 , the incidence of lc has been increasing .
gastric cancer is the most common cancer in korea ; however , lc associated with gastric cancer ( gc ) is relatively rare . in the korean medical literature ,
the incidence of lc with gc has been reported to be 0.06% of all gastric cancers3 ) . among cases with lc ,
gc is reported as the most frequent cause of cancer in korea4 , 5 ) .
however , these results are based on the experience of one institution , so these findings may not be generalized to the entire korean population .
the clinical manifestations of lc are very variable and depend on the site of involvement .
many patients complain of headache , nausea , vomiting , ataxia , and have a variety of neurological deficits .
lc is usually a manifestation of advanced cancer discovered during follow - up after a cancer diagnosis ; it is rarely the presenting symptom as it was in this case .
the median time to the diagnosis of lc , from the initial cancer diagnosis , is very variable ; reports have found a 76 day5 ) to 17 month4 ) time interval .. diagnosis is based on cytology examination of the csf and imaging studies ( especially , magnetic resonance imaging with gadolinium enhancement)8 ) .
the csf findings include an increased opening pressure , elevated protein , the presence of tumor markers ( for example , cea , ca19 - 9 in gastric cancer ) , decreased glucose , and pleiocytosis .
however , the diagnosis is frequently missed on the first trial but the diagnosis rate increases by 30% on a second one . in our case ,
reported on patients with breast cancer metastasized to the central nervous system over 20 years .
out of five lc patients , only one ( 20% ) patient was confirmed cytologically9 ) .
pathologically , poorly differentiated adenocarcinoma with signet ring cell features is the most frequent type of lc associated with gc in japan10 ) and this is likely true for korea3 ) .
lc was a more frequent finding in advanced disease with or without distant metastases ; it is rarely reported with early gastric cancer11 ) .
imaging studies ( mri with gadolinium enhancement ) show abnormalities in 67% of lc patients12 ) .
there are several treatment options for patients with lc including intrathecal ( it ) chemotherapy with or without radiotherapy ( rt ) .
most chemotherapeutic agents do not penetrate the blood - brain barrier ( bbb ) , and are given by direct intrathecal administration .
agents that can be used for it chemotherapy include methotrexate ( mtx ) , cytarabine , thiotepa , and steroids .
the benefit ( i.e. the prolongation of survival ) achieved by it chemotherapy ( single or multiple regimens ) in lc patients continues to be debated .
the median survival of lc is reported to be four to six months even with aggressive therapy . in one study ,
the objective response rate and median survival of it mtx were 20% and 11 weeks ; with sustainedrelease cytarabine they were 25% and 15 weeks .
therefore , the results did not confirm a definite benefit with the it chemotherapy13 ) .
however , in a korean study , there was a favorable outcome with combination it chemotherapy , compared to the single it chemotherapy14 ) .
mtx , one of most useful agents for it chemotherapy , penetrates the blood brain barrier poorly in low doses , but can penetrate in the higher dose range .
high dose intravenous mtx ( 3.5 g / m ) injection showed a 28% partial response and stable disease and 44% progressive disease .
the median survival of patients with lc ( with or without parenchymal disease ) was 12.6 weeks ; shorter than with isolated parenchymal disease ( 25.4 weeks)15 ) .
the clinical risk factors that influence the survival of patients with lc are performance status , underlying malignant disease and previous responsiveness to anticancer therapy .
the patients with disease that is more advanced , poor performance status ( ps ) , chemoresistant tumors , pronounced neurological clinical findings , and a heavy pretreatment status have a poor prognosis and additional management should be with conservative treatment16 ) .
in our case , the patient had a poor ps , pronounced abnormal neurological clinical findings and a very rapidly deteriorating clinical course . | leptomeningeal carcinomatosis occurs in approximately 5% of patients with cancer .
the most common cancers involving the leptomeninges are breast , lung cancer and melanoma .
however , gastric adenocarcinoma has been rarely reported with leptomeningeal carcinomatosis .
the presenting manifestations are usually headache , visual disturbances and seizures .
we report a case of leptomeningeal metastasis that presented as a gastric cancer .
a 49-year - old woman was admitted to our hospital with the symptoms of headache and melena for 10 days .
the endoscopy showed a thickening of the folds of the stomach compatible with the diagnosis of a borrman type iv gastric cancer .
the biopsy revealed a signet ring cell carcinoma .
the mri of brain showed no abnormal findings ; however , the patient complained of an intractable persistent headache , nausea and vomiting on admission day 6 .
the cytology examination of the cerebrospinal fluid supported the diagnosis of metastatic signet ring cell carcinoma . |
a 52-year - old male patient presented at our hospital with progressive unilateral proptosis since 2 years ago and vision loss in his left eye .
ocular examination revealed 2 + relative afferent pupillary defect in the left eye . computed tomography scan of the patient showed an intraconal mass sticking to superior ophthalmic fissure [ fig . 1 ] .
the procedure was done without complication , and the early postoperative vision was fine but about an hour later , patient developed sudden vision loss and became no light perception ( nlp ) . for ruling out postoperation hemorrhage ,
fundus examination with indirect ophthalmoscopy revealed typical view of crao with the presence of cherry - red spot on the macula , white ground - glass appearance of the retina , and optic disc edema [ fig . 2 ] . for decreasing intraocular pressure and establishing retinal reperfusion , immediate ocular massage and anterior chamber paracentesis as well as systemic therapy with mannitol were done .
two hours later , it was improved to 1 m counting finger , but he complained a large central scotoma .
his va improved to 20/200 the day after the surgery , and he complained of diplopia .
orbital computed tomography - scan of the patient shows well - defined dumble shaped intraconal orbital mass that pushed the optic nerve superomedially and extending to the supra orbital fissure fundus photograph 3 days after the surgery showing cherry - red spot on the macula and white ground - glass appearance of the retina and also optic disc edema medical workup did not show any evidence of collagen vascular or cardiovascular , hematologic , and neurologic disorders .
systemic investigations including fasting blood sugar , lipid profile , and homocysteine levels all were normal .
crao is usually caused by a thrombus or embolus that resulting to reduce blood perfusion of the retina .
it clinically presents as sudden painless acute unilateral or bilateral vision loss in the range of counting fingers to nlp .
the rate of spontaneously recanalization of the artery is about 15% with timely intervention , the prognosis is very poor as only 61% of patients can regain a final va of 6/120 or less .
one of the typical findings in crao is cherry - red spot that is found in about 90% of cases .
there is a golden time of 90120 min after occlusion to perform interventions to improve vision , however there is no approved modality to be effective in the treatment of crao , but some methods such as immediate ocular massage and anterior chamber paracentesis , use of drugs such as intravenous acetazolamide and mannitol or inhalation of a mixture of 95% oxygen and 5% carbon dioxide ( carbogen ) , all aimed to reduce intraocular pressure and improving blood flow to the eye . without doing these modalities , < 10% of patients can recover meaningful vision .
multiple theories for crao during surgical procedures have been proposed ; a prolonged hypotensive status and reduction in blood flow or also increasing intraocular pressure including ocular compression during certain ocular surgical procedures associated with ocular ischemia , causing ischemia of the retina and may lead to crao and vision loss after surgery .
although we did not face any significant drop in blood pressure ( bp ) during intervention but regarding differences in populations may be our patient was very sensitive to even small drop in bp that could lead to crao .
in another theory , krner - stiefbold explains that retrobulbar masses such as hematoma , neoplasms , and retrobulbar injections may compress central retinal artery and lead to arterial occlusion and decrease blood perfusion causing crao . a more likely explanation in that ocular vascular damage may be happened by surgical maneuvers , and it will results in activation and aggregation of platelets and activated platelets release serotonin ( 5-hydroxytryptamine ) .
serotonin is a vasoconstrictor and inducing transient arterial spasm , causing transient or complete arterial occlusion leading to ischemia of retina and creating crao .
we recommend that in all patients with orbital tumors that undergo tumor removal , the ocular exam should be performed immediately , and every 13 h after the surgery to rule out crao , as delay in diagnosis and intervention could eventuate to severe vision loss .
| a 52-year - old male patient presented at our hospital with unilateral proptosis and vision loss in his left eye .
imaging evaluations showed orbital tumor , so the patient underwent surgery .
about an hour later after tumor removal , patient developed sudden vision loss and became no light perception .
fundus evaluation revealed central retinal artery occlusion ( crao ) .
the patient was treated immediately with ocular massage and anterior chamber paracentesis as well as systemic therapy with mannitol and intravenous administration of acetazolamide .
after thirty minutes , he recovered perception to light and then hand motion and 2 h later , it was improved to 1 m counting finger .
crao following orbital tumor has not been reported before .
we recommend ocular examination in all patients that undergo orbital surgery immediately to 23 h after surgery . |
although splenic angiosarcoma has been presented as an extremely rare malignant tumour in medical literature spanning the last 20 years , an increasing number of cases have been identified over that same time period .
some of these cases are typically identified upon spontaneous rupture of the organ ( 32% ) , and as such are associated with a poor prognostic outcome for the patient . despite tremendous strides undertaken in medicine over the last two decades , both conservative and invasive treatment measures continue to produce low - yield results when it comes to prolonging patient survival .
this may likely be attributed to a high rate of distant metastasis , affecting organs such as the liver , breast and bone .
only hara et al . have reported long - term survival of a patient with angiosarcoma after splenectomy , and high - dose chemotherapy , and autologous peripheral blood stem cell transplantation .
we hereby report our experience with two splenic angiosarcoma cases , which despite being similar in their diagnosis , nonetheless presented with an entirely different clinical picture . both may be found in common practice , and ought to be highlighted for their significance .
a 54-year - old caucasian man was initially admitted to the clinic of gastroenterology with non - specific complaints of abdominal pain and loss of appetite over the last 6 weeks , which grew worse post - prodigally and with respiration .
laboratory results revealed normocytic anaemia ( haemoglobin 10.0 g / dl ) , reticulocytosis , thrombocytopenia ( platelets 68 10/l ) , a small increase in bilirubin ( 2.34 mg / dl ) , as well as elevated ldh ( 758 iu ) , ggt ( 200 iu ) and alp ( 336 iu ) levels .
ultrasound was inconclusive , but revealed structural changes that corresponded to either the presence of metastasis or a haemangioma .
a subsequent ct scan further elucidated an affected spleen , with possible metastatic or lymphoma - like changes ( fig .
1 ) . abdominal computed tomography scan demonstrating an en larged spleen with the metastatic liver tumours the patient was scheduled for explorative surgery , but prior to it being performed , he went into shock and presented with an ascitic abdomen .
his subsequent morphological parameters were increasingly depressed when compared to their previous counterparts , and emergency ultrasound and ct scans showed a significant amount of peritoneal fluid . during the emergency surgery , approximately 3 litres of both old and fresh blood
were discovered within the peritoneal cavity , along with a 16 cm cyst located in the spleen , which was also seeping blood .
both lobes of the liver were covered with numerous small tumours , 2 of which were bleeding , as they sat interspersed between multiple blood clots .
after suturing and a histological sample of the liver had been obtained , a splenectomy and peritoneal lavage with warm saline to evacuate the clots were performed , and the operation was concluded .
a cross - section of the spleen subsequently revealed a greyish - white discharge , with the organ itself measuring 16 12 10 cm ( fig .
a 77-year - old caucasian woman was admitted to our hospital due to haematochezia and fatigue .
she initially presented at the clinic with a number of concurrent illnesses , chief amongst them being normocytic anaemia , diabetes mellitus type 2 , hypertension , and paroxysmal atrial fibrillation .
having previously undergone a hysterectomy , the patient had now been referred for the operation . the spleen was visibly enlarged , and showed a number of superficial tumours .
purpura - like changes were further noted atop the greater omentum , as well as the wall and mesentery of the small bowel ( fig .
3 ) . at the sigmoid portion of the colon , immediately above the colorectal junction ,
4 ) . a resection of the sigmoid colon as well as the anterior portion of the rectum was undertaken , with an accompanying colorectal anastomosis .
subsequent histological examination of the obtained samples evaluated them to be an adenocarcinoma of the sigmoid colon ( g2 , t3 ) , an angiosarcoma of the spleen ( g3 ) , as well as lymph nodes that were affected by metastasis ( fig .
no complications were noted following the surgery , although the patient 's state was deemed to be less than ideal .
her age and the concurrent presentation of two independent cancers excluded her from the possibility of receiving adjuvant chemotherapy .
intraperitoneal dissemination of the splenic angiosarcoma carcinoma of the sigmoid colon the splenic angiosarcoma angiosarcoma with anastomosing vascular channels .
tumour cells are large , polygonal with abundant eosinophilic cytoplasm and large nuclei with prominent eosinophilic nucleoli
amongst the rarest of all neoplasms , primary splenic angiosarcoma has a frequency of 0.140.25 reported cases per one million persons annually [ 3 , 4 ] .
no previous article has made mention of primary splenic angiosarcoma existing independently of another primary neoplasm . although jimenez - heffernan et al .
( 1999 ) have outlined the rupture of a splenic angiosarcoma into the colon subsequently presenting as anal bleeding they made no mention of the event occurring as a result of synchronous presentation of two primary tumours .
more accurately , these results suggest that the affected splenic flexure was a site of a gi metastasis , rather than primary neoplastic involvement . as such
, our case would be the first one to highlight the co - occurrence of a simultaneous adenocarcinoma of the colon and an angiosarcoma of the spleen .
the clinical symptoms and diagnostic values associated with splenic angiosarcoma are extremely variable . nonetheless , the majority of patients ( 75% ) complain of abdominal pain , and a quarter to one - third present with rupture of the involved organ [ 312 ] .
anaemia , leucopoenia , elevated ldh levels and thrombocytopenia are also frequently reported , with anaemia being caused by damage to the red cell surface , whilst the other factors may be precipitated by severe haemolysis .
it has been increasingly postulated that thrombocytopenia may be the first manifestation of the disease , even in the absence of other laboratory irregularities .
interestingly , while both cases are characterized by similarities when it comes to the values obtained in their laboratory results , the first patient had never suffered from any major illness prior to his presentation of splenic angiosarcoma , while the second had long been affected by a number of other illnesses .
splenic angiosarcoma typically carries with it a grave prognosis for all but 20% of patients , who then manage to survive for a period longer than 6 months [ 4 , 6 ] .
survival statistics are thought to be increased in those instances where the diagnosis and/or the splenectomy are performed prior to the actual rupture of the organ [ 3 , 11 ] .
both cases were indeed marked by significant intraperitoneal bleeding , lending further support to the previously produced research .
as in the case of the second patient , the co - existence of two independent tumours then coupled with an intraperitoneal haemorrhage appears to have decreased the survival rate even further . although typically described as rare , primary splenic angiosarcoma nonetheless carries with it an extremely low survival rate upon infection .
it has an ever - changing clinical and laboratory profile , with no consideration for genetics , ethnicity , or geographical location .
its diagnostic evasiveness may be further compounded by the fact that although it is typically thought to occur between the 5 and 6 decade of life , it may also strike the paediatric population at virtually any age [ 4 , 7 ] . as such , the aforementioned two cases serve to contrast the possible circumstances in which the tumour may occur .
the second instance also highlights the first possible reported occurrence of a synchronous presentation of a splenic angiosarcoma alongside a colonic adenocarcinoma , further emphasizing the fact that the former may carry with it a neoplastic risk which extends well beyond distant metastasis .
| splenic angiosarcoma has been presented as an extremely rare malignant tumour . amongst the rarest of all neoplasms ,
primary splenic angiosarcoma comprises 0.140.25 of all annually reported cases per one million persons splenic angiosarcoma was first described in 1879 by t. langerhans .
the clinical symptoms and diagnostic values associated with splenic angiosarcoma are extremely variable .
nonetheless , majority of the patients ( 75% ) complain of abdominal pain , and a quarter to one - third present with rupture of the involved organ .
we hereby report our experience with two splenic angiosarcoma cases , which despite being similar in their diagnosis , nonetheless present with an entirely different clinical picture .
our first case , whereby the patient presented with a liver that was also affected by the angiosarcoma of the spleen . in the second case
however , although the patient did show evidence of metastasis to the abdominal cavity as well as the liver , she also suffered from primary adenocarcinoma of the colon .
to our knowledge , no previous article has made mention of primary splenic angiosarcoma existing independently of another primary neoplasm . both may be found in common practice , and ought to be highlighted for their significance as such . |
structural variants of -galactosylceramide ( gc ) that activate invariant natural killer t cells ( inkt cells ) are being developed as potential immunomodulatory agents for a variety of applications .
identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti - inflammatory cytokines is central to current efforts , but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds .
here we describe a fluorescence - based assay to identify functionally distinct gc analogues .
our assay is based on recent findings showing that presentation of glycolipid antigens by cd1d molecules localized to plasma membrane detergent - resistant microdomains ( lipid rafts ) is correlated with induction of interferon- secretion and th1-biased cytokine responses . using an assay that measures lipid raft residency of cd1d molecules loaded with gc , we screened a library of 200 synthetic gc analogues and identified 19 agonists with potential th1-biasing activity .
analysis of a subset of these novel candidate th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a th1 type bias .
these results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high - throughput assay for identification and functional classification of inkt cell activating glycolipids . | |
certain phenyl - substituted hydrocarbons of environmental concern have the potential to disrupt the endocrine system of animals , apparently in association with their estrogenic properties .
competition with natural estrogens for the estrogen receptor is a possible mechanism by which such effects could occur .
we used comparative molecular field analysis ( comfa ) , a three - dimensional quantitative structure - activity relationship ( qsar ) paradigm , to examine the underlying structural properties of ortho - chlorinated hydroxybiphenyl analogs known to bind to the estrogen receptor .
the cross - validated and conventional statistical results indicate a high degree of internal predictability for the molecules included in the training data set .
in addition to the phenolic ( a ) ring system , conformational restriction of the overall structure appears to play an important role in estrogen receptor binding affinity .
hydrophobic character as assessed using hydropathic interaction fields also contributes in a positive way to binding affinity .
the comfa - derived qsars may be useful in examining the estrogenic activity of a wider range of phenyl - substituted hydrocarbons of environmental concern.imagesfigure 1.figure 2 . | |
they are not only the powerhouses of the cell , they are also major triggers for the intrinsic pathway of apoptosis , regulators of calcium homeostasis and providers of various chemicals for the cell , such as phospholipids and iron - sulfur clusters ( indispensable for membrane biogenesis and for the assembly of many catalytic centers , respectively ) .
the vast majority of mitochondrial proteins are encoded in the nuclear genome and are imported post - translationally , while a dozen proteins are directly encoded in the mitochondrial genome . as both genomes contribute subunits to the respiratory chain complexes , coordination of the expression of the nuclear and the mitochondrial genome , as well as the rate of protein import , is crucial for the proper stoichiometric assemblies of these complexes .
there are a number of ways that this can go wrong . facing such problems ,
the cell is left with two options : repair the faulty mitochondria or get rid of them .
interestingly , mitochondrial quality control is tightly linked to medically important phenomena , such as neurodegeneration and aging . while parkinson s disease is a mostly sporadic disease characterized by the cytoplasmic accumulation of plaques containing aggregates of the protein alpha - synuclein in dopaminergic neurons , a small percentage of parkinson s disease
is strictly genetically inherited and its onset is earlier than sporadic parkinson s disease . besides pathological dominant alleles of the alpha synuclein - gene itself , most of these genetic cases of parkinson s disease are due to recessive mutations in genes encoding phosphatase and tensin homolog ( pten)-induced kinase 1 ( pink1 ) and parkin .
the activation of another mitochondrial quality control pathway extends the lifespan of worms and mice .
therefore the study of mitochondrial quality control holds promise to boost our understanding of these medically relevant conditions .
this pathway can be triggered by a sudden need for energetic resources , for instance in the case of a starvation response . in this case ,
random bits of the cytoplasm are engulfed in a double membraned organelle ( autophagosome ) and delivered to the lysosome for degradation and recycling . in other conditions ,
select parts of the cytoplasm , such as organelles , are sorted for engulfment in a selective autophagic process .
treating cells with harsh mitochondria damaging agents can cause the virtually complete mitophagic disappearance of mitochondria ( e.g. ) . in physiological conditions ,
mitophagy not only is specific for mitochondria but it also only targets a subset of mitochondria , that is , the terminally damaged ones .
pink1 is a serine / threonine kinase bearing a mitochondrial targeting sequence and a transmembrane domain . in the absence of damage , pink1
is quickly turned over and does not accumulate . however , pink1 is selectively stabilized on the surface of faulty mitochondria [ 8 - 10 ] .
pink1 accumulation then causes the recruitment of parkin , which is a ring - domain containing e3-ubiquitin ligase .
parkin broadly ubiquitinylates proteins on the cytosolic side of the outer mitochondrial membrane ( omm ) .
ubiquitin is then used as a flag to degrade omm proteins and to recruit the autophagic machinery .
mitochondria are highly dynamic , in that they undergo cycles of fusion and fission . as a result
, the number of mitochondria in a cell is not fixed , but fluctuates depending on whether mitochondria are rather elongated or fragmented .
the physiological role of mitochondrial dynamics is largely mysterious , but one of its important functions appears to be quality control . on the one hand
, a damaged mitochondrion may be able to segregate its damaged components into subcompartments and divide , giving rise to one healthy mitochondrion and one that presumably carries the damage load of the original mitochondrion .
this damaged mitochondrion has an opportunity to repair itself , or is targeted to mitophagy by the pink1-parkin pathway . on the other hand ,
fusion is inhibited in damaged mitochondria . the opa1 protein ( mutated in dominant optic atrophy )
is involved in the fusion of the inner mitochondrial membrane ( imm ) during mitochondrial fusion .
opa1 exists in various forms that are produced by proteolytic cleavage . among the proteases that process opa1 is oma1 .
damaged organelles are therefore prevented from contaminating healthy ones . moreover , preventing fusion also leads to a shortening of mitochondria , which are thereafter more easily engulfed in mitophagosomes .
another way to prevent fusion is the selective pink1-parkin - dependent proteasomal degradation of the mitofusins .
cells undergoing stress homeostatically adapt by changing their transcription program to cope with and overcome the stress .
such transcriptional stress responses include the cytoplasmic heat - shock response and the endoplasmic reticulum unfolded protein response ( upr ) . in both of these cases , the accumulation of misfolded proteins is sensed by one or more sensor proteins in the respective compartments , causing the activation of a nuclear transcription factor , which turns on a transcriptional program aimed at fixing the problem .
a more recently discovered pathway is the mitochondrial upr , which has been most extensively studied in the worm caenorhabditis elegans . at the core of this pathway is activating transcription factor associated with stress-1 ( atfs-1 ) .
atfs-1 is a basic leucine zipper ( bzip ) transcription factor , which bears a mitochondria targeting sequence at its n - terminus . in healthy cells
, atfs-1 is efficiently imported into the mitochondrial matrix , where it is quickly degraded . upon mitochondrial dysfunction ,
the import efficiency of atfs-1 drops and atfs-1 starts accumulating outside of mitochondria . because atfs-1 bears a nuclear localization sequence ( nls ) close to its bzip domain , it is imported into the nucleus where it starts a transcription program . among the transcriptional targets of atfs-1 are mitochondrial chaperones , which are upregulated to cope with the stress in mitochondria .
the pathways we have just described have almost completely opposite outcomes . both mitochondrial fragmentation and mitophagy aim at getting rid of the problem , while mitochondrial upr tries to fix it . when and why does the cell choose one over the other ?
this explanation , however , does not hold as the stresses eliciting mitophagy , mitochondrial upr and fusion inhibition appear to be identical : it s a loss of electrochemical potential in the imm , which is the cause of pink1 accumulation in the omm , of atfs-1 failure to enter the mitochondria and possibly also of oma1 activation .
the mechanisms underlying these various phenomena are actually very similar and relate to the fact that the electrochemical potential of the imm is crucial for the translocation of proteins across the imm . in healthy mitochondria ,
the mitochondrial targeting sequence of pink1 precursor crosses both translocases of the outer and inner membranes ( tom and tim ) , to reach the imm , where its transmembrane domain is inserted .
imm insertion of pink1 triggers a degradation reaction initiated by the intra - membrane cleavage of the transmembrane domain by the presenilin - associated rhomboid - like protease ( parl ) ( fig . 1 , left ) .
the final degradation reaction is pursued in the cytosol ( likely after retrotranslocation through the omm ) by the cytosolic proteasome because the n - terminal amino acid of cleaved pink1 is targeted by the n - end rule pathway .
compromised mitochondria are depolarized , therefore pink1 can reach tom but does not efficiently translocate into the tim complex . as a result ,
it is most likely released from the tom complexes and inserted laterally into the omm , where it can recruit parkin and trigger mitophagy ( fig .
1 , right ) . in healthy mitochondria , pten - induced kinase 1 ( pink1 )
is transported to the inner mitochondrial membrane ( imm ) where it is processed by the intramembrane presenilin - associated rhomboid - like protease ( parl ) .
this leads to the retrotranslocation of pink1 to the cytosol , where it is degraded by the proteasome via the n - end rule pathway . in depolarized mitochondria
, pink1 fails to reach the imm and instead inserts in the outer mitochondrial membrane ( omm ) , where it recruits the ubiquitin ( ub ) ligase parkin to ubiquitinylate omm resident proteins . in healthy cells ,
atfs-1 is imported into the mitochondrial matrix , thanks to its mitochondrial targeting sequence ( mts ) . in depolarized mitochondria , atfs-1 import
is inhibited . as a result , atfs-1 translocates to the nucleus where it acts as a transcription factor to induce the mitochondrial upr .
it is likely in an inactive form . as a consequence , a long non - processed form of its substrate the dynamin - related protein opa1 accumulates ( opa1-l ) . in depolarized cells
, oma1 is activated and cleaves opa1 to a short form ( opa1-s ) , thereby inhibiting mitochondrial fusion . the case of atfs-1 is more straightforward : a simple kinetic competition appears to take place between its mitochondrial targeting sequence and its nls . since the electrochemical gradient across the imm is crucial for mitochondrial protein import and , since depolarized mitochondria do not import proteins efficiently , loss of electrochemical potential will shift the equilibrium to favor nuclear import of atfs-1 and trigger mitochondrial upr ( fig .
it is proposed that oma1 inserts in the omm , where it is rendered inactive by proteolytic cleavage ( fig . 1 , left ) .
upon depolarization , oma1 might insert into the imm in a way that prevents its proteolytic cleavage and instead accumulates as an active precursor ( fig .
1 , right ) . thus , if the type of insult that triggers mitophagy or mitochondrial upr is not qualitatively different , then what decides between the two cellular pathways ?
. a single damaged mitochondria will be sufficient to expose pink1 on its surface and trigger its own degradation .
however a single faulty mitochondrion will likely not be able to release enough atfs-1 to mount a mitochondrial upr , as enough healthy mitochondria can compete with the nucleus for atfs-1 import . in genetics terms , one could say that mitophagy is dominant , while mitochondrial upr is recessive . as a result
, mitophagy is an organelle - specific response while mitochondrial upr is a systemic response .
therefore , the choice of one pathway over the other will depend on the overall degree of damage of the mitochondrial network : a few highly damaged mitochondria will trigger their selective elimination by mitophagy , while a low general level of damage to most mitochondria will cause mitochondrial upr .
of course , any intermediate situation could be dealt with by a combination of mitophagy and mitochondrial upr activation .
these two dominant and recessive types of response are actually a very neat way of fine tuning the cellular response to mitochondrial damage . on the one hand
, a single faulty mitochondrion might not necessitate the induction of a costly systemic program and can be dealt with mostly by mitophagy .
on the other hand , eating up the whole mitochondrial network is a bad idea .
pink1 and parkin are the most prevalent recessive mutations found in patients with early onset parkinson s disease , suggesting that a specific malfunction in the machinery that gets rid of damaged mitochondria can cause the disease .
the aetiology of the disease is not clear in pink1 and parkin - related early onset parkinson s disease .
sporadic parkinson s disease is characterized by the appearance of amyloid plaques of alpha - synuclein , which is not a mitochondrial protein .
moreover , most patients suffering from parkin- or pink1-related parkinson s disease do not show plaques .
since the original observations that calorie restriction could extend lifespan , a close connection exists between energy metabolism and aging .
mitochondria , as an important source of reactive oxygen species , have long been considered a prime suspect for causing cellular aging . however
, a different picture emerges from recent studies . in a screen for long - lived worm mutants , andrew dillin s lab found that creating imbalance in the assembly of respiratory chain subunits caused increased lifespan .
indeed , this increase in lifespan did not correlate to a reduced activity of the mitochondria , but to an activation of mitochondrial upr , since increase in lifespan is blunted in mutants incapable of mounting a mitochondrial upr .
thus , although direct causality between mitochondrial upr induction and lifespan extension is not clearly established , it seems that mitochondrial quality control , more than mitochondrial activity , contributes to aging regulation .
these observations , originally made in worms , could be extended to mice , where a large - scale genomic association study found that longevity was associated with variations in genes encoding mitochondrial proteins .
finally , besides aging and parkinsonism , other medically relevant conditions seem to involve mitochondrial biogenesis and turnover , and mitochondrial dynamics and quality control genes are often mutated in neurological diseases .
these include dominant optical atrophy ( opa1 ) , charcot - marie - tooth type 2a ( mitofusin 2 ) , leber hereditary optical neuropathy ( various mutations in mitochondrial dna ) among others ( reviewed in ) .
the unexpected relationship between mitochondrial quality control , neurodegeneration and aging thus stresses the importance of the global and detailed understanding of these pathways for human health .
| repairing or disposing of a malfunctioning object is an everyday dilemma .
replacing an item may be quicker than repairing it , but may also be more costly .
cells are faced with the same options when their organelles are challenged . ensuring the health of the mitochondrial network is of utmost importance for cellular health and , not surprisingly , mitochondrial quality control can take both the repair and disposal routes .
spectacular advances have been made in recent years and a picture is starting to emerge of what drives a cell to take one or the other path .
interestingly , mitochondrial quality control seems to be deficient in various medically relevant conditions , such as neurodegeneration and aging . |
in the united states , 3 million cases of suspected child abuse are filed each year , but only 30% of these lead to a conviction , indicating that diagnosing child abuse is difficult .
the presence of multiple bruises of different color suggests long term abuse , as different colors imply different stages of healing , and thus different times of origin for each bruise .
therefore , accurate determination of the age of bruises has the potential to contribute to an improved diagnosis of physical abuse , as it can confirm or exclude the presence of a possible perpetrator at the determined time point .
the color of the bruise changes over time [ 18 , 39 ] , hence color has become the choice for age determination .
these colors and their corresponding age are documented in standardized protocols available to the physician .
however , there is an enormous variability in color descriptions versus age of the bruise which seriously complicates the age determination of the bruise by visual inspection alone [ 3 , 18 , 22 , 26 , 32 , 33 , 39 ] .
therefore , it has been attempted to objectively determine the color of a bruise by applying reflectance spectroscopy [ 6 , 14 , 24 , 2830 ] . however , and perhaps more importantly , color is not uniquely characterizing for the age of a bruise since bonhert et al
. showed a relation between color and depth of the bruise : i.e. a deeper bruise has a more blue appearance than a superficial bruise of equal age .
in other words , two bruises of equal age but located in different depths in the skin have different appearances . unfortunately , therefore , accurate age determination is currently not possible .
following randeberg et al . , we hypothesize that an increased accuracy of age determination of bruises is only possible when the pathophysiology of bruise formation and healing is better understood than now .
randeberg et al . constructed an analytic model of formation and healing of bruises , based on darcy s law of convection and fick s law of diffusion , to describe fluid transport in tissue and distribution of blood and hemoglobin breakdown products , and computing the temporal distribution of blood and hemoglobin breakdown products integrated over the whole bruise area . by applying an integrating sphere to illuminate a bruise and measuring the spectrum of the remitted light , randeberg et al
the optical data were interpreted with an analytic model and an accuracy of 3 h for a fresh bruise ( less than 1 day old ) , and 1 day for a bruise up to 10 days old was reported . in randeberg s model , spatial variations in spectral properties are averaged out while , as demonstrated by hughes et al .
we hypothesized that a more comprehensive modeling of 3d spatial and time dependent transport of bruise chromophores may result in a more accurate determination of the age of the bruise .
therefore , our next generation model includes the possibility to model the spatial dependence of the chromophores in the bruise . also , we added horizontal convection and diffusion and michaelis menten kinetics of the enzymes involved in conversion of hemoglobin to bilirubin . apart from initial formation of the bruise
, such a model also needs to account for skin thickness , clearing time of bilirubin , and the different diffusivities of hemoglobin and bilirubin .
these different diffusivities imply that the bilirubin ( yellow ) part of bruises diffuses farther than the hemoglobin part . in this article
, we present the formulation and parameter analysis of a 3d finite distributed compartment model incorporating all these aspects .
we also include a typical natural bruise , photographed every day from 1 to 9 days , and we fitted our model to the measured chromophore areas , as a first quantitative validation of the model . finally , within the validity of our modeling , we show how the age of ( simulated ) bruises can potentially be determined from the time dependent differences between hemoglobin and bilirubin areas within the bruise .
the model of the skin consists of three layers ; the top layer of the dermis ( layer 1 ) , the bottom layer of the dermis ( layer 2 ) and the subcutaneous tissue layer ( layer 3 ) .
each layer is subdivided into 100 100 corresponding compartments as schematically shown in fig .
for ease of analysis , all compartments have the same lateral dimensions equal to the thickness of the total dermis , and the thickness of the layers can be varied to account for inter - individual variability or body location .
the initial condition of the bruise is modeled by a pool of hemoglobin of arbitrary shape and size in the subcutaneous tissue ( fig .
the bruise develops over time by conversion of hemoglobin into bilirubin and transport of these molecules via pressure driven flow , i.e. convection and concentration driven diffusion .
the concentration of hemoglobin within a compartment may change by three processes : ( 1 ) convection in vertical direction from the subcutaneous tissue layer into the dermis ( fig .
1b ) , ( 2 ) vertical diffusion between the layers , as well as horizontal diffusion within the layers ( fig .
darcy s law for transport of fluids describes the convection of hemoglobin in vertical direction , assuming no pressure gradient in horizontal direction , hence neglects flow in horizontal direction ( first right hand term in eq . 1 ) .
diffusion , in vertical and horizontal direction , follows concentration gradients and is described by the first law of fick ( second right hand term in eq . 1 ) .
we will first present the formula for compartments in layer 2 , from which the formulas for layers 1 and 3 follow.fig .
1a the skin model consists of 3 layers , the top layer of the dermis , the bottom layer of the dermis and the subcutaneous fat layer .
each layer consists of 100 100 compartmets ( for ease of presentation a smaller number is shown ) .
b and c a pool of hemoglobin is defined in the subcutaneous layer . via michealis
both hemoglobin and bilirubin flow inside the layers and between the layers a the skin model consists of 3 layers , the top layer of the dermis , the bottom layer of the dermis and the subcutaneous fat layer .
each layer consists of 100 100 compartmets ( for ease of presentation a smaller number is shown ) .
b and c a pool of hemoglobin is defined in the subcutaneous layer . via michealis
both hemoglobin and bilirubin flow inside the layers and between the layers the rate of change of hemoglobin in the compartments in layer 2 ( z = 2 ) is given by eq .
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\begin{document}$$ \begin{gathered } { \frac{{\updelta [ { \text{hb}}]_{i , j , z } } } { \updelta t } } = \left ( { { \frac{{[{\text{hb}}]_{i , j , z + 1 } } } { { v_{z + 1 } } } } \cdot { \frac{{k \cdot \updelta p_{z + 1,z } \cdot a_{z + 1 , } } } { { \updelta x_{z + 1,z } } } } } \right ) + \left ( { d_{\text{hb } } \cdot \left ( { \frac{1}{6}\sum\limits_{k = 1}^{6 } { \left ( { { \frac{{\updelta n_{\text{hb } } \cdot a}}{\updelta x } } } \right)_{ijz , k } } + \frac{1}{4}\sum\limits_{l = 1}^{4 } { \left ( { { \frac{{\updelta n_{\text{hb } } \cdot a}}{\updelta x \cdot \sqrt 2 } } } \right)_{ijz , l } } } \right ) } \right ) \hfill \\ - \left ( { { \frac{{v_{\max } \cdot [ hb]_{i , j , z}^ { * } } } { { k_{m } + [ hb]_{i , j , z}^ { * } } } } \cdot [ ho ] \cdot mw_{\text{hb } } } \right ) \hfill \\ \end{gathered } $ $ \end{document } for the compartments in the subcutaneous layer ( layer 3 , z = 3 ) , eq .
1 is valid if the first right hand term is negated to account for convection out of the subcutaneous layer . neglecting the convection from the subcutaneous layer to the top layer of the dermis ( layer 3 to layer 1 ) , the rate of change for the top layer of the dermis ( layer 1 ) is equal to eq . 1 if the first right hand term is zero . here ,
[hb]i , j , z is the change in the amount of hemoglobin in compartment i , j , z ( mg ) , 0 i < 100 , 0 j < 100 , 1 z 3 , t is the time step ( h ) , [ hb]i , j , z+1 is the amount of hemoglobin in corresponding compartment i , j in the layer z + 1(mg ) , vz+1 is the volume of the compartment in layer z + 1 , k is the hydraulic conductivity ( m / nh ) , pz+1,z is the pressure difference between layer z + 1 and layer z ( n / m ) , az+1,z is the contact surface between the two compartments of layers z + 1 and z ( m ) , xz+1,z is the distance from center to center of the two compartments in layers z + 1 and z ( m ) , dhb is the diffusivity of hemoglobin ( m / h ) . to calculate the contributions by diffusion ,
the average difference in hemoglobin density between compartment i , j , z and the surrounding compartments k and l is used , where k are compartments directly adjacent to compartment i , j , z , and l are compartments across compartment i , j , z , as clarified in fig . 2 .
the first sum specifies the difference between compartment i , j , z , and the 6 compartments k , whereas the second sum specifies the difference between compartment i , j , z , and the 4 compartments l. nhb is the difference in hemoglobin density between compartment i , j , z , and k or l ( mg / m ) , a is the contact surface between compartment i , j , z , and k ( m ) and x is the distance from the center of i , j , z , to the center of k ( m ) . for the compartments
l , we assumed an effective contact area between compartment i , j , z , and l of a , and a distance from center to center of x2 .
vmax is the maximum speed of reaction per mg heme oxygenase ( mol / h / mgho ) , [ hb]i , j , z , is the concentration of hemoglobin in compartment i , j , z , ( mol / l ) , defined as ; \documentclass[12pt]{minimal }
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\begin{document}$$ [ { \text{hb}}]^ { * } ( { \upmu } { \text{mol}}/{\text{l } } ) = { \frac{{[{\text{hb}}]({\text{mg}}/{\text{l}})}}{{{\text{mw}}_{\text{hb } } ( { \text{mg}}/{\upmu } { \text{mol } } ) } } } $ $ \end{document } , km is the affinity of the enzyme for the reaction ( m
= mol / l ) , [ ho ] is the amount of heme oxygenase in the compartment ( mg ) ( = concentration ( mg / l ) volume ( l ) ) , mwhb is the molecular weight of hemoglobin ; 65 mg/mol.fig .
2the average of adjacent compartments k and across compartments l are used in the calculations the average of adjacent compartments k and across compartments l are used in the calculations for the rate of change of bilirubin , a similar equation as for hemoglobin applies .
bilirubin behavior also depends on diffusion and enzymatic conversion ( first and second term in eq . 2 ) .
this clearance is not enzyme controlled and is modeled to follow zero order reaction kinetics ( third right hand term in eq . 2 ) .
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\begin{document}$$ \begin{gathered } { \frac{{\updelta [ { \text{b}}]_{i , j , z } } } { \updelta t}}\,=\,\left ( { d_{\text{b } } \cdot \left ( { \frac{1}{6}\sum\limits_{k = 1}^{6 } { \left ( { { \frac{{\updelta n_{\text{b } } \cdot a}}{\updelta x } } } \right)_{ijz , k } } \,+\,\frac{1}{4}\sum\limits_{l = 1}^{4 } { \left ( { { \frac{{\updelta n_{\text{b } } \cdot a}}{\updelta x \cdot \sqrt 2 } } } \right)_{ijz , l } } } \right ) } \right ) + \left ( { 4 \cdot { \frac{{v_{\max } \cdot [ { \text{hb}}]_{i , j , z}^ { * } } } { { k_{m } + [ { \text{hb}}]_{i , j , z}^ { * } } } } \cdot [ { \text{ho } } ] \cdot { \text{mw}}_{\text{b } } } \right ) \hfill \\ - \left ( { { \frac{{[{\text{b}}]_{i , j , z } } } { { \tau_{\text{b } } } } } } \right ) \hfill \\ \end{gathered } $ $ \end{document}where [b]i , j , z is the change in amount of bilirubin in compartment i , j , z ( mg ) , db is the diffusivity of bilirubin ( m / h ) , nb is the difference in bilirubin density between compartment i , j , z and k or l ( mg / m ) , the number 4 in the second hand term is to account for the fact that 4 mol of bilirubin is formed per mole of hemoglobin , mwb is the molecular weight of bilirubin ; 0.584 mg/mol , [ b]i , j , z is the amount of bilirubin in compartment i , j , z ( mg ) and b is the clearance time of bilirubin from the skin into the lymphatic system ( h ) .
for all simulations , the model calculates the spatial distribution of the amount of hemoglobin and bilirubin as a function of time by a forward difference method in space , using labview 8.6 professional development system .
the model can simulate a bruise of any given shape and size . for a time step of 0.1 h
, a simulation of 400 h takes 1 min on a standard laptop computer .
since the injured vessels in the subcutaneous layer close , the convection goes to zero in a short time period , assumed to be 12 h [ 18 , 30 ] .
diffusivity values for bilirubin in the skin are unknown , but since 4 mol of bilirubin is produced per mole of hemoglobin , diffusivity values are assumed to be four times larger than hemoglobin diffusivity .
the dermis is separated in two layers for two reasons ( 1 ) to enable vertical flow inside the dermis layer , ( 2 ) to enable a comparison between natural bruises and simulated bruises . since only natural bruises in the top layer of the dermis
the thickness of the top layer of the dermis was fixed at 400 m , representing the penetration depth of visible light , and the lateral dimensions are equal to the thickness of the total dermis .
other parameters ( table 1 ) were varied within biological ranges to account for variability within one individual and between different individuals and to assess the influence of these parameters on the formation and healing of the bruise.table 1parameters used in the modelbiological rangestandard parameters for simulationssi unitstarting concentration of hemoglobin150 g / l150 g / l150 g / lstarting diameter of blood pool2100 mm10 mm0.01 mhydraulic conductivity , k ( t = 0)5 10 m / nh5 10 m / nh1.4 10 m / nspressure difference , p2.6 10 n / m2.6 10 n / m2.6 10 n / mdiffusivity hemoglobin , dhb1 101 10 m / h1
10 m / h2.8 10 m / sdiffusivity bilirubin , db4 104 10 m / h4
10 m / h1.1 10 m / saffinity , km0.24 m0.24 m0.24 10 mol / lspeed of conversion , vmax3.4 mol / h / mgho3.4 mol / h / mgho9.4 10 mol / s / mghoconcentration of ho0.110 mg / l5 mg / l5 10 g / lclearance of bilirubin , b50400 h150 h54 10 sdermal thickness , x5002000 m , dependent on body site400 m ( top layer)4 10 m ( top layer)600 m ( bottom layer)6 10 m ( bottom layer ) parameters used in the model for the first simulation , a circular pool of hemoglobin was assumed with fixed standard parameters as provided in table 1 .
subsequently , we estimated how variation of the clinically most relevant parameters influences the maximal diameter of the bruise in the top layer of the dermis ( for both hemoglobin and bilirubin ) and the time it takes for the bruise to resolve .
we assumed a minimal detectable level of chromophores of 1 10 mg per compartment for both hemoglobin and bilirubin .
the time point at which the bruise is resolved was taken as the time at which the bilirubin concentration in all compartments is below its assumed critical detectable value .
the potential of the model is demonstrated by simulating a natural bruise , originating from a pinch and located on the upper arm of a 26 year old woman ( with assumed skin thickness of 1000 m ) , which was photographed almost every day for 2 weeks .
the shape of the non homogeneous starting hemoglobin pool in the subcutaneous layer was estimated from the shape of the bruise in the dermis at day 1 .
this shape was obtained using the rgb values of the image ; a small ( hemoglobin containing ) area in the bruise was selected and the range of rgb values in this area was determined .
pixels in the image of the total bruise falling within the rgb range of this small area were defined as containing hemoglobin and were given the starting hemoglobin concentration , whereas the other pixels were given a hemoglobin concentration zero . using this method , not only the shape but also the total areas of the hemoglobin and bilirubin parts of the bruise were determined ; the hemoglobin area was obtained by adding up all the hemoglobin containing pixels and multiplying with the area of one pixel . to determine the bilirubin area , a small yellow area was selected .
since bilirubin is only produced when hemoglobin is present , the bilirubin area of the real bruise is defined as the hemoglobin area + the yellow area .
these areas of the real bruise were determined to enable a comparison with the areas of the simulated bruise , which were defined by adding up all compartments having chromophore levels above the detection threshold and multiplying with the area of one compartment .
the bruise was then simulated using the hemoglobin containing pixels from the image at day 1 to define the non symmetric shaped blood pool .
diffusivity , relaxation time , and concentration of ho were varied until the simulated bruise at various time points resembled the natural bruise at corresponding time points , both in shape as in total area of the hemoglobin and bilirubin parts .
an enhanced false color image of the simulation was constructed , in which a high hemoglobin concentration is depicted as a bright red color , fading into a less intense red for lower concentrations , and a high bilirubin concentration is depicted as a yellow color , fading into less intense yellow for lower concentrations .
this false color image allows comparison between the simulated hemoglobin and bilirubin areas and the real bilirubin and bilirubin areas , as well as a comparison between high versus low concentrations of the chromophores .
the model of the skin consists of three layers ; the top layer of the dermis ( layer 1 ) , the bottom layer of the dermis ( layer 2 ) and the subcutaneous tissue layer ( layer 3 ) .
each layer is subdivided into 100 100 corresponding compartments as schematically shown in fig .
for ease of analysis , all compartments have the same lateral dimensions equal to the thickness of the total dermis , and the thickness of the layers can be varied to account for inter - individual variability or body location .
the initial condition of the bruise is modeled by a pool of hemoglobin of arbitrary shape and size in the subcutaneous tissue ( fig .
the bruise develops over time by conversion of hemoglobin into bilirubin and transport of these molecules via pressure driven flow , i.e. convection and concentration driven diffusion .
the concentration of hemoglobin within a compartment may change by three processes : ( 1 ) convection in vertical direction from the subcutaneous tissue layer into the dermis ( fig .
1b ) , ( 2 ) vertical diffusion between the layers , as well as horizontal diffusion within the layers ( fig .
darcy s law for transport of fluids describes the convection of hemoglobin in vertical direction , assuming no pressure gradient in horizontal direction , hence neglects flow in horizontal direction ( first right hand term in eq . 1 ) .
diffusion , in vertical and horizontal direction , follows concentration gradients and is described by the first law of fick ( second right hand term in eq . 1 ) .
we will first present the formula for compartments in layer 2 , from which the formulas for layers 1 and 3 follow.fig .
1a the skin model consists of 3 layers , the top layer of the dermis , the bottom layer of the dermis and the subcutaneous fat layer .
each layer consists of 100 100 compartmets ( for ease of presentation a smaller number is shown ) .
b and c a pool of hemoglobin is defined in the subcutaneous layer . via michealis
both hemoglobin and bilirubin flow inside the layers and between the layers a the skin model consists of 3 layers , the top layer of the dermis , the bottom layer of the dermis and the subcutaneous fat layer .
each layer consists of 100 100 compartmets ( for ease of presentation a smaller number is shown ) .
b and c a pool of hemoglobin is defined in the subcutaneous layer . via michealis
both hemoglobin and bilirubin flow inside the layers and between the layers the rate of change of hemoglobin in the compartments in layer 2 ( z = 2 ) is given by eq .
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\begin{document}$$ \begin{gathered } { \frac{{\updelta [ { \text{hb}}]_{i , j , z } } } { \updelta t } } = \left ( { { \frac{{[{\text{hb}}]_{i , j , z + 1 } } } { { v_{z + 1 } } } } \cdot { \frac{{k \cdot \updelta p_{z + 1,z } \cdot a_{z + 1 , } } } { { \updelta x_{z + 1,z } } } } } \right ) + \left ( { d_{\text{hb } } \cdot \left ( { \frac{1}{6}\sum\limits_{k = 1}^{6 } { \left ( { { \frac{{\updelta n_{\text{hb } } \cdot a}}{\updelta x } } } \right)_{ijz , k } } + \frac{1}{4}\sum\limits_{l = 1}^{4 } { \left ( { { \frac{{\updelta n_{\text{hb } } \cdot a}}{\updelta x \cdot \sqrt 2 } } } \right)_{ijz , l } } } \right ) } \right ) \hfill \\ - \left ( { { \frac{{v_{\max } \cdot [ hb]_{i , j , z}^ { * } } } { { k_{m } + [ hb]_{i , j , z}^ { * } } } } \cdot [ ho ] \cdot mw_{\text{hb } } } \right ) \hfill \\ \end{gathered } $ $ \end{document } for the compartments in the subcutaneous layer ( layer 3 , z = 3 ) , eq .
1 is valid if the first right hand term is negated to account for convection out of the subcutaneous layer . neglecting the convection from the subcutaneous layer to the top layer of the dermis ( layer 3 to layer 1 ) , the rate of change for the top layer of the dermis ( layer 1 ) is equal to eq . 1 if the first right hand term is zero . here ,
[hb]i , j , z is the change in the amount of hemoglobin in compartment i , j , z ( mg ) , 0 i < 100 , 0 j < 100 , 1 z 3 , t is the time step ( h ) , [ hb]i , j , z+1 is the amount of hemoglobin in corresponding compartment i , j in the layer z + 1(mg ) , vz+1 is the volume of the compartment in layer z + 1 , k is the hydraulic conductivity ( m / nh ) , pz+1,z is the pressure difference between layer z + 1 and layer z ( n / m ) , az+1,z is the contact surface between the two compartments of layers z + 1 and z ( m ) , xz+1,z is the distance from center to center of the two compartments in layers z + 1 and z ( m ) , dhb is the diffusivity of hemoglobin ( m / h ) . to calculate the contributions by diffusion ,
the average difference in hemoglobin density between compartment i , j , z and the surrounding compartments k and l is used , where k are compartments directly adjacent to compartment i , j , z , and l are compartments across compartment i , j , z , as clarified in fig . 2 .
the first sum specifies the difference between compartment i , j , z , and the 6 compartments k , whereas the second sum specifies the difference between compartment i , j , z , and the 4 compartments l. nhb is the difference in hemoglobin density between compartment i , j , z , and k or l ( mg / m ) , a is the contact surface between compartment i , j , z , and k ( m ) and x is the distance from the center of i , j , z , to the center of k ( m ) . for the compartments
l , we assumed an effective contact area between compartment i , j , z , and l of a , and a distance from center to center of x2 .
vmax is the maximum speed of reaction per mg heme oxygenase ( mol / h / mgho ) , [ hb]i , j , z , is the concentration of hemoglobin in compartment i , j , z , ( mol / l ) , defined as ; \documentclass[12pt]{minimal }
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\begin{document}$$ [ { \text{hb}}]^ { * } ( { \upmu } { \text{mol}}/{\text{l } } ) = { \frac{{[{\text{hb}}]({\text{mg}}/{\text{l}})}}{{{\text{mw}}_{\text{hb } } ( { \text{mg}}/{\upmu } { \text{mol } } ) } } } $ $ \end{document } , km is the affinity of the enzyme for the reaction ( m
= mol / l ) , [ ho ] is the amount of heme oxygenase in the compartment ( mg ) ( = concentration ( mg / l ) volume ( l ) ) , mwhb is the molecular weight of hemoglobin ; 65 mg/mol.fig .
2the average of adjacent compartments k and across compartments l are used in the calculations the average of adjacent compartments k and across compartments l are used in the calculations for the rate of change of bilirubin , a similar equation as for hemoglobin applies .
bilirubin behavior also depends on diffusion and enzymatic conversion ( first and second term in eq . 2 ) .
this clearance is not enzyme controlled and is modeled to follow zero order reaction kinetics ( third right hand term in eq . 2 ) .
2):2\documentclass[12pt]{minimal }
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\begin{document}$$ \begin{gathered } { \frac{{\updelta [ { \text{b}}]_{i , j , z } } } { \updelta t}}\,=\,\left ( { d_{\text{b } } \cdot \left ( { \frac{1}{6}\sum\limits_{k = 1}^{6 } { \left ( { { \frac{{\updelta n_{\text{b } } \cdot a}}{\updelta x } } } \right)_{ijz , k } } \,+\,\frac{1}{4}\sum\limits_{l = 1}^{4 } { \left ( { { \frac{{\updelta n_{\text{b } } \cdot a}}{\updelta x \cdot \sqrt 2 } } } \right)_{ijz , l } } } \right ) } \right ) + \left ( { 4 \cdot { \frac{{v_{\max } \cdot [ { \text{hb}}]_{i , j , z}^ { * } } } { { k_{m } + [ { \text{hb}}]_{i , j , z}^ { * } } } } \cdot [ { \text{ho } } ] \cdot { \text{mw}}_{\text{b } } } \right ) \hfill \\ - \left ( { { \frac{{[{\text{b}}]_{i , j , z } } } { { \tau_{\text{b } } } } } } \right ) \hfill \\ \end{gathered } $ $ \end{document}where [b]i , j , z is the change in amount of bilirubin in compartment i , j , z ( mg ) , db is the diffusivity of bilirubin ( m / h ) , nb is the difference in bilirubin density between compartment i , j , z and k or l ( mg / m ) , the number 4 in the second hand term is to account for the fact that 4 mol of bilirubin is formed per mole of hemoglobin , mwb is the molecular weight of bilirubin ; 0.584 mg/mol , [ b]i , j , z is the amount of bilirubin in compartment i , j , z ( mg ) and b is the clearance time of bilirubin from the skin into the lymphatic system ( h ) .
for all simulations , the model calculates the spatial distribution of the amount of hemoglobin and bilirubin as a function of time by a forward difference method in space , using labview 8.6 professional development system . the model can simulate a bruise of any given shape and size . for a time step of 0.1 h ,
a simulation of 400 h takes 1 min on a standard laptop computer .
since the injured vessels in the subcutaneous layer close , the convection goes to zero in a short time period , assumed to be 12 h [ 18 , 30 ] .
diffusivity values for bilirubin in the skin are unknown , but since 4 mol of bilirubin is produced per mole of hemoglobin , diffusivity values are assumed to be four times larger than hemoglobin diffusivity .
the dermis is separated in two layers for two reasons ( 1 ) to enable vertical flow inside the dermis layer , ( 2 ) to enable a comparison between natural bruises and simulated bruises . since only natural bruises in the top layer of the dermis
the thickness of the top layer of the dermis was fixed at 400 m , representing the penetration depth of visible light , and the lateral dimensions are equal to the thickness of the total dermis .
other parameters ( table 1 ) were varied within biological ranges to account for variability within one individual and between different individuals and to assess the influence of these parameters on the formation and healing of the bruise.table 1parameters used in the modelbiological rangestandard parameters for simulationssi unitstarting concentration of hemoglobin150 g / l150 g / l150 g / lstarting diameter of blood pool2100 mm10 mm0.01 mhydraulic conductivity , k ( t = 0)5 10 m / nh5 10 m / nh1.4 10 m / nspressure difference , p2.6 10 n / m2.6 10 n / m2.6 10 n / mdiffusivity hemoglobin , dhb1 101 10 m / h1
10 m / h2.8 10 m / sdiffusivity bilirubin , db4 104 10 m / h4
10 m / h1.1 10 m / saffinity , km0.24 m0.24 m0.24 10 mol / lspeed of conversion , vmax3.4 mol / h / mgho3.4 mol / h / mgho9.4 10 mol / s / mghoconcentration of ho0.110 mg / l5 mg / l5 10 g / lclearance of bilirubin , b50400 h150 h54 10 sdermal thickness , x5002000 m , dependent on body site400 m ( top layer)4 10 m ( top layer)600 m ( bottom layer)6 10 m ( bottom layer ) parameters used in the model for the first simulation , a circular pool of hemoglobin was assumed with fixed standard parameters as provided in table 1 .
subsequently , we estimated how variation of the clinically most relevant parameters influences the maximal diameter of the bruise in the top layer of the dermis ( for both hemoglobin and bilirubin ) and the time it takes for the bruise to resolve .
we assumed a minimal detectable level of chromophores of 1 10 mg per compartment for both hemoglobin and bilirubin .
the time point at which the bruise is resolved was taken as the time at which the bilirubin concentration in all compartments is below its assumed critical detectable value .
the potential of the model is demonstrated by simulating a natural bruise , originating from a pinch and located on the upper arm of a 26 year old woman ( with assumed skin thickness of 1000 m ) , which was photographed almost every day for 2 weeks .
the shape of the non homogeneous starting hemoglobin pool in the subcutaneous layer was estimated from the shape of the bruise in the dermis at day 1 .
this shape was obtained using the rgb values of the image ; a small ( hemoglobin containing ) area in the bruise was selected and the range of rgb values in this area was determined .
pixels in the image of the total bruise falling within the rgb range of this small area were defined as containing hemoglobin and were given the starting hemoglobin concentration , whereas the other pixels were given a hemoglobin concentration zero . using this method , not only the shape but also the total areas of the hemoglobin and bilirubin parts of the bruise were determined ; the hemoglobin area was obtained by adding up all the hemoglobin containing pixels and multiplying with the area of one pixel . to determine the bilirubin area , a small yellow area was selected . since bilirubin is only produced when hemoglobin is present , the bilirubin area of the real bruise is defined as the hemoglobin area + the yellow area .
these areas of the real bruise were determined to enable a comparison with the areas of the simulated bruise , which were defined by adding up all compartments having chromophore levels above the detection threshold and multiplying with the area of one compartment .
the bruise was then simulated using the hemoglobin containing pixels from the image at day 1 to define the non symmetric shaped blood pool .
diffusivity , relaxation time , and concentration of ho were varied until the simulated bruise at various time points resembled the natural bruise at corresponding time points , both in shape as in total area of the hemoglobin and bilirubin parts .
an enhanced false color image of the simulation was constructed , in which a high hemoglobin concentration is depicted as a bright red color , fading into a less intense red for lower concentrations , and a high bilirubin concentration is depicted as a yellow color , fading into less intense yellow for lower concentrations .
this false color image allows comparison between the simulated hemoglobin and bilirubin areas and the real bilirubin and bilirubin areas , as well as a comparison between high versus low concentrations of the chromophores .
a circular symmetric bruise simulated with the standard parameters shows spatial and temporal differences in hemoglobin and bilirubin concentrations . in the center ,
the kinetics differ from the edge , which has shifted due to diffusion from 5 mm at t = 0 to 8 mm from the center ( fig . 3 ) .
both the center and the edge of the bruise show a fast increase in hemoglobin concentration followed by a fast decrease , and a slower increase and decrease in bilirubin concentration .
please note that the peak concentration of hemoglobin in the center is a factor of 6 higher than the peak concentration of hemoglobin at the edge .
besides this maximum concentration difference , their temporal behavior is also different : the peak concentration of hemoglobin in the center is reached earlier than at the edge ( center peak at 7.5 h , vs. edge peak at 12 h ) .
the peak in bilirubin concentration in the center is also higher than that at the edge .
but in contrast to the hemoglobin concentrations , the bilirubin peak in the center is reached later than at the edge ( center peak at 40 h , vs. edge peak at 34 h ) .
furthermore , the ratio of hemoglobin over bilirubin in the center is much higher than at the edge.fig .
the concentration of hemoglobin in the center in the graph is divided by 2 , to display all chromophores more clearly in 1 graph .
the simulations are done with the standard parameters as given in table 1 kinetics in bruise in center and at 8 mm from center .
the concentration of hemoglobin in the center in the graph is divided by 2 , to display all chromophores more clearly in 1 graph .
the simulations are done with the standard parameters as given in table 1 figure 4 shows the influence of the diffusivity dhb , which is varied between 1 10 and 8 10 m / h for a circular symmetric bruise of a 10 mm starting diameter .
we kept db four times larger than dhb and hence dhb is varied between 4 10 and 3.2 10 m / h .
a higher diffusivity value ( representing less dense tissue , i.e. less collagen fibers ) leads to a larger bruise ( fig .
4a ) . also , the concentrations of hemoglobin and bilirubin per compartment are lower for higher diffusivity values ( not shown ) and the bruise resolves faster ( fig .
m / h , db remained 4 dhb and was varied between 4 103.2 10
the simulations are done with the standard parameters as given in table 1 , except diffusivity a final diameter of bruise for different diffusivities .
m / h , db remained 4 dhb and was varied between 4 103.2 10
the simulations are done with the standard parameters as given in table 1 , except diffusivity figure 5 shows the influence of skin thickness on the maximal diameter of a circular symmetric bruise in the top layer of the dermis and the time to resolve .
the top layer of the dermis was kept constant at 0.4 mm , and the bottom layer was varied between 0.6 and 1.6 mm . as depicted in fig .
5a , a larger dermal thickness causes a larger diameter of the hemoglobin and bilirubin areas , and a bruise in a thicker dermis takes longer to resolve ( fig .
the simulations are done with the standard parameters as given in table 1 , except dermal thickness a final diameter of bruise for different dermal thicknesses .
the simulations are done with the standard parameters as given in table 1 , except dermal thickness figure 6a shows the maximal diameter of a circular symmetric bruise for different starting diameters , ranging from 2 to 30 mm .
the bilirubin in the bruise diffuses faster than the hemoglobin , resulting in a larger area containing bilirubin than containing hemoglobin .
the maximal diameter of the hemoglobin area in the bruise ranges from 8 to 40 mm and the diameter of the bilirubin area ranges from 26 to 96 mm .
the concentrations per compartment of hemoglobin and bilirubin in the larger bruises are higher than in the smaller bruises and a larger bruise takes longer to resolve .
the diameter of the hemoglobin area of the bruise over time for different starting diameters is shown in fig .
the ratio between these two diameters over time for different starting diameters is shown in fig .
6b and c relates the diameter of the hemoglobin area uniquely with the ratio of the hemoglobin over the bilirubin area diameters ( fig .
6d ) , from which the age of the simulated bruise follows directly ; e.g. if the diameter of the hemoglobin area is 20 mm , and the ratio of the hemoglobin over bilirubin area is 0.25 , there is a unique solution for the age of the bruise ; the bruise must be 2.8 days old.fig .
b diameter of hemoglobin area of bruise over time for different starting diameters ( the steps in the hemoglobin kinetics are due to the resolution of the model ) .
c ratio of the diameter of the area containing hemoglobin over the diameter of the area containing bilirubin for different starting diameters .
the simulations are done with the standard parameters as given in table 1 , except for the starting diameter .
. ratios of hemoglobin over bilirubin ( color ) a final diameter of the bruise for different starting diameters .
b diameter of hemoglobin area of bruise over time for different starting diameters ( the steps in the hemoglobin kinetics are due to the resolution of the model ) .
c ratio of the diameter of the area containing hemoglobin over the diameter of the area containing bilirubin for different starting diameters .
the simulations are done with the standard parameters as given in table 1 , except for the starting diameter .
6b , c : diameter of the hemoglobin area ( gray ) . ratios of hemoglobin over bilirubin ( color ) for the non homogeneous shaped starting blood pool ( fig .
7b , day 0 ) , derived from the photos of a natural bruise ( fig .
7a , day 1 ) , a qualitative comparison of the temporal and spatial distributions of hemoglobin and bilirubin concentrations in this natural versus the simulated bruise is presented in fig .
small amounts of bilirubin are visible . on day 4 , the amount of hemoglobin is lower than on day 1 , and bilirubin has filled the areas between the different hemoglobin parts . on day 7 , the upper part of the bruise has almost resolved , where the lower part contains mainly bilirubin . figure 8 shows the total areas of the hemoglobin and bilirubin parts over time , both for the simulated bruise and for the natural bruise .
the total simulated hemoglobin area matches the total hemoglobin area of the natural bruise at several time points for a single set of parameters .
the total bilirubin area of the simulated bruise matches the total bilirubin area of the natural bruise in the first 100 h. after 100 h , the total bilirubin area of the simulated bruise is overestimated compared to the natural bruise.fig .
b simulations of a non homogeneous bruise ( starting blood pool extracted from photo day 1 ) dermal thickness = 1000 m , dhb 1.6 10 m / h , db 6.4 10 m / h , b 100 h , concentration ho = 10.5 mg / l .
b total bilirubin area of the simulated and natural bruise a non homogeneous natural bruise photographed on 3 different days ( cross sect . 40 mm ) .
b simulations of a non homogeneous bruise ( starting blood pool extracted from photo day 1 ) dermal thickness = 1000 m , dhb 1.6 10 m / h , db 6.4 10
m / h , b 100 h , concentration ho = 10.5 mg / l .
ballpoint stripes were placed on the same location for comparison a total area of hemoglobin area of the simulated and natural bruise .
to our best knowledge , our model is the first that allows simulating the temporal as well as spatial changes in hemoglobin and bilirubin concentrations as occurring during the lifetime of a bruise .
particularly , the spatial dependence of the chromophores , as observed in natural bruises , is an essential addition to previous modeling .
we showed that combining the hemoglobin area diameter with the ratio of hemoglobin over bilirubin diameters provides a unique solution for the age of circular symmetric simulated bruises ( fig .
we hypothesize that this concept can be extended to age determination of inhomogeneous natural bruises because of the faster diffusion of bilirubin compared to hemoglobin , i.e. it fills the gaps between different hemoglobin areas ( fig .
. a quantitative validation of the approach was presented , and showed that it was feasible to simulate a non homogeneous bruise with a single set of parameters ( fig .
a qualitative validation of this approach requires further study , particularly the assessment of crucial model parameters to match the simulations with the individual bruise under study .
we intend to perform this model optimization by combining simulations with spatially resolved reflectance measurements to determine chromophore concentrations extracted from the spectra using light propagation models [ 4 , 7 , 36 ] .
a bruise is formed when small vessels in the subcutaneous tissue layer ( in the order of 510 m ) rupture due to a mechanical impact .
although the dermis also contains small vessels , we assume that the blood comes from the subcutaneous layer , therefore , the contribution of ruptured dermal vessels is neglected .
we assume a starting concentration of hemoglobin of 150 g / l for all simulations .
l for women and 140175 g / l for men values of children are slightly lower , as are values for anemic patients ) , these small deviations in starting concentration will hardly influence the peak concentration , as well as the temporal behavior of the hemoglobin and bilirubin concentrations .
the pressure difference between the blood vessels and the interstitial fluid causes the blood to leak from the ruptured subcutaneous vessels into the surrounding tissue [ 17 , 18 ] .
we assume no flow to the epidermal layer because of the tight basal membrane ; therefore this layer is not incorporated in the model .
we assume an instantaneously established pool of blood in the subcutaneous layer , meaning no convection horizontally within this layer .
the vertical convection from the subcutaneous layer into the dermis is assumed to be slower than the horizontal convection , and is modeled with darcy s law , which uses the hydraulic conductivity k. the value for k is unknown across the subcutis / cutis border , which consists of retinacula , i.e. thin fibrous septa of collagen i + iii . in randeberg
et al . , a range of values is given for k in the dermis , horizontally along the collagen i + iii and elastin fibers , and discussed is how k , vertically across these structures , is smaller .
literature shows a broad range of values of k for different types of tissue , and also that k changes with 2 orders of magnitude between normal and edematous skin .
this makes it difficult to give an estimate for k. however , because ruptured vessels close ( assumed to be within 12 h ) , the convection gradually goes to zero and the influence of k on the total simulation is small .
k for vertical diffusion across the subcutis / cutis border is set to the smallest known value in the dermis at 5 10 m / nh . because of their small influence on the formation and healing , in our simulations k and p are not varied .
the free blood initiates an inflammatory reaction , which causes macrophages to extravasate and take up the erythrocytes , free hemoglobin and free heme .
the hemoglobin is broken down non - enzymatically into heme and globin [ 8 , 38 ] . inside the macrophages , heme oxygenase-1 ( ho-1 ) converts heme into biliverdin [ 11 , 23 ] .
the first step is the rate limiting step and is assumed to occur significantly slower than the second step .
menten kinetics , which takes into account the affinity of the enzyme for the reaction ( km ) and the maximum speed of the conversion ( vmax ) .
these parameters are standard for each specific enzyme and were , therefore , not varied in this article .
the rate of conversion is limited by the enzyme capacity of ho , which is reached even with low concentrations of hemoglobin ( the maximum speed of the reaction is already reached at concentrations a factor of 10,000 lower than the concentration in blood ) .
consequently , the kinetics of the hemoglobin and bilirubin do not change for slightly higher or lower starting concentrations of hemoglobin .
two other forms of heme oxygenase ( ho-2 and ho-3 ) , both with different enzyme kinetic values , are unlikely to play a role in the bruise and have , therefore , not been taken into account .
first , wounds ( e.g. bruises ) show a higher concentration of ho-1 [ 15 , 27 ] and ho-1 is the only ho form that is inducible by high heme concentrations , as occurring in bruises .
second , and in contrast , ho-2 is only inducible by adrenal glucocorticoids , implying it will not be upregulated in the bruise .
ho-3 has a too low activity in the human body to play a role in bruises [ 10 , 13 ] .
the concentration of ho-1 in these simulations is based on the normal serum value of ho-1 , the range is set to 10100 times this normal value , because of the upregulation of ho-1 in wounds .
the bilirubin is released from the macrophages into the extracellular space and flows inside the skin before being drained into the lymphatic system .
is in the order of 240 h. we varied this clearance time because we hypothesize there are inter - individual variabilities , e.g. more muscle movement will lead to faster lymphatic drainage .
we arbitrarily used clearance times between 50 and 400 h , and found this range to affect the kinetics ; a higher clearance time resulted in a longer time to resolve and a larger bilirubin area .
bruise , the peak concentration of hemoglobin in the center is higher and is reached earlier than at the edge ( fig .
3 ) , because the hemoglobin diffuses toward the edge and causes a time dependent gradient in concentration .
contrastingly , the peak concentration of bilirubin at the edge is reached earlier than at the center , because the pool of hemoglobin at the edge is depleted earlier . in
, who assume hemoglobin diffusion to be only in vertical direction , we assume diffusion in both the vertical and horizontal direction . this important addition to the model is based on the fact that diffusion is concentration driven , so irrespective of direction .
in addition , the inclusion of horizontal diffusion is the key to the method of age determination described here .
diffusivity values of hemoglobin in skin from literature are widespread , as they are adapted from diffusivities in other tissues , e.g. from the diffusivity of myoglobin in skeletal muscle ; 1.08 10 m / h . to account for this uncertainty and also to account for different locations on the body
, the diffusivity of hemoglobin dhb was varied between 1 10 m / h and 1 10 m / h , where a higher diffusivity is related to less dense tissue .
diffusivity values for bilirubin in the skin are unknown , but since 4 mol of bilirubin is produced per mole of hemoglobin ( as hemoglobin contains 4 heme groups ) , diffusivity values are expected to be larger ; values were varied between 4 10 and 4 10 m / h .
this factor between the dhb and db should be verified in future experiments , but our simulation of the natural bruise , where this factor 4 was used , shows at least that this factor gave promising results .
the validation of this factor will have to take into account solubility and possible conjugates of bilirubin in the skin .
the extent of the mechanical damage on the retinacula , which influences the diffusivity in vertical direction across the subcutis / cutis border , is unknown .
however , based on the previous discussion on convective flow across this structure , we assume that diffusivity in the horizontal direction is equal to diffusivity in vertical direction .
the diffusivities of hemoglobin and bilirubin are important factors that influence the kinetics of bruises . a higher diffusivity ( corresponding to less dense tissue such as around the eyes ) results in a larger bruise ( fig .
also , the higher diffusivity leads to faster kinetics , as the amount of hemoglobin per compartment is reduced and thus less time is needed to convert all the hemoglobin in that compartment to bilirubin .
these results imply that the type of tissue influences the rate of development and clearing of bruises .
a higher diffusivity may also occur in the case of a severe beating and resulting extensively damaged muscle and collagen fibers . since bilirubin is a small molecule , it diffuses more rapidly in the skin than hemoglobin ( db > dhb ) .
our assumption that db is not negligible , contrary to randeberg et al . , is an important addition to the modeling of bruises .
the assumed faster flow of bilirubin than hemoglobin causes the gaps between the hemoglobin areas to be filled with bilirubin , as shown for the non homogeneous , natural bruise in fig .
this phenomenon likely becomes important in future methodology to reliably determine the age of bruises .
if the skin thickness used in the model is underestimated compared to the actual thickness ( as simulated in fig .
5 ) , the simulated bruise will resolve faster , resulting in an incorrect prediction of kinetics .
the volume of the subcutaneous compartments was kept constant , but as the dermal thickness was varied , the volume and the concentration of the chromophores in the dermal compartments also varied .
for a thicker dermis , the concentration is lower , causing a slower flow to the edge of the bruise , and hence smaller maximal diameters of hemoglobin and bilirubin areas ( fig .
ideally , the skin thickness should be measured in every situation with high frequency ultra sound or optical coherence tomography .
the possible presence of edema , which accumulates in the skin after a traumatic impact and thickens the skin , has not been taken into account in the model .
. first , edema increases the skin thickness and consequently induces a slower bruise development and healing .
second , the diffusivity is larger because the density of the collagen fibers is reduced due to the increased fluid concentration , resulting in faster kinetics .
therefore , the effects of skin edema on the kinetics are expected to be small . increasing the size of the blood pool , as depicted in fig . 6 , results in a larger maximal diameter .
interestingly , whereas the maximal diameter of the hemoglobin component of the bruises increases almost linearly with starting blood pool diameter , the maximal diameter of the bilirubin component increases more rapidly for small starting sizes , before increasing linearly .
the latter behavior is caused by the lower concentration of bilirubin for the smaller starting sizes ; all the bilirubin becomes cleared before reaching its maximal possible diameter .
6b are due to the resolution of the model ; the lateral dimensions of the compartments in these simulations were 1 1 mm , so if the diameter of the bruise is 10 mm , 10 elements in the cross section of the bruise are above the detection threshold .
if the amount of hemoglobin in a compartment becomes lower than the threshold , a step of 1 mm in the computations is seen .
the determination of the age of the bruise at 2.8 days can be done under the idealized condition that all parameters are known .
if not all parameters are known , a series of measurements with a known time between each measurement can provide enough information to estimate the parameters , as was shown in fig .
8 , and with these known parameters the age of the bruise can be determined .
the resulting fit of the parameters for this natural bruise appeared to be within the previously assumed physiological range of parameter values .
the shape of the subcutaneous , starting blood pool for the real bruise was estimated using the shape of the visible bruise at day 1 .
using the shape at t = 0 is not possible , because at that time point the blood pool is located in the subcutaneous layer , and the penetration depth of visible light is only 400 m . at day 1 ,
the visible part of the bruise had already diffused upward from the subcutaneous layer into the upper 400 m of the skin .
although using this diffused part of the bruise as input could lead to an error in the shape and size , the simulation shows good resemblance to the shape of the bruise at day 1 . a good resemblance at the later time points
the non homogeneous simulated bruise shows important features that resemble the natural bruise ( fig .
7 ) : first in the shape of the bruise : the bilirubin fills the gaps between the hemoglobin areas in both the simulated and the natural bruise .
second , in the high versus low concentrations of chromophores : the amount of hemoglobin is highest on day 1 in both bruises . on day 7 , the upper part of the bruise had almost resolved in both the simulated and natural bruise , and the lower part shows mainly bilirubin .
differences between the simulated and the natural bruise exist mainly in the lower part of the bruise ; the amount of bilirubin on day 7 seems higher in the natural bruise .
a possible explanation is that during the formation of the bruise , the concentration of hemoglobin in the lower part became higher than the concentration in the upper part .
a lower concentration of hemoglobin in the upper part of the bruise could resolve this .
another explanation could be the influence of gravity on the flow of the chromophores in the skin , which is not taken into account . a bruise located on the forehead can sag downwards until the patient has one or two bruised eyes . on most parts of the body , where the skin is more dense then around the eyes , gravity is expected to play a less important role .
third , the simulated bruise shows resemblance to the natural bruise in total area : the total hemoglobin area of the simulated bruise matches the total area of the natural bruise ( fig . 8) .
since extracting the bilirubin area from the photos of the natural bruise proved more difficult than extracting the hemoglobin areas , the deviation from the simulated bilirubin area is larger than for the hemoglobin area .
we showed the simulation and age determination of a single bruise . in real abuse situations , where a child is beaten on several days
the model does allow addition of an extra ( new ) pool of blood after several days , but the influence of such a second bruise on the age determination requires further study .
our model allows simulating the spatial and temporal behavior of the different chromophores in bruises and was used to assess the influence of e.g. diffusivity and skin thickness on the kinetics .
varying the parameters in the model in such a way that properties of individual natural bruises are closely matched allows optimizing our model .
the spatio - temporal differences of hemoglobin and bilirubin diffusion may identify methodology for more accurate age determination of bruises than currently possible , which could contribute to an improved diagnosis of child abuse . | simulating the spatial and temporal behavior of bruises may identify methods that allow accurate age determination of bruises to assess child abuse .
we developed a numerical 3d model to simulate the spatial kinetics of hemoglobin and bilirubin during the formation and healing of bruises . using this model
, we studied how skin thickness , bruise diameter and diffusivities affect the formation and healing of circular symmetric bruises and compared a simulated bruise with a natural inhomogeneous bruise .
healing is faster for smaller bruises in thinner and less dense skin .
the simulated and natural bruises showed similar spatial and temporal dynamics .
the different spatio - temporal dynamics of hemoglobin and bilirubin allows age determination of model bruises .
combining our model predictions with individual natural bruises may allow optimizing our model parameters .
it may particularly identify methods for more accurate age determination than currently possible to aid the assessment of child abuse . |
autoerythrocyte sensitization syndrome ( aes ) was first described by gardner and diamond in 1955 , when four women with painful bruising were depicted .
patients with aes typically present with the development of recurrent , spontaneous , painful ecchymosis , frequently preceded by a prodrome of pain or itching of the skin . over 100 cases have been reported since then , and nearly all cases were in women [ 3 , 4 ] .
the patients are sensitive to their own red blood cells injected intradermally , and underlying coagulopathies are thought to be absent .
psychophysiologic complaints are so frequent that aes has also been referred to as psychogenic purpura . here
, the authors described a 50-year - old woman with aes with a history of general chronic neurodermatitis recently studied in the authors institution , who was subjected to extensive investigations over a period of 6 months before the final diagnosis .
the 50-year - old woman had recurrent episodes of painful bruising over the neck , thorax , abdomen , lumbus , and the four extremities for 8 months , whilst the scalp , face , nucha , back , and buttocks were not affected ( fig . 1 ) .
the patient complained that the bruises appeared spontaneously and were not associated with any inducement , but were accompanied by a gradual increase in pain for approximately 2 days .
the patient had a 2-year history of general chronic neurodermatitis without any bleeding manifestations , such as melena , hematuria , and menorrhagia .
the neurodermatitis had usually recurred approximately every 46 months with severe itching over the lesions , and the last relapse was approximately 3 months prior to the study .
symptoms of itching could be relieved by oral antihistamines or corticosteroids ; however , the ecchymosis was unaffected .
nonetheless , the patient did not frequently use topical or systemic corticosteroids for fear of potential side effects .
a detailed inquiry revealed that prick - acupuncture management was usually performed by the patient to relieve the pruritus since 9 months prior to the study when the penultimate episode of neurodermatitis initially broke out.fig .
1a 50-year - old woman with recurrent ecchymosis over the neck , thorax , abdomen , lumbus , and the four extremities ( a ) , while the scalp , face , nucha , back , and buttocks were unaffected ( b ) .
erythra of chronic neurodermatitis can also been seen following the same distribution a 50-year - old woman with recurrent ecchymosis over the neck , thorax , abdomen , lumbus , and the four extremities ( a ) , while the scalp , face , nucha , back , and buttocks were unaffected ( b ) .
erythra of chronic neurodermatitis can also been seen following the same distribution physical examination at the time of bruising revealed scattered ecchymosis with lesions varying in size from 0.5 to 10.0 cm .
dispersive , noticeable excoriation could be seen on the same regions as the ecchymosis lesions of the neck , thorax , abdomen , lumbus , and the four extremities , whilst the scalp , face , nucha , back , and buttocks were not affected by either .
the amount of erythrocytes , leukocytes , hemoglobin , and platelets in a blood routine examination were normal .
extensive serum chemical analyses showed all of the following indexes were negative : platelet function , von willebrand factor , blood clotting , antibodies against platelets , erythrocyte sedimentary rate , rheumatoid factor , antinuclear antibodies , anti - dsdna , proliferating cell nuclear antigen ( pcna ) , anticardiolipin antibodies , treponema pallidum particle agglutination ( tppa ) , and anti - hiv .
tests of tuberculin - purified protein derivative on the right forearm and a capillary fragility experiment over both forearms also remained negative .
no clinical gastrointestinal or genitourinary hemorrhaging was evident by the occult blood test in feces and urine , and no organic diseases were revealed by a urine routine examination , chest x - ray , and type - b ultrasonic examination . an intradermal test for autoerythrocyte sensitization
was performed , with 0.1 ml of the patient s washed red blood cells injected in the left forearm , and a saline control on the opposite side .
approximately 24 h later , the patient developed a painful ecchymotic reaction on the left forearm , similar to the spontaneous lesions , whilst no reaction was observed on the control side ( fig . 2 ) .
skin biopsies of hemorrhage lesions were performed on the right forearm and the left abdominal wall , the former showed a slight perivascular inflammatory infiltration ( fig .
3 ) , whilst the latter showed intense erythrocytes extravasation or hemorrhage in the dermis and subcutis ( fig . 4).fig .
2the test for aes on the left forearm with painful ecchymosis within 24 h after injection ( a ) , and the negative result of a saline control on the opposite side ( b)fig .
3biopsy of right forearm showed a slight perivascular inflammatory infiltrate ( original magnification 40)fig .
4biopsy of left abdominal wall revealed an intense erythrocytes extravasation or hemorrhage in the dermis and subcutis ( original magnification 40 ) the test for aes on the left forearm with painful ecchymosis within 24 h after injection ( a ) , and the negative result of a saline control on the opposite side ( b ) biopsy of right forearm showed a slight perivascular inflammatory infiltrate ( original magnification 40 ) biopsy of left abdominal wall revealed an intense erythrocytes extravasation or hemorrhage in the dermis and subcutis ( original magnification 40 ) apart from the general chronic neurodermatitis , the patient had no other important dermopathic history .
however , the patient complained of multiple stressors in daily life , and anxiety and depression often disturbed her .
a high score of falsehood was revealed by psychiatric examinations using the minnesota multiphasic personality inventory .
the ecchymosis reduced and finally diminished approximately 4 weeks later , with a follow - up of stationary phase for more than 18 months .
groch et al . tried to identify a single biochemical agent that could be responsible for the characteristic skin lesions .
proposed phosphatidylserine , which could be isolated from the erythrocyte membrane , to be the substance causing the disease .
however , patients who were injected with phosphatidylserine showed no reaction . thereafter , most investigators proposed psychosomatic components to be one of the important contributors eventually leading to the outbreak of the disease .
agle and ratnoff suggested that aes might be a psychophysiological entity , in which the reactions to psychosomatic instabilities involve the autonomic nervous system , and is a way of relieving anxiety .
such psychosomatic instabilities include emotional stress , hysteria , and masochism , which tended to erupt and become more severe during the course of the disease , while antipsychotic drugs , antidepressants , or psychotherapy were often helpful .
agle and ratnoff [ 9 , 10 ] believed that these patients always presented with hysterical and masochistic traits , depression , anxiety , and the inability to deal appropriately with their own hostile feelings , and hypothesized that psychogenic purpura appearing in the context of emotional stress was an interesting manifestation of a conversion reaction .
jacobi , who had questioned why certain people reacted with vascular symptoms instead of more common conversion symptoms under certain stress situations , stated that specific psychological mechanisms could influence the central nervous system , and this mechanism might be expressed as bleeding episodes or stigmata .
apart from the hypothesis of autoimmunity and psychophysiology , some investigators still highlight the mechanical injury of the skin .
these investigators proposed that the mild trauma to the skin could at least be partially responsible for the onset of ecchymosis .
a case of aes was reported by levin et al . as early as 1969 . in this case , a teenage girl presented with recurrent , painful ecchymosis and autoerythrocyte sensitization was finally proved to be factitious purpura by means of plaster casts .
hersle and mobacken also demonstrated another case in which an intra - uterine copper device caused marked exacerbation of the ecchymosis .
additional cases also showed the onset of autoerythrocyte sensitization could be preceded by surgical procedures or other forms of trauma .
analogously , the correlation between the ecchymosis and acupuncture in the present patient created doubts with regards to a diagnosis of aes .
firstly , the ecchymosis was almost identically distributed over the neck , thorax , abdomen , lumbus , and extremities , where the prick - acupuncture management had been performed , whilst the other sites were not affected .
this suggested the possibility that the ecchymosis were just factitious purpura resulting from the mechanical injury of the prick - acupuncture .
this coincides with the fact that it was more convenient to perform the acupuncture on the regions of the neck , thorax , abdomen , lumbus , and extremities than the buttocks , back , and scalp .
furthermore , the autoerythrocyte sensitivity autoimmunity reaction should arouse the lesions symmetrically and uniformly over the body .
secondly , the coincidental timing seems to be more persuasive : the ecchymosis occurred directly following the administration of acupuncture and disappeared gradually after the acupuncture was stopped . according to sawhney and arora , in factitious purpura , there were usually no premonitions before the onset of the lesions . the results of skin sensitivity tests tended to follow the patient s perception of their doctor s expectations , and were positive only if the injected areas were accessible to them . in view of the present patient ,
the sensation of intermittent mild itching was definite , but whether it was actually a neurodermatitis symptom or one of the premonitions of ecchymosis is unknown .
moreover , the patient was blinded to the purpose of the injections , and the injection areas were overlaid by sterile carbasus to avoid access .
the close observation following intradermal injections confirmed that the patient had not interfered with the dressings .
as a result , it was still difficult to differentiate aes from the factitious purpura , especially in the absence of hematological abnormalities . in conclusion ,
one of the reasonable interpretations for the present patient probably lies on the hypothesis of synergism between psychoimmunology and mechanical injury of the skin . as for the authors inference
, the psychosomatic instabilities might have initially disturbed the skin s immunological function and weakened the skin s blood capillaries , so that even a mild trauma of the skin would damage the capillary walls and eventually result in the permeation of red corpuscles . | autoerythrocyte sensitization syndrome ( aes ) is a disorder characterized by recurrent , spontaneous , painful bruising in patients with underlying psychosis and neurosis .
its pathogenesis is uncertain .
purpuric lesions reproduced by injections of washed autologous erythrocytes suggest an autoimmune etiology .
the authors described a 50-year - old woman who presented with recurrent episodes of painful bruising for 8 months , along with a 2-year history of general chronic neurodermatitis .
a diagnosis of aes was made on the basis of the clinical symptoms and laboratory examinations results : the positive autoerythrocyte sensitization test and psychophysiologic derangement . however , the noticeable symptoms , simultaneously identical distribution of the ecchymosis and neurodermatitis lesions , as well as concomitant symptom of itching over the ecchymosis , created doubts regarding the real etiopathogenesis of the ecchymosis and whether the ecchymosis in the present patient were just factitious purpura .
the authors discuss the rarity of this case , which to the authors knowledge , had not been previously reported in the literature . |
this case represents the first report of a novel hemoplasma species in a human in association with doxycycline - responsive pyrexia , hemolytic anemia , and a history of chronic moderate neutropenia .
candidatus mycoplasma haemohominis is proposed for this novel organism should further characterization be possible .
this case is unique in several ways : first in describing human infection with a novel hemoplasma species that is distinct from described veterinary hemoplasma species , second in associating human hemoplasma infection with clinically significant hemolysis , and third in the use of qpcr to quantify hemoplasma dna in human diagnostic samples and thus monitor response to treatment .
diagnosis of hemoplasma infection in this patient was based on pcr analysis of bone marrow derived dna , but peripheral blood samples could have been an alternative source of dna for diagnosis , as performed in veterinary practice .
human hemoplasma - like infections have been reported occasionally in immunocompromised patients by means of cytological diagnosis , which is known to be very unreliable [ 1 , 3 ] .
although human hemoplasma infections have been reported in china [ 5 , 6 ] , these descriptions have not described clinical disease , pcr methodology , or infecting species , making interpretation difficult .
other studies have described the presence of existing veterinary hemoplasma species dna in humans : mycoplasma suis [ 7 , 8 ] , mycoplasma haemofelis and/or mycoplasma haemocanis [ 9 , 10 ] , and mycoplasma ovis . however , any association between hemoplasma infection and clinical signs in these reports is difficult to ascertain due to either lack of clinical information provided [ 7 , 8 , 10 ] or concurrent b. henselae infection [ 9 , 11 ] .
it is unlikely that co - infection with bartonella spp was present in this case due to the negative serological and pcr results for bartonella spp obtained and the fact that only hemoplasma dna was detected by 16s rdna pcr .
it is possible that the patient s neutropenia played a role in the initial development of clinical hemoplasmosis through immunocompromise .
it is less likely that a chronic hemoplasma infection played a role in causing neutropenia , as evidenced by the long history of neutropenia prior to any illness and return to low wbc counts with effective treatment and clearance of infection on specific qpcr , but this remains a possibility .
it is possible that it represents a zoonotic infection acquired , for example , from blood - sucking arthropods such as ticks , fleas , and lice or by direct transmission from another mammalian host through parenteral or oral inoculation of blood , but no such transmission events were recalled by the patient before onset of disease .
it is possible that this novel hemoplasma represents a species of which the primary host is humans .
hemoplasma infections exist in veterinary species in both the united kingdom and australia so transmission could have occurred before , during , or after the patient s vacation . besides the patient s husband and pet dog ( both of which were tested for hemoplasma dna ) there was no other significant animal or mammal contact reported .
as described elsewhere for veterinary hemoplasma infections , the patient described here showed a remarkable clinical response to doxycycline .
subsequently , a 6-month course of doxycycline and moxifloxacin was associated with negative qpcr results for hemoplasma dna , and the patient then remained qpcr negative following cessation of treatment .
the low levels of hemoplasma dna detected by qpcr at some time points during doxycycline and moxifloxacin treatment could reflect amplification of nonviable hemoplasma dna that had not been cleared from the circulation , although in animal studies rapid clearance of hemoplasma dna from the circulation is usually reported following effective antibiotic treatment . in conclusion
, hemoplasmosis should be considered as a differential diagnosis in patients with hemolytic anemia and pyrexia .
pcr testing for hemoplasma dna should be included in the investigation of such patients to enable the rapid detection of this infection , which may be more common than previously realized .
this case represents the first report of a novel hemoplasma species in a human in association with doxycycline - responsive pyrexia , hemolytic anemia , and a history of chronic moderate neutropenia .
candidatus mycoplasma haemohominis is proposed for this novel organism should further characterization be possible .
this case is unique in several ways : first in describing human infection with a novel hemoplasma species that is distinct from described veterinary hemoplasma species , second in associating human hemoplasma infection with clinically significant hemolysis , and third in the use of qpcr to quantify hemoplasma dna in human diagnostic samples and thus monitor response to treatment .
diagnosis of hemoplasma infection in this patient was based on pcr analysis of bone marrow derived dna , but peripheral blood samples could have been an alternative source of dna for diagnosis , as performed in veterinary practice .
human hemoplasma - like infections have been reported occasionally in immunocompromised patients by means of cytological diagnosis , which is known to be very unreliable [ 1 , 3 ] .
although human hemoplasma infections have been reported in china [ 5 , 6 ] , these descriptions have not described clinical disease , pcr methodology , or infecting species , making interpretation difficult .
other studies have described the presence of existing veterinary hemoplasma species dna in humans : mycoplasma suis [ 7 , 8 ] , mycoplasma haemofelis and/or mycoplasma haemocanis [ 9 , 10 ] , and mycoplasma ovis . however , any association between hemoplasma infection and clinical signs in these reports is difficult to ascertain due to either lack of clinical information provided [ 7 , 8 , 10 ] or concurrent b. henselae infection [ 9 , 11 ] .
it is unlikely that co - infection with bartonella spp was present in this case due to the negative serological and pcr results for bartonella spp obtained and the fact that only hemoplasma dna was detected by 16s rdna pcr .
it is possible that the patient s neutropenia played a role in the initial development of clinical hemoplasmosis through immunocompromise .
it is less likely that a chronic hemoplasma infection played a role in causing neutropenia , as evidenced by the long history of neutropenia prior to any illness and return to low wbc counts with effective treatment and clearance of infection on specific qpcr , but this remains a possibility .
it is possible that it represents a zoonotic infection acquired , for example , from blood - sucking arthropods such as ticks , fleas , and lice or by direct transmission from another mammalian host through parenteral or oral inoculation of blood , but no such transmission events were recalled by the patient before onset of disease .
it is possible that this novel hemoplasma represents a species of which the primary host is humans .
hemoplasma infections exist in veterinary species in both the united kingdom and australia so transmission could have occurred before , during , or after the patient s vacation . besides the patient s husband and pet dog ( both of which were tested for hemoplasma dna ) there was no other significant animal or mammal contact reported .
as described elsewhere for veterinary hemoplasma infections , the patient described here showed a remarkable clinical response to doxycycline .
subsequently , a 6-month course of doxycycline and moxifloxacin was associated with negative qpcr results for hemoplasma dna , and the patient then remained qpcr negative following cessation of treatment .
the low levels of hemoplasma dna detected by qpcr at some time points during doxycycline and moxifloxacin treatment could reflect amplification of nonviable hemoplasma dna that had not been cleared from the circulation , although in animal studies rapid clearance of hemoplasma dna from the circulation is usually reported following effective antibiotic treatment . in conclusion
, hemoplasmosis should be considered as a differential diagnosis in patients with hemolytic anemia and pyrexia .
pcr testing for hemoplasma dna should be included in the investigation of such patients to enable the rapid detection of this infection , which may be more common than previously realized .
ethics approval from the university of bristol was obtained for analysis of the human samples .
we thank hal neimark and chris helps for helpful discussions during the preparation of this manuscript , and sarinder day , daniel thomas , and andre buckeridge for technical support .
this work was supported by the wellcome trust ( grant 077718 ) ; the university of bristol ( postgraduate research scholarship to e. n. b. ) ; and pfizer animal health ( support for e. n. b. ) .
conflicts that the editors consider relevant to the content of the manuscript have been disclosed . | a patient with chronic moderate neutropenia , acute hemolysis , and pyrexia was found to be infected with a novel hemoplasma species . a clinical response to doxycyline
was noted , and moxifloxacin was added subsequently to aid infection clearance .
this represents the first report of hemolysis in association with confirmed hemoplasma infection in a human . |
the support vector machine ( svm ) algorithm is
among the most widely used supervised machine learning methods in
chemoinformatics and computer - aided drug discovery .
the popularity
of svm modeling primarily stems from generally high predictive performance
in compound classification and virtual screening .
although svms have been applied to investigate a variety
of class label prediction and also regression tasks in chemoinformatics
and drug discovery research , so far only very few
studies have addressed the issue of training set composition and size
for svm modeling and other machine learning
methods .
especially the choice of negative training
examples is often little considered in machine learning . typically ,
to train models for compound classification , a subjectively chosen
number of molecules are randomly selected from chemical databases
to serve as negative training instances , without further analysis .
two previous studies have investigated the choice of negative training
examples in greater detail . for svm modeling ,
the
use of experimentally confirmed negative training compounds from screening
assays and randomly chosen compounds from the zinc database was compared in the prediction of active compounds .
it was shown that the source of negative training
instances affected the performance of svm classification . perhaps
surprisingly , randomly selected zinc compounds often resulted in better
models than screening compounds that were confirmed to be inactive
against a target for which active compounds were predicted .
negative training sets were assembled from different
databases for compound classification using different machine learning
approaches .
these calculations revealed
a notable influence of negative training examples on the predictions
and a preference for randomly selected zinc compounds over compounds
from other sources . in this case , the
size of negative training sets was varied when building models using
different machine learning methods including svms with polynomial
kernels .
performance relationships
for varying numbers of negative and positive training examples were
not investigated .
in other studies , positive and negative training
examples were balanced to improve the performance of machine learning
models , addressing the issue of data imbalance in
machine learning .
herein , we report an analysis
of the influence of training set
composition and size on svm classification and ranking by systematically
varying the number of negative and positive training examples and
determining how these variations affect the prediction of active compounds
and stability of the calculations .
for svm classification , training compounds
are defined by a feature vector x and a class
label { 1 ,
1 } and projected into the reference space . svms
solve a convex quadratic optimization
problem to find a hyperplane h = { x|w , x + b = 0 } that separates
the positive and negative class .
the hyperplane h is defined by a normal vector w and a bias b and maximizes the margin between the two classes . to achieve
model generalization , non - negative slack variables i
in addition , the cost hyperparameter c controls the
trade - off between margin maximization and permitted training errors ,
and its value can be optimized by cross - validation .
once the decision boundary is defined , test instances
are projected into the feature space .
new compounds of unknown class
label are classified according to the side of the hyperplane on which
they fall or , alternatively , ranked according to the value of g(x ) = w , x. the latter strategy is equivalent
to changing the bias of the hyperplane , sliding it from the most distant
points on the positive side toward the negative side , and ranking
compounds in the order they pass through the plane . in the case
of
nonlinearly separable training data in a given reference
space , the scalar product < , > can be replaced by
a
kernel function k( , ) , which is known as the kernel
trick.(14 ) using kernel functions ,
the scalar product of two feature vectors can be computed in a higher
dimensional space where the
data may be linearly separable
without the need to explicitly compute the mapping of into . in svm - based
compound classification ,
the tanimoto kernel is one of the most frequently used kernel functions
for binary fingerprints . for imbalanced
data sets , different class weights can be assigned
to put relative weights on misclassification of positive and negative
training instances and avoid orienting the hyperplane toward the minority
class .
accordingly , c+ and c balance
the weight on slack variables for the positive and negative class ,
respectively .
ten sets with
at least 600 active compounds ( positive instances ) were obtained from
chembl version 22 . only compounds with
numerically specified equilibrium constants ( ki values ) for single human proteins
were selected , while omitting
borderline active compounds ( pki <
5 ) that might often represent artifacts .
table 1 reports the accession number , target name ,
number of compounds and mean pki values
for these 10 compound data sets . as background set ( pool of negative
instances ) ,
random subsets of these compounds were used as
negative training and test examples . for model building , all active
and inactive compounds
were represented as standard maccs fingerprints consisting of 166 bits monitoring the presence
( bit set on ) or absence ( set off ) of predefined structural fragments
or patterns .
although we deliberately selected the simplistic and
easy to rationalize maccs fingerprint for our proof - of - concept investigation ,
control calculations were also carried out using the folded version
of the extended connectivity fingerprint with bond diameter 4 ( ecfp4 ) .
ten compound
data sets were selected
from chembl and used for svm modeling . for each activity class ,
the
chembl accession no . , target name , number of compounds , and mean pki value are reported .
( 1)each activity class was randomly divided
into training and test ( prediction ) sets .
training set size was varied
across values # i = { 10 , 50 , 100 , 500 , 1000 } for the negative ( inactive )
class and # a = { 10 , 50 , 100 , 250 , 500 } for the positive ( active ) class .
test sets always consisted of 10 000 inactive and 100 active
compounds.(2)preprocessing
of the fingerprints
of the training and test data was carried out by removing zero - variance
features and applying centering and unit variance scaling to all features
on the basis of the training set for each trial.(3)for each of the 25 training set combinations ,
svm models were built using the linear and tanimoto kernel with class
weights c+ and c. in addition , cost factors c controlling
the influence of individual support vectors were optimized using values
of 0.01 , 0.1 , 1 , and 10 .
for cost factor optimization , 10-fold cross - validation
was carried out with training data splits of 60% ( model derivation )
and 40% ( testing , internal validation ) .
models with best cost factors
were selected on the basis of largest area under the roc curve ( auc).(4)the optimized svm model
was used to
rank test set compounds in the order of decreasing probability of
activity based upon the signed distance from the hyperplane ( positive
to negative side ) .
model performance was assessed by determining the
recall rate of active compounds within the top 1% of ranked test compounds .
in addition , balanced accuracy ( ba ) was calculated , defined as(tp , true positives ; tn , true negatives ; fp ,
false positives ; fn , false negatives).(5)for each activity class and combination
of a kernel function and training set size , the modeling process was
carried out 50 times to obtain a distribution of recall rates.(6)the results were compared
using hypothesis
testing . the nonparametric kolmogorov
smirnov test was employed to account for differences between
cumulative recall distributions and the levene test to compare the variance of these distributions . in addition ,
the bonferroni correction was introduced
for multiple testing.the calculation protocol
was implemented in r , and the kernlab package was used for
svm modeling . each activity class was randomly divided
into training and test ( prediction ) sets .
training set size was varied
across values # i = { 10 , 50 , 100 , 500 , 1000 } for the negative ( inactive )
class and # a = { 10 , 50 , 100 , 250 , 500 } for the positive ( active ) class .
preprocessing
of the fingerprints
of the training and test data was carried out by removing zero - variance
features and applying centering and unit variance scaling to all features
on the basis of the training set for each trial .
for each of the 25 training set combinations ,
svm models were built using the linear and tanimoto kernel with class
weights c+ and c. in addition , cost factors c controlling
the influence of individual support vectors were optimized using values
of 0.01 , 0.1 , 1 , and 10 . for cost factor optimization , 10-fold cross - validation
was carried out with training data splits of 60% ( model derivation )
and 40% ( testing , internal validation ) .
models with best cost factors
were selected on the basis of largest area under the roc curve ( auc ) .
the optimized svm model
was used to
rank test set compounds in the order of decreasing probability of
activity based upon the signed distance from the hyperplane ( positive
to negative side ) .
model performance was assessed by determining the
recall rate of active compounds within the top 1% of ranked test compounds .
in addition , balanced accuracy ( ba ) was calculated , defined as(tp , true positives ; tn , true negatives ; fp ,
false positives ; fn , false negatives ) . for each activity class and combination
of a kernel function and training set size ,
the modeling process was
carried out 50 times to obtain a distribution of recall rates .
smirnov test was employed to account for differences between
cumulative recall distributions and the levene test to compare the variance of these distributions .
for different
activity classes , svm classification and ranking
models were built under systematic variation of training set composition
and size and active compounds were predicted .
specifically , the number
of negative and positive training examples was varied in the ranges
of 101000 and 10500 , respectively , and all possible
combinations were explored .
in addition , cost factors were optimized
by cross - validation and class - specific weights were used to account
for data imbalance in the training set .
figure 1 compares
balanced accuracy of the predictions in the
presence or absence of class weights for two representative activity
classes .
consideration of class - specific weights consistently improved
the accuracy of the predictions for imbalanced training sets , except
for three cases of large training sets with at least 250 actives and
500 inactives for which the performance was comparable . hence , the
explicit consideration of different class weights for positive and
negative training instances produced more accurate classification
models . under these conditions ,
the derived hyperplane was not skewed
toward the minority class , resulting in improved model generalization ,
especially in the presence of large training data imbalance .
these
effects were outweighed only for the largest and least imbalanced
training sets . given the demonstrated relevance of class weights for
prediction accuracy , a factor
that is not always considered in svm
modeling , results reported in the following included class weight
settings
heat map representations
show balanced accuracy over 50 independent trials ( using a two - color
gradient ) for training sets of varying composition and size : ( top )
melanocortin receptor 4 ( mc4r ) ligands , ( bottom ) thrombin inhibitors .
in addition , optimization of cost
factors was carried out using
cross validation .
the best cost factors often varied depending on
training set composition , but for well - performing training sets ( i.e. ,
those with large numbers of actives and inactives ) , there was an overall
preference for c values of 0.01 for both the linear
and tanimoto kernels . for highly imbalanced data sets ,
larger cost
factors were frequently selected , indicating that adjusting margin
softness ( stability ) also contributed to model generalization .
it
is noteworthy that for different training set compositions and regardless
of the cost factor chosen the hyperplanes generated by the svms were
very frequently able to separate the training data without error and
thus resulted in a hard margin classifier .
figure 2 reports
compound recall for alternative
kernel functions under systematic variation of inactive and active
training instances for two representative activity classes .
figure 3 shows corresponding
density plots for recall rate distributions over multiple trials .
the results in figure 2 and 3 reveal generally higher recall performance for the tanimoto
than the linear kernel , frequently reaching a recall level of 0.9 .
however , even for the linear kernel , satisfactory recall was observed ,
often approaching a recall level of 0.75 .
differences in recall performance
between the linear and tanimoto kernel were quantitatively assessed
for all activity classes and statistically compared using the two - sided
and paired kolmogorov
the results confirmed that
the tanimoto kernel generally performed significantly better than
the linear kernel for training instances of # a = { 100 , 250 , 500 } and
#
i = { 100 , 500 , 1000}. however , there was no significant difference
in the cases of # a = { 10 } and # i = { 50 , 100 , 500 , 1000 } where prediction
accuracy was limited . furthermore , as shown in figure 3 , svm models derived using the tanimoto kernel
were generally more robust , i.e. , corresponding recall rate distributions
were sharper for the tanimoto than for linear kernel .
the presence
of narrow distributions indicated that models derived from different
training sets had comparable prediction accuracy for alternative test
instances .
as a control , svm calculations were also repeated using
the radial basis function ( rbf ) kernel , another popular
kernel function , with a sigma setting , corresponding to the inverse
kernel width , of 0.01 .
the results obtained
using the rbf kernel were , on average , nearly indistinguishable from
those obtained using the tanimoto kernel discussed in the following .
as an additional control , the calculations were also carried out using
ecfp4 instead of maccs to compare the trends observed for training
set variation . with both fingerprints , the same trends were observed
( with the typical slightly better recall performance of ecfp4 relative
to maccs ) . recall performance .
the median value and interquartile range of
the recall rate of active compounds among the top 1% of the ranking
is reported for 50 trials with the linear ( blue dashed line ) or tanimoto
( red solid line ) kernel .
results monitor the evolution of recall for
a constant number of inactives ( or actives ) and increasing number
of actives ( or inactives ) in the training set : ( a ) melanocortin receptor
4 ligands , ( b ) thrombin inhibitors .
the distribution of recall rates over 50 trials
is given for 100 ( top ) and 1000 ( bottom ) inactive and increasing numbers
of active training compounds : ( a ) melanocortin receptor 4 ligands ,
( b ) thrombin inhibitors .
the results
in figures 2 , 3 , and 4 revealed two key
findings ; ( i ) recall performance and model generalization consistently
improved with increasing size of training sets and ( ii ) the ratio
of active vs inactive training examples significantly influenced prediction
accuracy .
the increases in recall performance observed in figure 2 were detected for
all activity classes .
when the number of active training instances
was kept constant , recall rates increased with increasing numbers
of inactive instances , except in the case of 10 actives , where prediction
accuracy was generally low even over the range of 1001000
negative instances .
thus , a minimum number of active training compounds
was required for training sets of increasing size .
similar observations
were made when the number of inactive training compounds was kept
constant and the number of active examples was increased .
ten negative
examples were consistently insufficient for building effective models
and 50 negative training instances were often insufficient ( figure 2 ) .
however , in the
presence of at least 100 negative training instances , high prediction
accuracy was consistently achieved when the number of active examples
was increased ( figure 3 ) .
density estimates obtained from the distribution of recall rates over
50 trials are presented for training sets of varying size and composition .
for a constant number of 10500 active training compounds ,
recall distributions are shown for 10 ( pink ) , 100 ( green ) , and 1000
( blue ) inactive training compounds : ( a ) melanocortin receptor 4 ligands ,
( b ) thrombin inhibitors .
for all compound classes , incremental increase in the number
of
negative ( positive ) training instances led to systematic performance
enhancements when at least 50 positive ( 100 negative ) training compounds
were used , as confirmed by the one - sided kolmogorov
while overall highest prediction accuracy was achieved for training
sets consisting of 500 active and 1000 inactive examples , similar
accuracy was already observed for 100 active and 500 inactive training
compounds .
furthermore , recall generally began to reach a plateau
when at least 100 active and 500 inactive training instances were
used ( figure 2 ) .
however ,
with further increasing training set size , recall rate distributions
became narrower , as illustrated in figure 3 and 4 , which was
indicative of models with consistent prediction accuracy despite training
set variations , as mentioned above .
table 2 compares
the recall performance over all activity classes for one of the worst
and the best performing training set compositions of 10 actives/100
inactives and 500 actives/1000 inactives , respectively . in the bad
case scenario , recall rates of compounds were with one exception
lower
than 50% with large standard deviations and balanced accuracy was
around the 80% level .
by contrast , for the best performing large training
sets , recall rates were consistently high , with a mean of 87% , and
balanced accuracy was approaching 100% with very low standard deviations
( table 2 ) .
interestingly ,
training set imbalance only limited the accuracy of predictions in
the case of small but not large training sets , as illustrated in figure 4 , an effect that
can be ascribed to the use of class weights for svm models , as detailed
above .
for example , while an inactive vs active ratio of 10:1 produced
inaccurate predictions for training sets comprising 100 inactive and
10 active training examples , prediction accuracy was high when 1000
inactive and 100 active training compounds were used .
are the mean ( )
and standard deviation ( ) of recall of active compounds and
balanced accuracy after 50 independent trials for differently composed
training sets : 10 active and 100 inactive compounds
( low performance ) and 500 active and 1000 inactive compounds
( high performance ) .
results are shown for 10 compound classes , referred
by accession no . , according to table 1 .
taken together , the results in figure 3 and 4 clearly indicate
that the predictions became stable with increasing
size of training sets , another key finding .
figure 5 reports the variance of recall rates over
independent predictions using training sets of increasing size and
provides confirmatory evidence .
furthermore , levene tests for all
activity classes confirmed that the variance of recall distributions
significantly differed in 38 of 40 cases ( resulting from 10 compound
classes and four training set conditions ) when training sets with
at least 50 active and 10 or 1000 inactive examples were used . by
contrast , no statistically significant differences in variance of
recall rate distributions were detected when the svm models were trained
with 100 or 1000 inactive examples , regardless of the number of actives .
heat map representations show variance of recall rates over 50 independent
trials ( using a two - color gradient ) for training sets of varying composition
and size : ( left ) melanocortin receptor 4 ( mc4r ) ligands , ( right ) thrombin
inhibitors .
herein ,
we have systematically analyzed the influence of training
set composition and size on the prediction accuracy of svm classification
models .
different from earlier studies , our calculations have stressed
the importance of considering class weights and optimizing cost factors
when imbalanced training sets are used .
furthermore , the ratio of
active vs inactive training examples substantially affected the ability
of svm models to correctly predict active compounds . however , recall
rates and balanced accuracy consistently improved for training sets
of increasing size for all compound classes under study . increasing
size of training sets also compensated for inherent data imbalance .
moreover , large training sets led to robust predictions and the accuracy
was essentially constant when different training sets of the same
size were used . taken together , our findings have implications for
practical applications of svm classifiers .
best performing svm models were obtained when 250500
active and 5001000 randomly selected inactive training instances
were used . moreover , as long as 50 known active compounds
are available for training , increasing numbers of 5001000
randomly selected negative training examples improve and stabilize
model performance when class weights are taken into consideration ,
which provides a clear guideline for virtual compound screening . finally , we note that large numbers of active compounds may not
always be available for training .
however , since svm classification
and ranking models do not take compound potency as a parameter into
account , in contrast to support vector regression , large numbers of
hits often obtained from confirmatory screening assays might be readily
used for svm model building . | support
vector machine ( svm ) modeling is one of the most popular
machine learning approaches in chemoinformatics and drug design .
the
influence of training set composition and size on predictions currently
is an underinvestigated issue in svm modeling . in this study , we have
derived svm classification and ranking models for a variety of compound
activity classes under systematic variation of the number of positive
and negative training examples .
with increasing numbers of negative
training compounds , svm classification calculations became increasingly
accurate and stable .
however , this was only the case if a required
threshold of positive training examples was also reached .
in addition ,
consideration of class weights and optimization of cost factors substantially
aided in balancing the calculations for increasing numbers of negative
training examples . taken together , the results of our analysis have
practical implications for svm learning and the prediction of active
compounds . for all compound classes under study ,
top recall performance
and independence of compound recall of training set composition was
achieved when 250500 active and 5001000 randomly selected
inactive training instances were used . however , as long as 50
known active compounds were available for training , increasing numbers of 5001000
randomly selected negative training examples significantly improved
model performance and gave very similar results for different training
sets . |
sunitinib is a small molecule that inhibits multiple tyrosine kinase receptors . in 2006 , the united states food and drug administration approved this drug for treating clear cell metastatic renal cell carcinoma . in patients given sunitinib ,
median progression - free survival is significantly longer than in patients given the standard treatment with interferon - a , and some patients exhibit tumor regression .
its known adverse effects include fatigue , malaise , diarrhea , skin abnormalities , and cytopenia .
this report is the first to describe a case of nephrotic syndrome and ischemic acute tubular necrosis apparently related to sunitinib therapy .
we report the case of a 67-year - old man who was treated with sunitinib for metastatic clear cell renal cell carcinoma . in august 2008 ,
he was diagnosed with right huge renal tumor with inferior vena cava compression in the chang gung memorial hospital , kaohsiung branch , and then he visited our urology outpatient department for a tumor biopsy and management .
after admission in october 2008 , he refused the tumor biopsy , but agreed to tyrosine kinase inhibitor sunitinib for suspected metastatic renal cell carcinoma . before beginning the sunitinib therapy , blood pressure was 116/61 mm hg with medical control for hypertension , serum creatinine was 1.1 mg / dl , albumin level was 4.0 g / dl , and cholesterol was 57 mg / dl , triglyceride was 114 mg / dl and the patient had no proteinuria . after starting the sunitinib therapy with 37.5 mg daily at the end of november , he began to exhibit foamy urine and gradually decreasing urine excretion as well as a 21-kg weight gain .
his response to furosemide and water with salt restriction was poor , and general edema , dyspnea on exertion , and oliguria developed .
after 3 weeks of therapy , sunitinib was discontinued on december 18 , 2008 . however , urine excretion and edema showed no improvement 2 weeks after discontinuing sunitinib ( fig .
1 ) . the patient was re - admitted in january 2009 with serum creatinine 5.1 mg / dl , albumin level 1.5 g / dl , and sugar level 107 mg / dl .
no non - steroid anti - inflammatory drugs , antibiotics , or herbal supplements were used throughout the course of the sunitinib therapy .
physical examination revealed high blood pressure 161/83 mm hg , jugular venous pressure 10 cm , bilateral basal fine crackle breathing sounds , severe lower leg and scrotal edema without skin rash and arthralgia .
serum complement , anti - neutrophil antibody , cryoglobulin , protein electrophoresis , and rheumatoid factor were negative , and no hematological features of thrombotic microangiopathy were observed .
renal sonography revealed a large ( 14.8 13.3 cm ) tumor and normal left renal size without hydronephrosis .
response to high - dose furosemide and albumin supplement was poor , and temporal hemodialysis was done for 2 weeks due to respiratory distress . after a discussion with the patient and his family , a right tumor and left renal biopsy was performed on january 23 , 2009 .
immunofluorescence revealed negative igg , iga , igm , c1q , c3 , kappa and lambda light deposition , and glomeruli with no remarkable change other than mild mesangial expansion .
electron microscopy showed normal thickness of the glomerular basement membrane and segmental subendothelial widening , mild small dense deposits in the mesangial area , marked and extensive epithelial cell foot process effacement , moderate epithelial cell microvillous transformation ( fig .
2a ) , attenuation of the brush border , and sloughing tubule cell ( fig .
serum creatinine also improved ( creatinine 2.8 mg / dl [ 213.53 mol / l ] ) . however , daily protein loss increased to 48.1 g despite having discontinued sunitinib for 10 weeks .
the urine protein loss decreased to 9.85 g / day and serum creatinine to 1.8 mg / dl after the patient was given prednisolone 30 mg daily for 4 weeks .
the patient had taken no other over - the - counter medications , and all other possible causes of minimal change nephropathy were excluded . before beginning the sunitinib therapy , he was proteinuria - free and had normal renal function .
the naranjo adverse drug reaction probability scale score for this event was 6 , indicating a probable association of the event with the drug .
sunitinib is an oral , small - molecule , multi - targeted receptor tyrosine kinase inhibitor of targets such as vascular endothelial growth factor ( vegf ) and platelet - derived growth factor receptor .
the drug is administered orally once daily in a 4-weeks - on/2-weeks - off cycle .
the vegf is important for maintaining normal glomerular endothelial function . in normal mice and rats ,
circulating vegf - a can reportedly be neutralized by injection of anti - vegf - a antibodies , or soluble vegf - r1 induced proteinuria , glomerular endothelial cell detachment , and suppression of nephrin , an important protein for maintaining the glomerular slit diaphragm .
such findings implicate the role of vegf in the maintenance of the glomerular filtration barrier that prevents leakage of plasma proteins into urine .
proteinuria is common in mice treated with soluble vegf - r1 and in humans treated with anti - vegf antibody [ 3 , 4 ] .
however , the patient described in this report developed persistent proteinuria and required dialysis even after discontinuing sunitinib .
although a few reports have described renal dysfunction induced by sunitinib , including acute interstitial nephritis and thrombotic microangiopathy , this study is the first to report nephrotic syndrome with acute renal failure and ischemic acute tubular necrosis associated with this agent , and this is the first report of diagnosis by histological study .
we suspect that sunitinib may induce nephrotic syndrome by an immune reaction combined with amplification of the underlying pathogenesis of minimal change nephropathy , which may present with a transient episode of circulatory insufficiency during diuretic treatment and induce ischemic acute tubular necrosis .
oral anticancer agents such as sunitinib are generally considered safer than other anticancer agents and more tolerable by patients with poor condition or by patients with organ failure that is untreatable by standard cytotoxic chemotherapy .
this case report suggests that oncologists and nephrologists should be vigilant for possible minimal change nephropathy and ischemic acute tubular necrosis related to sunitinib treatment , especially in patients with renal cell carcinoma . until now ,
we recommend monitoring of serum creatinine and proteinuria immediately after initiating sunitinib therapy for early diagnosis of minimal change nephropathy and ischemic acute tubular necrosis and to avoid renal damage . | we report a case of nephrotic syndrome and acute renal failure apparently induced by sunitinib . a 67-year - old man with a history of metastatic renal cell carcinoma presented with progressive kidney dysfunction with proteinuria , general edema , and body weight gain of 21 kg after undergoing 3 weeks of sunitinib therapy .
the patient had taken no other over - the - counter medications , and all other possible causes of nephrotic syndrome were excluded .
the naranjo adverse drug reaction probability scale score for this event was 6 , indicating a high probability that the observed presentations were associated with use of the drug .
however , despite the discontinuation of sunitinib , his condition deteriorated , and hemodialysis was initiated for respiratory distress .
a renal biopsy was performed , which revealed ischemic acute tubular necrosis with minimal change nephropathy . in conclusion
, nephrologists and oncologists should be aware that nephrotic syndrome with ischemic acute tubular necrosis is a possible adverse effect of sunitinib .
for early diagnosis of this condition and to avoid renal damage , we recommend differential diagnosis of serum creatinine and proteinuria in patients undergoing sunitinib therapy . |
multidetector coronary computed tomography angiography ( ccta ) is a highly accurate noninvasive method for the diagnosis of coronary artery disease .
advances in computed tomography ( ct ) allow us to acquire high - resolution images and reduced in time and contrast material needed to scan the target organs .
image quality is affected by different parameters included motion artifact , coronary calcification , and coronary arterial enhancement which is related to the contrast material protocol and patients related factors . contrast enhancement protocol is determined by a variety of contrast medium - related factors , including contrast material dose , concentration , injection rate , injection duration , and the scan delay used after contrast material injection . for determination of an optimal scanning delay time during computed tomography angiography ( cta ) , it is important to predict the aortic enhancement pattern - especially peak aortic time ( pat ) and peak aortic enhancement ( pae ) - after bolus injection of a contrast material for each patient .
multiple patient - related factors such as weight , height , heart rate , and cardiac output ( co ) influence the condition of enhancement on the aorta and coronary arteries . among the patient factors ,
it has been observed that reduced co and cardiac function results in delayed and increased arterial enhancement . to the best of our knowledge
, only a few reports have been published regarding relationship between cardiac hemodynamic parameters and coronary artery opacification , and no literature about influence of ejection fraction ( ef ) measured by echocardiography on the coronary arterial enhancement .
the purpose of this study was to investigate the effect of ef on coronary arterial enhancement in a group of patients with narrow range of body weight which is also received adjusted contrast material dose according to the body weight .
this cross - sectional study was performed on 120 patients whom underwent coronary ct angiography for the evaluation of ischemic heart disease with a measurement of ef within 3 months of coronary cta from 2014 to 2016 at alzahra hospital in isfahan .
patients with stable angina pectoris , history of balloon angioplasty and recurrent symptoms after ballooning , atypical and typical chest pain , and high risk for coronary artery disease were included in our study .
clinical exclusions were renal insufficiency , unstable angina pectoris , acute myocardial infarction , history of allergy to an iodinated contrast material , high - grade heart failure , hemodynamic instability , and heart rate above 65 beats / min during cta .
the study protocol was approved by the local ethics committee ( research project number : 395065 ) .
all scans were performed with a 64-slice multidetector ct ( mdct ) scanner ( medical health care ge work station rdw 4.3 , ge , usa ) .
technical characteristics of mdct were as following : 64 mm 0.6 mm collimation , 350 ms gantry rotation time , kv of 120 , and mas of 600 .
almost all patients were given 40 mg of metoprolol orally to achieve a resting heart rate < 65 beats / min .
after calcium scanning , a bolus of 80100 ml of nonionic iodinated contrast agent ( scanlux-370 ) followed by 5060 ml of normal saline was injected by the way of an 18-gauge catheter at 46 ml / s .
a dose of 15 ml of contrast was used during the bolus timing scan calculated ( by the apparatus software ) at the level of the ascending aorta ( aa ) .
the region of interest ( roi ) was drawn in the aa at the level of the aortic root [ figure 1 ] .
image acquisition was timed to peak contrast enhancement in the aa in the test bolus study .
transverse coronary computed tomography angiography showed placement of region of interests in ascending aorta at the level of aortic root all data , including age , weight , height , heart rate , pat , pae and density of major coronary arteries ( right coronary artery [ rca ] , left coronary artery [ lca ] , left anterior descending [ lad ] , and left circumflex artery [ lcx ] ) and ef , were measured and recorded coronary arterial enhancement was measured at the aortic root , proximal of lca , lad , lcx , and rca .
pearson 's correlation coefficient analysis was used to investigate the relationships between ef , pat and pae , and peak attenuation ( these parameters collected as continuous variables ) of all coronary arteries .
pearson 's correlation coefficient was also used to verify how a patient 's heart rate , height , patient 's body weight and time to peak attenuation , and peak attenuation of aa on test bolus affected pae during coronary cta .
this cross - sectional study was performed on 120 patients whom underwent coronary ct angiography for the evaluation of ischemic heart disease with a measurement of ef within 3 months of coronary cta from 2014 to 2016 at alzahra hospital in isfahan .
patients with stable angina pectoris , history of balloon angioplasty and recurrent symptoms after ballooning , atypical and typical chest pain , and high risk for coronary artery disease were included in our study .
clinical exclusions were renal insufficiency , unstable angina pectoris , acute myocardial infarction , history of allergy to an iodinated contrast material , high - grade heart failure , hemodynamic instability , and heart rate above 65 beats / min during cta .
the study protocol was approved by the local ethics committee ( research project number : 395065 ) .
all scans were performed with a 64-slice multidetector ct ( mdct ) scanner ( medical health care ge work station rdw 4.3 , ge , usa ) .
technical characteristics of mdct were as following : 64 mm 0.6 mm collimation , 350 ms gantry rotation time , kv of 120 , and mas of 600 .
almost all patients were given 40 mg of metoprolol orally to achieve a resting heart rate < 65 beats / min .
after calcium scanning , a bolus of 80100 ml of nonionic iodinated contrast agent ( scanlux-370 ) followed by 5060 ml of normal saline was injected by the way of an 18-gauge catheter at 46 ml / s .
a dose of 15 ml of contrast was used during the bolus timing scan calculated ( by the apparatus software ) at the level of the ascending aorta ( aa ) .
the region of interest ( roi ) was drawn in the aa at the level of the aortic root [ figure 1 ] .
image acquisition was timed to peak contrast enhancement in the aa in the test bolus study .
transverse coronary computed tomography angiography showed placement of region of interests in ascending aorta at the level of aortic root
all data , including age , weight , height , heart rate , pat , pae and density of major coronary arteries ( right coronary artery [ rca ] , left coronary artery [ lca ] , left anterior descending [ lad ] , and left circumflex artery [ lcx ] ) and ef , were measured and recorded coronary arterial enhancement was measured at the aortic root , proximal of lca , lad , lcx , and rca .
pearson 's correlation coefficient analysis was used to investigate the relationships between ef , pat and pae , and peak attenuation ( these parameters collected as continuous variables ) of all coronary arteries .
pearson 's correlation coefficient was also used to verify how a patient 's heart rate , height , patient 's body weight and time to peak attenuation , and peak attenuation of aa on test bolus affected pae during coronary cta .
a total of 120 patients ( 64 men , 56 women ; mean age 57.61 11 years ; age range 2880 years ; mean weight 75.03 3.7 kg ; weight range , 7079 kg ) were included in the study .
the mean age of the male patients was 57.63 11.9 years , and the mean age of the female patients was 57.58 11.25 years .
the range of ef was ( 25%70% ) ( mean : 55 7.7 ) .
the range of pat and pae of aa on bolus test was 1331 s ( mean : 19.3 2 ) and 153435 hu ( mean : 235 40.6 ) , respectively .
mean peak attenuation of the aorta , rca , lca , lcx , and lad was ( 561 119 ) , ( 476 109 ) , ( 505 108 ) , ( 467 113 ) , and ( 473 104 ) , respectively [ table 1 ] .
there was a negative correlation between ef and pat ( r = 0.266 , p = 0.003 ) [ figure 2 ] ; however , there was no significant correlation between ef and pae ( r = 0.027 , p = 0.767 ) [ figure 3 ] also , there was no significant correlation between ef and the peak attenuation of coronary arteries ( rca , lca , lcx , and lad ) .
there was a significant negative correlation between the patient body weight and pae ( r = 0.255 , p = 0.005 ) ; however , there was no significant correlation between the patient body weight and pat ( r = 0.081 , p = 0.379 ) .
there was no significant correlation between the heart rate and pat ( r = 0.274 , p = 0.101 ) or pae ( r
patient demographic data and attenuation of arterial coronary scattergram shows the relationship between ejection fraction and peak aortic time .
there was a negative correlation between two parameters ( r = 0.266 , p < 0.003 ) scattergram shows the relationship between ejection fraction and peak aortic enhancement .
there was no significant correlation between two parameters ( r = 0.027 , p < 0.767 )
optimal contrast enhancement of coronary arteries is crucial for achieving best image quality and more conclusive results and contrast density of 300400 hu are preferable for ccta . by performing test bolus study ,
we can estimate pat for adjusting main study according to it but unfortunately despite considering appropriate timing ; suboptimal arterial enhancement was obtained .
the most possible explanation is that the magnitude of enhancement in test is not usually under attention of technologist , and this enhancing magnitude could be influenced by some patient 's related factors such as cardiac function .
the previous studies agree with the relationship between aortic and coronary enhancing time and magnitude with cardiac function , but co measured by thermodilution as used by sakai et al .
another study by husmann et al . used measured stroke volume and ef by reconstructed the image of ccta .
ef also could be measured by radionuclide technique , mdct , cardiac magnetic resonance imaging composite valve graft . to the best our knowledge ,
there are no clear clinical studies in which the relationship between ef measured by echocardiography and aortic enhancement on ct has been investigated .
we used echocardiography for estimating ef as a complete safety , noninvasive , available , and cost - benefit technique . in the other hand ,
echocardiographically reported ef could be a course estimator of co and considering that it is available before starting the study , we aimed to test if any possible relationship between echocardiographically reported ef and result of aortic and coronary enhance timing and magnitude .
ef in this study had a significant correlation with pat as been expected according to the previous studies , but no significant correlation was seen with peak attenuation in this study . although the similar result was also reported by husmann et al . , but we had expected different result due to the larger study population . in addition , we limited our included cases in narrow weight range and relative narrow heart beat range in the purpose of subsiding their confounding effect as far as possible .
one of the explanations of this nonsignificant correlation could be roughly estimating reported ef in reporting sheets of patient 's echocardiography .
experienced echocardiographers usually estimate ef by looking at the overall size and contractility of the various segments of the lv walls without taking exact measurements and visually estimate ef in intervals of 5%10% .
in addition , we included patients with narrow weight and heart rate range for decrease their bias effect but maybe other parameters of patients , which was not taken into account had interfered with the attenuation measurements and might have been a confounding variable for result .
it is true that ef could also be measured precisely by postprocessing images of ccta using dedicated software , but the cause that we choose echocardiogrphically to decide about adjusting protocol before its performance .
despite pat was related to ef but there was no relationship between pae and ef one of the explanation is that the left ventricular ef used for our study was assessed with echocardiography which is used roughly estimation of ef with interval of 5%10% and may cause confounding results .
mm contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work , ph contributed in the conception of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work mm contributed in the conception of the work , drafting and revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . | background : the purpose of this study was to evaluate the influence of ejection fraction ( ef ) on peak aortic time ( pat ) and peak aortic enhancement ( pae ) during coronary computed tomography angiography ( cta).materials and methods : one - hundred and twenty patients ( 64 men , 56 women ) underwent measurement of coronary cta with a measurement of ef within 3 months of coronary cta .
pearson 's correlation coefficient analysis was used to investigate the relationships between ef , pat and pae , and peak attenuation of all coronary arteries.results:the range of ef was ( 25%70% ) ( mean : 55 7.7 ) .
the range of pat and pae of ascending aorta on bolus test was 1331 s ( mean : 19.3 2 ) and 153435 hu ( mean : 235 40.6 ) , respectively .
mean peak attenuation of ascending aorta , right coronary artery , left coronary artery , left circumflex artery , and left anterior descending were ( 561 119 ) , ( 476 109 ) , ( 505 108 ) , ( 467 113 ) , and ( 473 104 ) , respectively .
there was a negative correlation between ef and pat ( r = 0.266 , p = 0.003 ) ; however , there was no significant correlation between ef and pae ( r = 0.027 , p = 0.767 ) .
in addition , there was no significant correlation between ef and the peak attenuation of coronary arteries.conclusion:pat was related to ef , but there was no relationship between pae and ef .
one of the explanation is that the left ventricular ef used for our study was assessed with echocardiography which is used roughly estimation of ef with interval of 5%10% and may cause confounding results . |
the subject of tumors of the nervous system is often looked upon with apprehension by medical students and practitioners , with some justification .
the plethora of terminologies and systems of grading and classification , the bewildering numbers of separately named lesions , and their apparently endless histologic variations provide ample basis for their perception .
central nervous system ( cns ) tumors are the neoplasms constituting 1 - 2% of all the neoplasms .
gliomas constitute 38.7% of cns tumors in which high - grade gliomas are 59.5% and low - grade gliomas are 33.1% .
the incidence of metastasis from lung carcinoma is 18 - 60% , breast carcinoma 5 - 21% , melanoma 4 - 16% , genitourinary 310% , while gastrointestinal malignancies constitute 5 - 12% .
immunohistochemistry ( ihc ) has become an important tool in the diagnosis of brain tumors .
although conventional hematoxylin and eosin ( h and e ) staining is the mainstay for pathologic diagnosis , ihc has played a major role in differential diagnosis and in improving diagnostic accuracy not only in general surgical pathology but also in neuro - oncologic pathology .
the judicious use of a panel of selected immunostains is unquestionably helpful in diagnostically challenging cases .
in addition , ihc is also of great help in predicting the prognosis for certain brain tumors .
ihc using monoclonal or polyclonal antibodies has greatly influenced the diagnosis of various neurological disorders . using this technique ,
the presence of characteristic antigen can be precisely defined in a sensitive and reproducible manner , thereby providing a better tool for making an accurate diagnosis of brain tumors .
glial fibrillary acidic protein ( gfap ) was first isolated from old multiple sclerosis plaques by roy and sarkar .
it is normally present in the astrocytes ( mature and developing ) , ependymal cells , and radial glia of developing the brain .
positive reaction to gfap has been demonstrated in astrocytomas , ependymoma , and astrocytic cells of mixed gliomas , subependymal giant cell astrocytoma , pleomorphic xanthoastrocytoma , astroblastoma , and gliosarcoma .
cytokeratins ( cks ) monoclonal antibodies are useful in identification of the epithelial nature of the neoplasm .
these antibodies are most useful in the differentiation of poorly differentiated epithelial malignancies from those other nonepithelial neoplasm .
group a ( acidic ) and group b ( basic ) keratins are separated into 19 members by molecular weight , charge specificities , and immunoreactivity with monoclonal antibodies with restricted specificities .
it has been shown to react with one group a keratin ( molecular weight 50 kd ) and two group b keratins ( molecular weight 43 kd and 39 kd ) .
high - grade gliomas are difficult to differentiate from metastatic tumors on the basis of light microscopy alone .
the role of cam5.2 being highly specific is emerging as a specific marker to diagnose metastatic carcinoma .
hence , this study is planned to differentiate glial tumors from metastatic tumors with the help of ihc ( cam5.2 and gfap ) .
the present study was conducted over a period of 2 years , from december 2010 to december 2012 in department of pathology , pandit b.d .
sharma pgims , rohtak and comprised of total 80 cases of cns tumors including metastatic tumors to the cns .
special ihc markers ( gfap and cam5.2 ) were used to differentiate high - grade gliomas from metastatic tumors .
sections of the human brain were run with each batch of ihc stains to act as a positive control for gfap and skin biopsy as a positive control for cam5.2 .
a negative control was obtained by substituting the primary antibody with an antibody of unrelated specificity .
over a period of 2 years , we examined total 80 cases of cns tumors including metastatic tumors to the cns [ figure 1 ] . in our study , the average age for the glial tumor was 35 years and for the metastatic tumor was 55 years . among glial tumors ,
71% were male , and 29% were female in a ratio of 2.5:1 , whereas in metastatic tumors male to female ratio was 2:1 showing male preponderance .
radiological findings based on contrast - enhanced computer tomography ( ct ) revealed that the maximum cases of glial tumors were nonenhancing as compared to metastasis , which were enhancing and hypodense .
frequency of various histologic subtypes of glial and metastatic tumors in the present study , glial tumors were widely reactive for gfap [ figures 2 and 3a b ] except few oligodendrogliomas [ figure 3c and d ] and mixed glial tumors and metastatic tumors were negative for gfap and reactive for cam5.2 [ figures 46 ] .
gfap expression in glial tumors was statistically significant ( sensitivity 81.1% and specificity 100% ) as compared to metastatic tumors .
cam5.2 expression in metastatic tumors was also statistically significant ( sensitivity 100% and 100% specificity ) [ table 1 ] . grade i astrocytoma ( a ; h and e , 200 ) and gemistiocytic astrocytoma ( c ; h and e , 200 ) . glial fibrillary acidic protein was positive in both the tumors ( b ; immunohistochemistry , 100 ) ( d ; immunohistochemistry , 200 ) glioblastoma multiforme ( a ; h and e , 40 ) revealing glial fibrillary acidic protein positivity ( b ; immunohistochemistry , 100 ) .
however , oligodendroglioma ( c ; h and e , 100 ) did not show positivity for glial fibrillary acidic protein ( d ; immunohistochemistry , 400 ) metastatic adenocarcinoma ( a ; h and e , 100 ) revealing cam5.2 positivity ( b ; immunohistochemistry , 200 ) metastatic renal carcinoma ( a ; h and e , 200 ) revealing cam5.2 positivity ( b ; immunohistochemistry , 200 ) and glial fibrillary acidic protein negativity ( c ; immunohistochemistry , 200 ) photomicrograph showing cam5.2 positivity ( a;ihc;200 ) and gfap negativity ( b;ihc;200 ) in metastatic follicular carcinoma thyroid correlation of gfap and cam5.2 staining in glial and metastatic tumors
some of the ihc markers are relatively sensitive and specific for some tumors ( e.g. , gfap for astrocytomas ) .
gfap is currently used as a routine antigenic marker for normal developing and mature astroglial cells and for pathologically altered astrocytes .
this protein is typically absent in primitive or neoplastic neuroepithelial cells , ganglion cells , oligodendrocytes , vascular endothelium , meningeal cells , fibroblasts , and other mesenchymal elements .
the value of immunoperoxidase stain for gfap is relevant in documenting astrocytic differentiation in the tumor growing outside the cns parenchyma .
the false negative reaction may occur as a consequence of delayed or improper fixation , presumably because much of the gfap is in the soluble form and that can leach from the tissue before it is processed appropriately .
gfap found to be highly specific and sensitive marker when compared with other special stains and markers .
it was found of particular importance in demonstration of astrocytic cellular differentiation in primitive or highly anaplastic cns tumors , in study of mixed glial tumors , demonstration of glial nature of tumors , and diagnosing glial tumors invading meninges and extraneural sites .
glial filament immunoreactivity had been observed in tumor cells of glial origin and in tumor cells with foci of glial differentiation arising within cns .
the value of the immunoperoxidase stain for gfap is particularly relevant in documenting astrocytic differentiation in tumors growing outside the cns .
it is , therefore , required for each center to determine the sensitivity and specificity of gfap staining under their own conditions .
cam5.2 is an igg2a murine monoclonal immunoglobulin composed of lower molecular weight ck proteins ( 50,000 , 43,000 , and 39,000 daltons ) .
comparison of cam5.2 with other epithelial markers showed that it was most effective in discriminating between benign and malignant lesions .
it is critical to keep this point in mind when faced with the differential diagnosis of high - grade gliomas versus metastatic tumors in brain biopsies
. it would be appropriate to go with highly specific low molecular weight ck ( cam5.2 ) , which are positive in metastatic tumors and do not stain glial tumors
. the wide variation in different series as shown in table 1 may be due to various factors such as difference in source of material that is surgical or autopsy , the type of the concerned hospital that is neurological or surgical and the hospital concerned may serve a selective population of older patients .
it also depends on the tendency of a neurosurgeon to avoid a biopsy if a primary site is known and multiple lesions are seen in the brain in ct scan .
high - grade astrocytic malignant neoplasms are difficult to distinguish from poorly differentiated metastatic carcinomas , particularly on small open biopsies .
metastatic tumors seen in cns may occasionally be difficult to distinguish histologically from high - grade gliomas .
glioblastoma multiforme ( gbm ) is characterized histologically by the presence of nuclear atypia , high mitotic rate and presence of necrosis , all characteristics often encountered in poorly differentiated metastatic carcinoma .
the absence of well differentiated foci of metastatic carcinoma may make a diagnosis of metastatic carcinoma versus gbm difficult by light microscopic examination alone .
early detection of cancer and effective treatment increases the longevity of cancer patients . in the present study , astrocytomas , as expected ,
besides helping in the origin of histopathologically difficult cases of anaplastic astrocytoma , gfap immunostain helped in the objective assessment of the degree of differentiation in these neoplasms .
astrocytic nature of the tumor cells was confirmed by demonstration of gfap in less anaplastic areas .
positive gfap immunostaining helped to overcome histopathological diagnostic problems in some undifferentiated tumors and to categorize these tumors as gbm .
use of gfap and cam5.2 immunomarker easily distinguished these two lesions in the occasional cases where light microscopic evaluation proven insufficient .
gfap is a superior marker to s-100 and vimentin as observed by few authors based on immunostaining pattern of gfap , vimentin , and s-100 using ihc techniques in human glial tumors .
perry et al . studied the diagnosis of metastatic adenocarcinomas to the brain of unknown primary .
sixty - eight cases of metastatic adenocarcinomas to the brain with known primaries were immunostained with antibodies to ck 7 , ck 20 , and cam5.2 .
cam5.2 is a useful confirmatory stain in suspected metastatic adenocarcinoma to the brain . unlike nonspecific ae1/3
ae1/3 antibody should be avoided in the brain because of the common staining of both normal and neoplastic astrocytes , but cam5.2 does not suffer this drawback and it is expressed in metastatic tumors to the brain .
concluded that normal and malignant astrocytes can be positive with nonspecific cks ae1/3 along with gfap but not with monoclonal antibody cam5.2 .
an understanding of the pathology of cns tumors plays a vital role in the management of patients and in clinical and biological research .
although ct and magnetic resonance imaging allow accurate localization of intracranial and spinal lesions , and often serve as a very good guide to the nature of the lesion , the final diagnosis of a tumor relies almost exclusively on histological evaluation of tissue taken at biopsy or autopsy .
pathology , radiology , and clinical evaluation all play a key role in the diagnosis and prognosis of tumors of the nervous system . an accurate diagnosis of brain tumors is usually possible after a careful assessment of routine microscopic features with sufficient clinical and radiological information .
high - grade gliomas such as gbm are fairly encountered in routine surgical neuropathology and it is crucial to differentiate them from metastatic tumors .
categorization is more problematic in such cases due to various parameters including the presence of necrosis and small sample size due to stereotactic biopsies . from the present study
, it is concluded that ihc is a valuable technique and the best effective combination in differentiating high - grade gliomas from metastatic tumors of cns are gfap and cam5.2 .
| background and objectives : immunohistochemistry ( ihc ) has become an important tool in the diagnosis of metastatic brain tumors .
the judicious use of a panel of selected immunostains is unquestionably helpful in diagnostically challenging cases . in our study , the best combination of immune markers useful in differentiating metastatic carcinoma from high - grade gliomas in central nervous system ( cns ) are glial fibrillary acidic protein ( gfap ) and cytokeratin ( ck ) ( cam5.2).materials and methods : the study was conducted on 80 cases of glial tumors including metastatic tumors to the cns .
histopathological diagnosis was established on routine hematoxylin and eosin staining of the sections .
special ihc markers , gfap , and cam5.2 were used to differentiate glial from metastatic tumors.result:of total 80 cases , 40 cases of astrocytic tumors , 2 cases of ependymoma , 2 cases of mixed glial tumors , and 16 cases of glioblastoma multiforme were positive for gfap .
twelve cases of oligodendroglioma were negative for gfap .
the sensitivity of gfap in glial tumors was statistically significant as 81.1% and specificity 100% , whereas sensitivity and specificity of cam5.2 in metastatic tumors were 100%.conclusion : ihc plays an important role in diagnosing tumors of cns and markers such as gfap and ck ( cam5.2 ) are quite effective in differentiating glial tumors from metastatic tumors of cns . |
hematopoietic stem cell transplantation ( hsct ) is an effective treatment modality , for a variety of hematologic malignancies . despite improvement in the prophylaxis and treatment strategies and supportive care measures ,
infection still is considered as a substantial cause of morbidity and mortality in patients undergoing hsct , by the advances achieved in the control of bacterial infections after hsct , invasive fungal infections ( ifis ) are chief infectious complications in these patients .
additionally , infections with less common fungi like fusarium and trichosporon species have been reported in this patient population .
the incidence of ifi after allogeneic hsct is estimated to be 10% to 25% in high - risk patients .
there are many risk factors that have been evaluated for the development of ifi following hsct .
some of them are neutropenia duration , receiving glucocorticoids , donor type , age , underlying disease , graft - versus - host disease ( gvhd ) , organ dysfunction ( renal , hepatic , or respiratory failure ) , hyperglycemia , cytomegalovirus or hiv infections , the function of immune system , breakdown of the gut mucosa due to chemotherapy and radiation therapy , use of indwelling devices and iron overload .
routinely four strategies including prophylactic , empiric , preemptive , and targeted therapy are applied for the management of fungal infections . as a common practice , high risk patients receive prophylaxis against fungal infections .
for prophylaxis against candida infections in patients undergoing hsct , fluconazole has been administered during the neutropenic period . whenever possible to identify the infectious fungi , targeted treatment can be implemented .
according to eortc / msg consensus group , the proved diagnosis of fungal infection in a patient with findings consistent with an endemic mycosis needs at least positive culture obtained from blood or the affected site or evidences from histopathology or direct microscopic observation . however , the diagnosis of fungal infections is difficult due to several factors .
moreover , according to idsa guideline for neutropenic patients with 5 days of persistent fever after the initiation of broad spectrum antibiotics , in whom no specific cause has been determined ; starting empiric antifungal treatment can be considered . despite the advances achieved in the diagnosis and treatment of fungal infections , unfortunately there are still centers in which the proved diagnosis of fungal infections can not be made and other strategies have been used for the treatment of suspected and less well documented infections . since the introduction of amphotericin b in 1958 , the availability of lipid - based formulations of amphotericin b , echinocandins and extended - spectrum triazoles were major advances towards the treatment of fungal infections and provided the clinicians with wider treatment options .
nowadays these agents are prescribed increasingly for the treatment of fungal infections due to increased number of immune compromised and critically ill patients .
in iran ,
different studies have addressed the prevalence of ifis in the setting of solid organ transplantation . but to the best of our knowledge , there is no data available on the prevalence of antifungal drugs administration in hsct setting .
so the aim of this study was to assess the prevalence of treatment with antifungal agents in allogeneic hsct recipients .
in this study , we present the retrospective review of records of patients who received allogeneic hsct in the hematology - oncology , bone marrow transplantation center at shariati hospital in tehran , iran , between august 2009 and august 2010 .
patients received different conditioning regimens according to the underlying disease and based on the hospital protocols .
none of the patients in this center received total body irradiation ( tbi ) as a part of conditioning regimen .
the day of the hsct procedure was considered to be day 0 and the days after and before that , were named to be + and respectively .
engraftment was considered when anc counts of 500 10/l and platelet counts of 2000010/l were achieved for three consecutive days without transfusion .
phenytoin was administered to patients receiving conditioning regimens containing busulfan for seizure prophylaxis additionally ; patients received prophylaxis against nausea and vomiting .
gvhd prophylaxis consisted of cyclosporine and low dose methotrexate in patients who received allogeneic hsct .
all patients received low dose acyclovir on admission which continued throughout hospitalization for prophylaxis of herpes zoster .
management of fever in neutropenic patients was according to the guideline of infectious diseases society of america ( idsa ) on the use of antimicrobial agents in neutropenic patients with cancer .
for neutropenic febrile patients an appropriate blood cultures were obtained to determine the source of infection before initiation of broad spectrum antibiotics empirically . if the fever episode was persistent after 48 hours and no etiology for the infection
was identified , vancomycin was added to the previous medications . whenever fever continued for more than 5 days after initiating antibiotics and
cross - checking of extracted data with the original data sheets and patients charts was done rigorously .
tables , absolute frequency , percentage , median , mean , standard error of the mean , maximum and minimum values were used to summarize different types of data as appropriate . to determine the relationship between distributions of demographic or clinical covariates in allogeneic hsct patients whether or not receiving treatment with antifungal agents , we used chi - square test or fisher 's exact test for categorical data , independent sample t - test for quantitative data with parametric distributions , and mann - whitney u test for quantitative data with non - parametric distribution .
the day of the hsct procedure was considered to be day 0 and the days after and before that , were named to be + and respectively .
engraftment was considered when anc counts of 500 10/l and platelet counts of 2000010/l were achieved for three consecutive days without transfusion .
phenytoin was administered to patients receiving conditioning regimens containing busulfan for seizure prophylaxis additionally ; patients received prophylaxis against nausea and vomiting .
gvhd prophylaxis consisted of cyclosporine and low dose methotrexate in patients who received allogeneic hsct .
all patients received low dose acyclovir on admission which continued throughout hospitalization for prophylaxis of herpes zoster . also patients received cotrimoxazole for the prevention of pneumocystis carinii infection .
management of fever in neutropenic patients was according to the guideline of infectious diseases society of america ( idsa ) on the use of antimicrobial agents in neutropenic patients with cancer . for neutropenic febrile patients
an appropriate blood cultures were obtained to determine the source of infection before initiation of broad spectrum antibiotics empirically . if the fever episode was persistent after 48 hours and no etiology for the infection
was identified , vancomycin was added to the previous medications . whenever fever continued for more than 5 days after initiating antibiotics and
cross - checking of extracted data with the original data sheets and patients charts was done rigorously .
tables , absolute frequency , percentage , median , mean , standard error of the mean , maximum and minimum values were used to summarize different types of data as appropriate . to determine the relationship between distributions of demographic or clinical covariates in allogeneic hsct patients whether or not receiving treatment with antifungal agents , we used chi - square test or fisher 's exact test for categorical data , independent sample t - test for quantitative data with parametric distributions , and mann - whitney u test for quantitative data with non - parametric distribution .
all the analyses were performed using spss version 17.0 ( spss inc . , chicago , il , usa ) .
during the one - year study period , eighty two patients were admitted to the ward .
patients consisted of 41 men ( 68.3% ) and 19 women ( 31.7% ) with mean age of 26.3 ( 1.2 ) years .
thirty two patients ( 53.3% ) received hsct due to hematologic malignancies ; the rest of them received hsct because of other underlying diseases such as thalassemia , aplastic anemia and a few other infrequent diseases .
in fact thalassemia was the most prevalent condition ( 26.7% ) leading to hsct followed by acute myeloid leukemia ( aml ) ( 23.3% ) in this set of patients .
median time to engraftment , median duration of neutropenia and median recovery period were 12 ( range : 9 - 32 ) , 8 ( range : 2 - 22 ) and 21 ( range : 12 - 48 ) days respectively .
all patients ( except one ) in this study received antifungal prophylaxis with fulconazole 100 mg twice daily orally .
one patient was under treatment with posaconazole and amphotericin b before hospitalization . in 28 patients ( 46.7% )
the prophylaxis of fungal infection was switched from oral fluconazole to low dose intravenous amphotericin b ( 0.3 mg / kg / day ) due to the severity of mucositis which made the oral administration of medications impossible for the patients . among all of allogeneic hsct recipient in this study , 15 patients ( 25% ) received treatment with systemic antifungal medications .
the prophylactic agent was discontinued upon the initiation of the treatment . among patients under prophylaxis with amphotericin b , in 4 patients
the dose of amphotericin b increased to the therapeutic dose due to the suspected infections .
the median duration of antifungal treatment for patients who received the therapeutic doses of amphotericine b was 10 ( 2 - 23 ) days .
neither positive cultures nor histopathologic or direct microscopic evidences were available from the specimen obtained from patients .
empiric treatment with antifungal agent was initiated with amphotericin b in 14 patients however , later in 3 patients ; treatment was switched from amphotericin b to voriconazole due to the suspicious of aspergillosis infection .
characteristics of hsct recipients and the frequent regimens used for pre transplantation conditioning are summarized in table 1 and table 2 respectively .
characteristic of allogeneic hsct recipients who received treatment with antifungal agents and those who did not receive treatment aml : acute myelogenous leukemia , all : acute lymphoblastic leukemia , aa : aplastic anemia , cml : chronic myeloid leukemia , mm : multiple myeloma , mds : myelodysplastic syndrome , cr : complete remission other conditions consisted of one patient with the diagnosis of paroxysmal nocturnal hemoglobinuria and one patient with myelofibrosis in each group p values are based on mann - whitney u test frequent conditioning regimens used for allogeneic hsct in patients who received treatment with antifungal agents and those who did not receive treatment
all patients ( except one ) in this study received antifungal prophylaxis with fulconazole 100 mg twice daily orally .
one patient was under treatment with posaconazole and amphotericin b before hospitalization . in 28 patients ( 46.7% )
the prophylaxis of fungal infection was switched from oral fluconazole to low dose intravenous amphotericin b ( 0.3 mg / kg / day ) due to the severity of mucositis which made the oral administration of medications impossible for the patients . among all of allogeneic hsct recipient in this study , 15 patients ( 25% ) received treatment with systemic antifungal medications .
the prophylactic agent was discontinued upon the initiation of the treatment . among patients under prophylaxis with amphotericin b , in 4 patients the dose of amphotericin b increased to the therapeutic dose due to the suspected infections .
the median duration of antifungal treatment for patients who received the therapeutic doses of amphotericine b was 10 ( 2 - 23 ) days .
neither positive cultures nor histopathologic or direct microscopic evidences were available from the specimen obtained from patients .
empiric treatment with antifungal agent was initiated with amphotericin b in 14 patients however , later in 3 patients ; treatment was switched from amphotericin b to voriconazole due to the suspicious of aspergillosis infection .
characteristics of hsct recipients and the frequent regimens used for pre transplantation conditioning are summarized in table 1 and table 2 respectively .
characteristic of allogeneic hsct recipients who received treatment with antifungal agents and those who did not receive treatment aml : acute myelogenous leukemia , all : acute lymphoblastic leukemia , aa : aplastic anemia , cml : chronic myeloid leukemia , mm : multiple myeloma , mds : myelodysplastic syndrome , cr : complete remission other conditions consisted of one patient with the diagnosis of paroxysmal nocturnal hemoglobinuria and one patient with myelofibrosis in each group p values are based on mann - whitney u test frequent conditioning regimens used for allogeneic hsct in patients who received treatment with antifungal agents and those who did not receive treatment
among patients received allogeneic hsct , acute gvhd appeared in 32 ( 53.3% ) patients and the most frequent involved site was skin ( 46.9% ) .
table 3 shows the acute gvhd severity and the affected organs in patients who underwent treatment with antifungal agents and those who did not receive antifungal treatment .
severity of acute gvhd and the involved site in hsct recipient p values are based on chi - square test
hematology - oncology wards and intensive care units are among the highest consuming antifungal agents in hospitals .
it is important to know the data about antifungal agents prescribed in these wards . in this retrospective study
we evaluated the prevalence of antifungal medications administered to patients receiving hsct during early post transplant period in a referral hospital in tehran . during this one - year study
almost all patients received fluconazole for the prophylaxis of fungal infections ; however , in 28 patients the switch to intravenous antifungal prophylaxis was inevitable and they received low dose amphotericin b for this purpose . although due to the potential toxicity , amphotericin b was more used as a treatment option rather than prophylaxis , the approach of using this drug as a prophylactic agent goes back to the studies conducted in 1990s . in a prospective randomized trial , koh et al . ,
compared the efficacy of prophylactic use of fluconazole and low - dose amphotericin b against fungal infections in recipients of hsct .
overall , the study showed that the incidence of proven , suspected or superficial fungal infections was not significantly different between the two groups .
for example martino et al . , evaluated 395 recipients of allogeneic hsct in spain and reported that 73% of patients received fluconazole , 17% itraconazole and 4% amphotericin b for prophylaxis and 6% did not receive any antifungal agent for this purpose .
none of the patients in our study received itraconazole for prophylaxis . also there are reports of poor tolerability of itraconazole .
for example in a cohort of 549 high risk haematology and hsct recipients barnes et al .
, found that itraconazole was poorly tolerated for prophylaxis and therapeutic serum levels were achieved only in 70% of patients .
fifteen patients ( 25% ) in our study received antifungal medications due to the suspected ifis .
none of the patients received azoles ( neither fluconazole nor itraconazole ) for the therapeutic purposes .
the empiric antifungal drug administered for patients in our study was amphotericin b. however , in a study on sales data of five university hospitals in germany , de with et al .
, showed that amphotericin b consumption decreased during the period of 2001 - 2003 in the hematology - oncology wards which was primarily attributed to increase in voriconazole administration . additionally , as it is expected the pattern of empirical antifungal administration is variable in different centers .
for example in a retrospective study of four - year antifungal prescription in hematologic patients , chan et al . , reported that echinocandins were accounted for 62% of all antifungal administrations . in contrast none of the patient in our study received this category of antifungal agents .
this was probably due to the unavailability of micafungin , and anidulafungin and restricted availability of caspogungin in iran .
additionally , the cost of the latter drug was not covered by the medication insurance systems at the time of the study . in a meta - analysis ,
goldberg et al . , compared empirical or preemptive antifungal treatment with placebo , no intervention , or another antifungal treatment in patients with hematologic malignancies .
the results showed that azoles were associated with lower mortality compared with amphotericin b. also administration of liposomal amphotericin b decreased the mortality and ifis more than other amphotericin b formulations .
voriconazole is the first line agent for the treatment of aspergillosis infection and during our study it was prescribed when there was a suspicion of aspergillosis infection and due to the considerable high cost it was not used as an option for empiric treatment .
this is the same as the study of chen et al in which they found that voriconazole was infrequently used for empirical therapy and it was prescribed in 23% of all antifungal drug administrations .
additionally , most often it was used for the cases of high clinical suspicion of invasive aspergillosis .
none of the patients in our study were proven cases of ifi and only in one patient the positive results of serum galactomannane test was available .
although it is proposed that empiric treatment did not result in considerable decrease in mortality , it led to a significant decrease in the documented or probable ifis and is considered to be the standard practice in special patient populations .
one of the patients in our study had a positive serum galactomannane test . in the absence of stronger evidences for proving fungal infections , serum galactomannane test and computed tomography ( ct )
are helpful measures for the initiation of antifungal treatment and can predict response . for example
ji et al . , retrospectively studied 124 patients who received empirical antifungal agents following allogeneic hsct and concluded that patients with positive serum galactomannane test and/or chest ct scan had significantly higher response rate to empirical antifungal treatment compared with all patients .
we did not observe any significant differences in neutropenia duration ( p value : 0.54 ) , length of hospital stay ( p value : 0.27 ) and number of patients developed graft versus host disease ( p value : 0.07 ) between patients received antifungal agents with those who did not receive treatment .
the most important restriction in our study was the lack of microbiologic confirmation of the ifis in this center which is almost the same as most other centers in iran .
another limitation is the short time patients follow up which did not allow us to assess the long - term outcomes .
also it should be noted that the number of patients included in this study was limited . so it is suggested that researchers conduct a prospective larger trial for better assessment of the fungal infections and their treatment .
the most important restriction in our study was the lack of microbiologic confirmation of the ifis in this center which is almost the same as most other centers in iran .
another limitation is the short time patients follow up which did not allow us to assess the long - term outcomes .
also it should be noted that the number of patients included in this study was limited .
so it is suggested that researchers conduct a prospective larger trial for better assessment of the fungal infections and their treatment .
none of the patients in this study received targeted treatment with antifungal agents and the treatment was implemented empirically . | backgroundinvasive fungal infections ( ifis ) are chief infectious complications in patients undergoing hematopoietic stem cell transplantation ( hsct ) . however , the diagnosis of fungal infections is difficult , and often empiric treatment initiates . since there is no data available on the prevalence of antifungal drugs administration in allogeneic hsct recipients in iran , we decided to conduct this study.methodsthis study was a retrospective review of records of patients who received allogeneic hsct in the hematology - oncology , bone marrow transplantation center at shariati hospital in tehran , between august 2009 and august 2010.resultssixty ( 73.1% ) patients consist of 41 men ( 68.3% ) with mean age of 26.3 ( 1.2 ) years received allogeneic hsct .
patients received prophylaxis with fulconazole however ; in 28 patients ( 46.7% ) it was switched to low dose amphotericin b. fifteen patients ( 25% ) received treatment with antifungal agents .
amphotericin b was the empiric agent administered . in 3 patients treatment
was switched to voriconazole .
neither positive culture nor direct microscopic evidence was available from the obtained specimen . only in one patient
the result of serum galactomannan assay was positive .
there were no significant differences in neutropenia duration ( p value : 0.54 ) , length of hospital stay ( p value : 0.27 ) and number of patients developed graft versus host disease ( p value : 0.07 ) between patients received antifungal agents with those who did not receive treatment.conclusionin this study hsct recipients received antifungal agents for prophylaxis .
twenty five percent of patients received treatment with antifungal agents empirically .
improvement in diagnosis of these infections can be helpful and lead to targeted therapy .
we suggest larger prospective trials for better assessment of antifungal agent administration . |
basilar artery occlusion ( bao ) has been associated with a high morbidity and mortality , compromising posterior cerebral blood flow to the occipital lobes , medial temporal lobes , the thalami , the cerebellum , and the brain stem .
clinical recovery is virtually impossible in the absence of recanalization . a medical emergency , bao calls for aggressive therapies to address clot dissolution , via intra - arterial thrombolysis ( iat ) , intravenous thrombolysis ( ivt ) , and/or endovascular removal .
we report a case in which recanalization on mra occurs in the absence of aggressive therapy .
a 58-year old patient with global left ventricular hypokinesis and wall motion abnormalities presented with acute ataxia , hemiparesis , and decreasing alertness .
initial mri revealed critically decreased basilar arterial flow likely secondary to a tight stenosis versus complete occlusion of the vessel ( figure 1 ) , and multiple associated strokes in the posterior circulation distribution ( figure 2 ) .
intervention with intra - arterial clot lysis and thrombolytic therapy were initially considered but the patient and family declined an interventional approach , thrombolytic therapy , or angiography .
the patient was subsequently admitted to the icu , kept flat under close observation for several days , received unfractionated iv heparin , and subsequently was administered warfarin over multiple days .
the patient had a follow - up mri / mra approximately 3.5 months later illustrating no new strokes and complete recanalization of the basilar artery ( figure 3 ) .
he remains on warfarin , is ambulatory , lives independently , and has only a mild residual hemiparesis and ataxia .
figure 1magnetic resonance angiography at onset of symptoms revealed basilar stenosis persistent with basilar artery occlusion ( arrow ) .
magnetic resonance angiography at onset of symptoms revealed basilar stenosis persistent with basilar artery occlusion ( arrow ) .
figure 2at the time of presentation , t2 weighted imaging showed infarcts in the cerebellum ( a ) and axial flair imaging showed infarcts in the brainstem ( b ) .
at the time of presentation ,
t2 weighted imaging showed infarcts in the cerebellum ( a ) and axial flair imaging showed infarcts in the brainstem ( b ) .
figure 3repeat magnetic resonance angiography 3.5 months after symptom onset revealed a complete recanalization of the previously occluded basilar artery on mra ( arrow ) .
repeat magnetic resonance angiography 3.5 months after symptom onset revealed a complete recanalization of the previously occluded basilar artery on mra ( arrow ) .
bao can have morbid outcomes , and mortality rate has been reportedly 8598% even with fibrinolytic , and/or endovascular therapy , if not recanalized . morbidities with thrombolysis include extracerebral hemorrhage , asymptomatic or symptomatic acute intracerebral hemorrhage , and neurological damage , most concerning of which is the locked - in state may occur if these therapies are not considered and bilateral infarction of the brainstem occurs .
the outcome of acute bao depends on the length and location of occlusion , degree of recanalization , and time between onset of symptoms and treatment . hospital based factors are also important , in that there are a limited number of centers with interventional neuroradiologists capable of endovascular therapy .
various studies have been carried out comparing the efficacy of iat versus ivt therapy , and/or endovascular management in the setting of bao , all resulting in remarkable rates of recanalization .
the aggressive treatment options are associated with a significant improvement in occlusion ; ivt or iat results in about 4065% recanalization with 2224% favorable outcome .
the lower improvement in morbidity and the serious risks of these therapies may be grounds for further investigation into conservative management via anti - coagulants potentially resulting in spontaneous recanalization .
our patient s decision to elect medical therapy may be supported by the data of the basics study which failed to show any special benefit to ia therapy over iv therapies , with more aggressive therapies entailing greater risks .
early spontaneous recanalization has been reported in the anterior cerebral circulation , whereas late spontaneous recanalization has been documented only in the extracerebral circulation .
the mr angiography changes in our patient illustrate that the use of conservative treatment with unfractionated heparin in an acute setting of basilar thrombosis was associated with complete recanalization and restoration of blood flow on mra by an unknown mechanism within 3.5 months , with a good clinical outcome .
there are limited , if any , documented reports of late spontaneous recanalization of the posterior cerebral circulation similar to our case in the english language literature .
reports of this nature call for further investigation into the underlying mechanism of spontaneous recanalization . | basilar artery occlusion may be associated with a poor prognosis in the absence of recanalization .
choices in aggressive treatment for this potentially fatal condition vary from intra - arterial or intravenous thrombolysis , endovascular removal , or a combination of the two , with adjunct anti - coagulation therapy .
these therapies have proven to be effective in recanalization , whereas conservative management with anti - coagulants alone has had more limited success in the literature .
we report a case of basilar artery occlusion managed conservatively with unfractionated heparin , resulting in complete recanalization 3.5 months after symptom onset .
conservative management of basilar artery occlusion with unfractionated heparin was associated with complete recanalization long after symptom onset . |
amyloid peptides are derived from the amyloid precursor protein ( app ) by sequential proteolytic cleavages by - and -secretases [ 1 , 2 ] .
amyloid peptides , particularly the longer a42 species , aggregate into various and different chemical or physical entities , which are now considered the primary pathological agents in ad .
controversial experimental data leave nevertheless many questions open with regards to the exact composition and size of the amyloid oligomers in vivo [ 35 ] .
moreover , the precise molecular actions of the amyloid peptides and their aggregates remain unknown and enigmatic , largely because their molecular targets , or their specific receptors remain undefined .
this issue raises more fundamental questions . for one , while the proteolytic enzymes and their mechanisms responsible for the generation of the amyloid peptides become well - known and understood at the molecular level , the eventual physiological function of the peptides is still questioned and remains questionable .
the original pathological importance ascribed to amyloid plaques was weakened , if not eliminated based on information gathered in transgenic models expressing mutant app .
we have advocated this hypothesis since we discovered early defects in cognition and ltp in app.v717i mice [ 611 ] .
matters are , however , complicated further by recent data originating from several clinical trials that identified a large fraction of individuals in the control groups with considerable brain amyloid load using pet - imaging [ 1214 ] .
obviously , this means that high - amyloid concentration in the brain is not per se incompatible with normal cognitive functioning in old age .
this calls for other factors to be at least coresponsible for the cognitive demise in ad - forwarding protein tau as the most obvious candidate .
primary tauopathy is recognized in a vast and still increasing number of cns - disorders .
the pathology is visualized postmortem as intraneuronal aggregates of protein tau , known as neuropil treads and neurofibrillary tangles .
these are similar if not identical to those observed in ad brain and mostly present without other associated pathological hallmarks , except inflammation markers .
the large clinical variability among the primary tauopathies stems from the presence of the tau pathology in different types of neurons and in different brain - regions [ 1518 ] .
biochemically , all tauopathies consist of smaller and larger aggregates and fibrils that become intertwined to form larger tangles in the soma as well as in axons and dendrites of affected neurons .
the aggregates consist almost exclusively of protein tau , albeit as different isoforms in different diseases , that is , either tau.3r or tau.4r or as a variable admixture .
their widely different grading of phosphorylation is usually referred to as hyperphosphorylated tau , denoted here as hp - tau .
importantly , no accurate definition based on either the level or the specific phosphorylated residues can typify any tauopathy exactly , because of the large variability with each primary disease .
. rare familial cases of frontotemporal dementia ( ftd ) are linked to exonic and intronic mutations in the mapt gene coding for protein tau ( chromosome 17 ) .
although rare , their identification marked a major breakthrough and boosted interest in tauopathy , because of the evident implication that tauopathy itself is sufficient to cause cognitive decline and dementia [ 15 , 16 , 1821 ] .
both intronic and exonic mutations have important mechanistical implications : ( i ) most expressed mutations are in the microtubule binding domain of tau ; ( ii ) intronic mutations promote or hinder splicing of exon 10 coding for the second repeat domain in the microtubule binding domain , skewing expression of isoforms to either tau3r or tau4r , but both with normal sequences and consequently normal binding - affinity for microtubules .
the pathology resulting from intronic mutations in the mapt gene can only be explained by effects on rna splicing , eventually resulting in changes in absolute or relative levels of tau isoforms that otherwise have normal wild - type protein sequences .
the overexpression or overrepresentation of one or the other tau isoform can then be extrapolated to the primary tauopathies that are sporadic , as well as to sporadic ad as the most prominent secondary tauopathy involving normal , wild - type tau - isoforms .
the major unresolved problem in ad is the mechanistic relation of amyloid and tau pathology .
amyloid oligomers are proposed to precede , and eventually trigger the intracellular tauopathy , likely by increasing the phosphorylation of protein tau [ 11 , 15 , 17 , 22 ] . while this hypothesis remains at this moment in time impossible to prove in sporadic ad cases ,
indeed , even in the most early onset familial ad that are caused by mutant presenilin or mutant app , and therefore by definition are amyloid - triggered , tauopathy is always evident and codiagnostic .
experimentally , double and triple transgenic mouse models with combined amyloid and tau - pathology prove the same intimate relation [ 2326 ] .
the genetic data imply that deranged tau - microtubule interactions , caused either directly by the mutation , by phosphorylation , by increased absolute or by disturbed relative concentrations of tau , all can contribute or even be sufficient to cause neurodegeneration in primary tauopathies , that is , in the absence of amyloid pathology .
importantly , experimental decrease of the level of tau in amyloid transgenic mice can actually ameliorate several clinical symptoms and defects .
the combined data further corroborate the hypothesis that protein tau is essentially contributing to amyloid - induced cognitive defects . not resolved
are the identity and exact nature of the receptors and associated signaling pathways that lead from amyloid to tau .
these pathways obligatory involve kinases such as gsk3 [ 25 , 26 ] that contribute , directly or indirectly to the phosphorylation of tau at residues that affect its binding to microtubuli .
this is thought , but not proven , to instigate its eventual aggregation into fibrils that are deposited locally as neuropil treads or transported to the soma and form the neurofibrillary tangles , and both types of aggregates occur in the same neurons .
nevertheless , tauopathy does not per se cause immediate or even delayed neuronal death , as tangled and tau - loaded neurons are observed in humans as well as in experimental models , that is , transgenic mice , zebra - fish , and flies [ 26 , 28 , 29 ] .
the pertinent question to be answered then is : what chemical or physical form of protein tau causes synapto- and neurodegeneration ?
invaluable insight in the problems that we are concerned with , have resulted from analyzing different transgenic mouse models .
our research - group generated , characterized , and validated many different transgenic mouse strains and their bigenic combinations as preclinical models over the last two decades .
so far none of the models recapitulates robustly the evident neurodegeneration observed in the brain of ad patients .
we explored and reported on additional models to increase our understanding of this aspect of the pathogenesis of ad [ 11 , 30 ] . here , we review data and insights obtained with adeno - associated viral vectors that were engineered to express wild - type or mutant app or protein tau in pyramidal neurons of the hippocampus of wild - type mice .
thereby , we aimed to recapitulate aspects of ad pathology and related features that are not attainable in transgenic mice , particularly neurodegeneration . among the different viral vectors
available , we selected aav because these have been demonstrated as excellent and safe tools for gene delivery into the cns .
recent technical advances have improved aav - based vectors as tools in neuronal research [ 31 , 32 ] .
the expression of the human transgenes used in our studies was controlled by the human synapsin1 gene promoter , with constructs packed in chimeric capsids aav1/2 , expressing efficiently in pyramidal neurons of the ca1/2 subfield of the hippocampus ( figure 1 ) .
control aav vectors expressed egfp in the designated neurons and areas that persisted for months without negative signs or symptoms ( figure 1 ) and [ 31 , 32 ] .
we first constructed aav vectors to recapitulate the amyloid pathology in the limbic region of wild - type mice .
the constructs contained app695 as the most abundant neuronal isoform , either as wild type sequence or as the engineered triple mutant ( denoted app.sla ) .
the incorporated swedish , london and austrian mutations are each associated with familial forms of ad [ 3335 ] . following intracerebral injection into wild - type mice , the app.sla mutant produced a gradual accumulation of app and amyloid peptides in pyramidal neurons in the hippocampal ca1/2-region , as well as in the deeper layers of the neighboring cortex .
similar expression of wild - type human app695 failed to produce any deposition of amyloid or of the related effects observed with mutant app.sla . initially , from 3 weeks to 3 months postinjection ( p.i . ) , amyloid immunoreactivity appeared as intracellular inclusions or vacuolar bodies inside pyramidal neurons . at 6 months
, amyloid plaques developed as immunoreactive and thioflavins positive deposits in the hippocampus and cortex ( figure 2 ) . at this late time - point
, we also observed increased phosphorylation of endogenous mouse protein tau , particularly at residues t181 and t231 , both known to be substrate for gsk3 .
these phosphoepitopes , defined by antibodies at270 and at180 were differentially expressed , that is , in hippocampal pyramidal neurons ( t231 ) and in dystrophic neurites around amyloid plaques ( t181 ) .
in contrast , intracerebral injection of aav-app.sla not in wild - type but in tau.p301l mice led to the formation of neurofibrillary tangles ( nft ) , demonstrating that the viral assault did not prevent protein tau from forming tangles ( figure 2 ) .
moreover , the combination demonstrated that besides the primary hit , also the receiving genetic background is of utmost importance .
most interesting for the interpretation of the data obtained with protein tau , discussed in the next section , was the observation that at 6 months p.i .
the phosphorylation of endogenous mouse tau was increased and coincided with a minor but significant reduction in the number of neurons in the ca1 region of aav-app.sla injected mice . in second instance , we similarly expressed either wild - type tau.4r or mutant tau.p301l by aav - vectors in wild - type mouse brain . in sharp contrast to
app - sla , aav - mediated expression of protein tau resulted already at 3 weeks p.i .
these findings were rather unexpected and we analyzed them in different directions , yielding novel and important insight into the mechanisms causing neuronal death by protein tau .
most remarkably , we could not detect any major form of aggregates of protein tau in pyramidal neurons that expressed either wild - type or mutant protein tau .
extensive analysis by histochemistry and immunohistochemistry with a panel of indicator compounds and monoclonal antibodies to protein tau , did not reveal any appreciable signs of formation of intraneuronal tau - aggregates , either before or during or after the pyramidal neurons in ca1/2 succumbed .
the actual levels in hippocampal extracts were near - physiological and only about twice those of endogenous mouse tau4r .
note that also in the aav-app.sla model described in the previous section , the hippocampal levels of human app.sla were only about twice those of endogenous murine app .
in addition , expression of egfp by the same aav - type vectors proved harmless to pyramidal neurons over long time - periods ( figure 1 ) and [ 30 , 31 ] .
intracerebral injection of 3- and 10-fold less aav - tau , with consequently less expression of human protein tau , produced a graded , lesser loss of hippocampal neurons .
the aav - tau - induced neurodegeneration is thereby further demonstrated to be directly caused by and proportional to the near - physiological levels of protein tau .
consequently , the observed pyramidal cell - death is qualified as specific for human protein tau .
this conclusion holds up for wild - type tau.4r and for mutant tau.p301l , making the model at least conform the observations in familial ftd .
indeed , as discussed above , intronic and exonic mutations yield very similar clinical outcome , despite the expression of mutant or wild - type tau4r , respectively .
the actual contribution of the mutation to the mechanism underlying the pathogenesis requires a rational explanation in human ftd patients , as well as in the aav - models .
extensive analysis to define the mechanisms that underlie the observed tau - mediated pyramidal neurodegeneration are described , summarizing reported data and preliminary data of work in progress .
similar aav - mediated expression of a truncated version of protein tau.4r , lacking the microtubule binding c - terminal domain , proved completely harmless and did not cause any neurodegeneration over similar time - periods .
the inherent conclusion must be that microtubule binding of protein tau is essentially involved in the neurotoxic degenerative mechanism .
consequently , we must orient further analysis to mechanisms that involve axonal and dendritic transport over microtubuli , which is of paramount importance for all cells but especially for neurons with their intricate branching . of note
, microgliosis was observed to be intense , and spatially and temporally closely associated with aav - tau induced degenerating neurons .
this was most recently also reported in a similar model based on aav vectors but in rats and for a different pathology in a different brain - region .
previously , we have observed a similar relation of microgliosis to neurodegeneration in an unrelated model for hippocampal sclerosis , caused by conditional expression of p25 , the truncated activator of cdk5 .
also , in that model , intense neurodegeneration was not marked by aggregation of protein tau , further strengthening our conclusion that cdk5 is not a major tau - kinase in vivo [ 3941 ] .
the data originating from the aav - models led us to conclude that not large aggregates of protein tau cause neurodegeneration .
this confirms our observations in transgenic mice that show aggregation of tau leading to tauopathy in somata and neuropil , but not accompanied by marked neurodegeneration [ 25 , 26 , 36 ] .
we proposed that neurons affected by tauopathy can either engage in aggregation of tau and thereby try to decrease the toxic species and hope to survive , or to enter the path to death by failing to aggregate protein tau .
clearly , the viral models comply with the transgenic models that aggregation of tau and neurodegeneration are not closely linked .
thereby , questions are raised that are addressed in more detail in the next sections . the classic concept that tau - fibrils or tangles are neurotoxic in tauopathies can be abandoned .
we advocate that neurofibrillary tangles , like amyloid plaques are the final pathological hallmarks , but not the neurotoxic agents .
the outcome fits independent observations that the extent of neuronal loss exceeds the number of nft in patients with ad [ 42 , 43 ] . in inducible tg4510 mice , memory decline and neuronal loss
are dissociated from tangle formation in time and brain region [ 44 , 45 ] . in the aav - tau injected mice ,
phosphorylation of tau was evident at many pathological epitopes , including those defined by antibodies at8 , at100 , at180 , and at270 . despite the increased phosphorylation
no aggregates of protein tau were deposited in degenerating pyramidal neurons , analyzed histochemically or immunohistochemically .
biochemical analysis indicated the formation of low molecular weight aggregates which need and deserve further analysis .
intriguingly , experiments whereby aav-app.sla was injected intracerebrally not in wild - type mice but in transgenic tau.p301l mice , produced not only amyloid plaques but also intracellular tau aggregates . nevertheless , no substantial neuronal loss was evident , substantiating our previous data of absence of marked neurodegeneration in tau.p301l mice and tau.p301lxgsk3b bigenic mice ( bigt ) , despite extensive or even dramatic tauopathy [ 26 , 36 ] .
the combined results from our experimental models and from a drosophila model of tauopathy consolidates the thesis that neurotoxicity is not exerted by large aggregates of protein tau but rather by tau - species that are intermediate between normally phosphorylated protein tau and the hyper - phosphorylated fibrils .
the identity of the toxic tau species , which we have termed tau - p * [ 11 , 30 ] remains to be defined a challenge equaling that of the identification of the amyloid receptor .
it must be remembered that tau is subject to other posttranslational modifications , besides phosphorylation .
indeed , tau can be ubiquitinated , truncated , glycosylated , glycated , oxidized , and , moreover , undergoes isomerization at proline residues . at any given moment protein tau is present in various molecular forms , which most likely differ subtly in properties of binding to microtubuli , interaction with other proteins , binding to membranes , being transported for normal duty or for degradation .
the decision whether neurons will enter the cell death path or protect itself by forming tauopathy , must depend on the actual levels of protein tau and on its modifications .
the rapid accumulation of tau - p * will lead to cell death , while more gradual accumulation of tau - p * would result in formation of aggregates .
both pathways will be affected by various external factors , for example , inflammation , amyloid , stress , and so forth . in this concept ,
evidence from patients , whereby ca1 hippocampal neurons survive for decades despite neurofibrillary tangles and experimental animals show tangle bearing neurons survive , even with lost membrane integrity .
two major pathways are responsible for removal of damaged , unfolded , or aggregated cellular proteins : the ubiquitin / proteasome system and the autophagy / lysosomal system that also can remove aged or damaged organelles .
malfunction of autophagy can result in accumulation of protein aggregates that may contribute to the disease pathology in ad and other neurodegenerative disorders [ 4952 ] .
stimulating autophagy pharmacologically , for example , by inhibition of mtor , ameliorates cognitive deficits and reduces amyloid and tau pathology in the 3xtg mouse model for ad .
autophagosomes form in a stepwise process that requires transport over the microtubular network , which is known to be affected by protein tau , a major microtubule - associated protein . in our mouse models ,
degenerating neurons in the aav - tau.p301l injected mice displayed as dark neurons with extensive vacuolization that could be taken to indicate a defective autophagic process . a similar facet of neurons we observed in an unrelated mouse model , that is , the p25 inducible mice that recapitulate hippocampal sclerosis .
the combined data do lend more support to the hypothesis that vacuolization is a correlate or consequence of the degenerative process , as an intrinsic mechanism needed to clean up defective organelles , protein aggregates or even entire dead or dying neurons .
consistent with this view is our observation in the aav - tau.p301l mice that vacuoles do not form early in the disease process but only after the neurons are already degenerating [ 11 , 30 ] . finally , we searched but did not find biochemical evidence for the conversion of lc3-i to lc3-ii , which is generally accepted as the necessary step in and read - out of the increased formation of autophagosomes .
after differentiation from neuronal precursors , neurons enter their post - mitotic state , while attempts to reenter the cell cycle is considered pathological and eventually result in cell death . in ad and other tauopathies , cell - cycle related events
we tested this hypothesis in the aav - tau model by examining cell cycle - related markers .
various markers were increased , that is , cyclind2 , phosphorylated retinoblastoma protein , proliferating cell nuclear antigen ( pcna ) , cyclin b1 . however , intracerebral injection of aav - tau in cyclind2 deficient mice showed no extra effect on neurodegeneration , suggesting that cell cycle reentry is not a pre - requisite for neuronal death .
microtubules ensure cell shape and constitute roads of transport , a feature prominent in neurons with the most complex architecture of all cells .
microtubule dependent transport is ensured by families of motor proteins dyneins and kinesins , respectively for retrograde transport from distal processes towards soma and as plus - end directed motor for anterograde transport .
first , mutant tau and hp - tau can cause it to detach from the microtubules and decrease its ability to control microtubule dynamics . on the other hand ,
increased levels of protein tau can saturate microtubules and hinder the foot - stepping of the motor proteins needed for axonal and dendritic transport .
both aspects of tau - related transport deficits have been observed and both can fit into a model leading to starving synapses that eventually culminates in neuronal death . in this hypothesis ,
tau - mediated neurodegeneration is likely to start in distal neuronal processes , that is , axons and apical dendrites , which degenerate progressively towards the cell soma .
synaptic pathology could be an early defect in neurodegenerative tauopathies as proper functioning of distal synapses is totally dependent on adequate transport of essential cargos and uninterrupted provision of energy .
impaired oxidative phosphorylation and mitochondrial dysfunction has been observed in mouse models expressing mutant tau , which was potentiated by mutant app and presenilin1 [ 56 , 57 ] . in tau.4r transgenic mice , we previously documented axonopathy and motor deficits that were rescued by coexpression of constitutively active gsk3b [ 37 , 58 ] .
the data explain how excess tau in first instance blocks transport by covering microtubuli , while gsk3 acting as major tau - kinase i phosphorylates tau to detach it from the microtubules and relieves the transport blockade .
interestingly , treatment of tau.4r mice with lithium salts significantly increased the axonopathy , an effect that can be ascribed to its inhibition of gsk3b .
as discussed above , aav - mediated expression of truncated tau , lacking the microtubule binding domain did not cause neurodegeneration , in marked contrast to full length protein tau ( figures 3 and 4 ) and .
the combined data from various approaches allow us to conclude that tau mediated pathology is , at least in part , mediated by its microtubule binding properties .
chronic inflammation is evident in brain of ad patients and proposed to contribute essentially to the vicious circle leading eventually to neuronal death , brain atrophy and severe dementia .
cns - related features of the immune system remain hardly understood , and supposed to be janus - faced , that is , providing protection as well as causing damage , steered by complex mostly unknown control mechanisms [ 60 , 61 ] .
protection could be impaired in ad , fueled by and fueling debates on possible prevention or therapeutic benefits of antioxidants , radical scavengers , nonsteroidal anti - inflammatory drugs ( nsaid ) , and so forth , although clinical trials do not substantiate the claims .
amyloid deposits attract microglia in a phagosomal attempt of their elimination , but activated microglia release proinflammatory cytokines , chemokines , reactive oxygen species , prostaglandins , and other mediators that harm neurons .
much attention has been paid to neurotoxicity mediated by free radicals , both reactive oxygen and nitrogen species , released by microglia .
astrocytes are the most common cell - type in the brain , involved in many functions and increasingly appreciated also in synaptic signaling and integrity .
astrocytes co - localize with microglia at damage - sites , but remain to be explored in detail in ad . strictly speaking ,
neurons must also be considered when discussing neuroinflammation because they ( can ) produce active factors , that is , complement , interleukins , tumor necrosis factor , acute - phase proteins .
we observed and discussed that degenerating neurons were temporally and spatially closely associated with activated microglia in two independent models , that is , the aav - tau viral model and the p25 transgenic model [ 30 , 39 ] .
too many unknowns prevent us from answering the question how tau - mediated damage can lead to neurodegeneration by involving activation of microglia .
we maintain the most straightforward hypothesis , that is , that the expression of protein tau , either wild - type or mutant by the viral vectors , is imposing stress on the pyramidal neurons that can not adapt rapidly enough to produce tau - aggregates as a means of temporarily detoxification .
the impact on various neuronal functions is evident , including disturbances in post - mitotic state and cell - cycle control , as well as disturbing microtubule - mediated transport .
the latter is most destructive in axons and larger dendrites , compromising the major synapses that become dys- or nonfunctional
. secreted neuronal proteins then activate nearby microglia that counter - react by secreting proinflammatory and potentially neurotoxic factors , fueling the viscous cycle that eventually leads to neuronal death .
based on the combination of transgenic and viral models , and taking into account pathological and clinical data from human patients , we defend the thesis that neurodegeneration is not caused by a single defect but by at least dual actions ( figure 4 ) .
the first is proposed to be intrinsically neuronal , for example , damage by accumulation of endogenous proteins in various molecular forms . in the case discussed here , protein tau is the cause in primary tauopathies . in ad ,
the upstream triggers are the amyloid peptides that accumulate because of mutations or other imposed defects in the proteolytic machinery that controls their formation and turnover .
the second factor is also essential , and can be neuronal or microglial in origin , related to any type of stress the brain can experience or has to endure , that is , partial hypoxia , glucose overload or shortage , problems with lipid or cholesterol , or other essential metabolites .
protein - aggregates , that is , plaques and tangles , are not the most essential in causing cognitive defects and dementia .
the challenge to define the neurotoxic tau - species and its mode of action does nevertheless become not less complicated .
the combination of transgenic and viral models illustrates eloquently the power of the approach as well as its weaknesses .
we still have more modeling to do , taking into account the data and indications that are provided by ongoing clinical studies , because only the human patient can eventually confirm any hypothesis based on experimental models . | patients suffering from alzheimer 's disease ( ad ) are typified and diagnosed postmortem by the combined accumulations of extracellular amyloid plaques and of intracellular tauopathy , consisting of neuropil treads and neurofibrillary tangles in the somata .
both hallmarks are inseparable and remain diagnostic as described by alois alzheimer more than a century ago .
nevertheless , these pathological features are largely abandoned as being the actual pathogenic or neurotoxic factors .
the previous , almost exclusive experimental attention on amyloid has shifted over the last 10 years in two directions .
firstly , from the concrete deposits of amyloid plaques to less well - defined soluble or pseudosoluble oligomers of the amyloid peptides , ranging from dimers to dodecamers and even larger aggregates .
a second shift in research focus is from amyloid to tauopathy , and to their mutual relation .
the role of tau in the pathogenesis and disease progression is appreciated as leading to synaptic and neuronal loss , causing cognitive deficits and dementia .
both trends are incorporated in a modified amyloid cascade hypothesis , briefly discussed in this paper that is mainly concerned with the second aspect , that is , protein tau and its associated fundamental questions . |
bio2010 calls for faculty in biology , mathematics , and the physical and information sciences to work collaboratively to find ways of integrating mathematics , the physical sciences , and the life sciences in courses and to recognize that faculty development is a crucial component to doing so .
it also calls for increased opportunities for undergraduate research as early in a student 's career as possible ( national research council , 2003 ) . at university of richmond ( ur )
, we are discovering how undergraduate research can be a driving force in achieving interdisciplinary collaboration .
as we increase the number of students participating in undergraduate research , we have observed just how often elements of research projects find their way into the classroom , particularly research projects that have engaged students and/or faculty from different areas to work on interdisciplinary questions .
a prime case study at university of richmond is in the development of our new supercourse called integrated quantitative science ( iqs ) , which combines and integrates the concepts from the first course in each of mathematics , physics , computer science , biology , and chemistry .
the course development was funded by a science education grant from the howard hughes medical institute ( hhmi ) and is being taught for the first time during the 20092010 academic year . here ,
we describe four recent undergraduate research projects involving students and faculty in biology , chemistry , physics , mathematics , and computer science and how each contributed in significant ways to the conception and implementation of the iqs course .
ur is a private liberal arts institution of approximately 2800 undergraduate students that is focused on undergraduate research across campus . in summer 2009
, there were 150 students involved in undergraduate research in science and mathematics , 35% of whom were funded internally and 65% of whom were funded externally by various grants and awards ( e.g. , hhmi , merck - american association for the advancement of science , beckman , national institutes of health , national science foundation [ nsf ] , american chemical society - petroleum research fund ) .
richmond faculty values the one - on - one interactions and opportunities for teaching outside of a classroom that occur when mentoring undergraduate students in research projects .
these opportunities add a new dimension to the students ' understanding of the field , providing them with practical skills , opportunities to solve problems , motivation to refine and improve oral and written communication skills , and exposure to the joys and frustrations of scientific research .
as one student put it , doing research will turn you into a scientist faster than a class will . preparing our students to be twenty - first century scientists was also a motivating factor behind the conception and design of iqs .
iqs is a two - semester course for first - year students that integrates the material currently taught in the first course of the major in each of biology , chemistry , physics , mathematics , and computer science .
although it is called a course , it is really a new way of teaching the concepts normally covered in these five courses .
students enroll in the course for a full year , and it replaces two courses in their schedules in each of the fall and spring semesters .
the course meets for lecture 300 min per week ; it has a weekly 3-h laboratory component and a weekly 1-h workshop / laboratory .
the course substitutes for the first course in the major in each of the five disciplines involved . after completing both semesters of iqs , the students will complete a traditional major in at least one of the contributing disciplines .
these students will have the option to have an interdisciplinary minor in integrated quantitative science by completing additional upper - level courses that combine two of the disciplines and by completing an undergraduate research project .
the course development was originally inspired by models from harvey mudd 's interdisciplinary lab ( national research council , 2003 ) and the integrated science program at princeton university ( lewis - sigler institute for integrative genomics , 2010 ) .
the themes were chosen because they related to the research interests of the faculty involved and because of their relevance to all the disciplines .
the theme for the first semester is antibiotic resistance , and the theme for the spring semester is signaling and cell cell communication .
the laboratory component of the first semester included creating an agent - based computer simulation model to study the evolution of antibiotic resistance in a macrolevel population , modeling of molecular dynamical behavior of antibiotics , and a semester - long experiment using a novel animal model system to study bacterial symbionts and the selection of antibiotic resistant species .
the antibiotic resistance study generated primary research data that allowed students to gain experience using a variety of molecular and bioinformatic techniques .
similar to iqs-1 , the laboratory associated with iqs-2 is a discovery - oriented experience composed of both experimental and computer simulation approaches .
students focus on five separate projects during the semester : 1 ) understanding signaling in the immune system , 2 ) spot detection / automated generation of protein profiles from two - dimensional gel electrophoresis , 3 ) brownian motion : experimental and simulation approaches , 4 ) kinetics of drug binding to wild - type and multidrug - resistant human immunodeficiency virus-1 protease , and 5 ) signal propagation and waves .
we are assessing the first year and making changes for its second offering in 20102011 .
more information about the iqs course as it becomes available can be found at http://iqscience.richmond.edu .
the course is team - taught by five faculty members , one from each of the disciplines involved .
the faculty teaching the first semester are different from the faculty teaching the second semester , so in all , 10 faculty are involved .
the development of the course was funded by hhmi and was done via a yearlong seminar in 20082009 , with the 10 faculty members involved .
participation in the seminar and developing the course constituted all of each faculty member 's teaching assignment for the second semester .
most of the faculty who participated in the seminar had previous experience directing , collaborating , consulting , or a combination on interdisciplinary research projects .
with funding from the nsf , including its innovative interdisciplinary training for undergraduates in biological and mathematical sciences ( ubm ) program and its enhancing the mathematical sciences workforce in the 21st century program and from the hhmi , these faculty had experienced first - hand the power of undergraduate research projects in motivating students to study techniques and methods outside of their majors .
four undergraduate research projects that fed the iqs course development are described below . the first research project described inspired the semester - long experiment used in the laboratory component of the first semester of iqs .
the conception of this experiment was a defining moment early in the iqs course development seminar that set the tone of integrating the discipline - specific material by applying it to solve a modern , real - life problem .
the experiment uses a novel animal system developed with undergraduates in the lab of the biology member of the first - semester team , dr .
the nsf - funded project is an interdisciplinary endeavor between an evolutionary ecologist , a biochemist , a microbiologist , and a developmental geneticist .
one aim of the grant is to develop marine sponges and their microflora as model systems to study host
symbiont associations . over the past few years , one student ( who was originally an hhmi - funded prefreshman in dr .
hill 's lab ) piloted methods to use sponge stem cell cultures to study sponge - associated bacterial symbionts . she found that changing the class of antibiotics used in the experiment led to shifts in the bacterial community , often enriching for different bacterial species .
several of the bacterial species that increased in representation after antibiotic treatment belonged to known marine symbiont families .
building on the data from this project , dr . hill and her research student developed a hypothesis that some of the novel antibiotic - resistant bacteria also might be producers of antimicrobial compounds themselves .
thus , they developed protocols for the iqs students to perform investigations on potential new species of antibiotic - resistant bacteria isolated from marine sponge cultures .
the iqs students used cell culture , dna isolation , polymerase chain reaction , and molecular cloning techniques as well as electron microscopy and antimicrobial activity assays to analyze antibiotic - selected marine sponge bacteria .
students used molecular modeling methods based on chemical and physical principles to study the structures and properties of antibiotics used for selection of resistant strains .
they used mathematical regression analyses to calculate mutation rates for selected bacteria , and they coded computer programs in java to compare dna sequences from newly isolated antibiotic - resistant bacteria .
moreover , this experiment led to further research projects for students in the iqs course .
hill and barry lawson , a computer science faculty member , continued portions of this interdisciplinary work in summer 2010 .
two of the students will pursue genes involved in host / symbiont integration , will further analyze the antibiotic - resistant bacterial species , and will isolate and characterize more species of bacteria from each of the antibiotic selected pools .
two other students will use bioinformatics and other computer science methods to perform analysis on sponge genome sequences as well as symbiont genomes .
these two students will also develop and modify bioinformatic software programs to enhance the analysis of the data .
the next example occurred during summer 2008 ( the summer before the iqs course development seminar began ) and contributed to the development of the first semester of the iqs course .
lester caudill , biomathematician , directed a team of four undergraduates ( three freshmen and one sophomore ) in a research project that integrated microbiology , epidemiology , and mathematics .
three of the students were funded by the nsf 's enhancing the mathematical sciences workforce in the 21st century program , through a grant entitled , long - term undergraduate research experiences ( lure ) , and the fourth student was funded by a grant from the hhmi .
this project was entitled models of antibiotic resistance and hospital - acquired infections and focused on the development and analysis of a mathematical model of bacterial infection dynamics within a hospital ward , including the rise and spread of antibiotic - resistant pathogens .
the overall goal was to use this model to test the potential effectiveness of several different strategies ( control strategies ) proposed in the medical literature for preventing and controlling the rise of antibiotic - resistant bacteria .
like the ubm project , because the students involved were first- and second - year students , dr .
caudill provided background instruction on key aspects of development and analysis of differential equation models , bacterial growth dynamics , and principles of epidemiology and pharmacokinetics , and training on several salient topics , including the following :
principles of mathematical modelingpotential benefits and limitationsbasics of model developmentthe biology and chemistry of antibiotic - resistant hospital - acquired infectionsprimer on bacterial infectionsmechanisms of antibiotic resistanceways to combat resistanceprinciples of antibiotic pharmacokineticsbuilding differential equation systems models via compartment diagramsanalysis of differential equation systems modelsanalytical solution formulasqualitative analysis of long - term behavior via eigen - analysisnumerical analysis with mathematica principles of mathematical modeling potential benefits and limitations basics of model development the biology and chemistry of antibiotic - resistant hospital - acquired infections primer on bacterial infections mechanisms of antibiotic resistance ways to combat resistance principles of antibiotic pharmacokinetics building differential equation systems models via compartment diagrams analysis of differential equation systems models analytical solution formulas qualitative analysis of long - term behavior via eigen - analysis numerical analysis with mathematica simultaneously , the students conducted a search of the medical research literature to produce a list of proposed control strategies .
the students then read and analyzed a research paper from the proceedings of the national academy of sciences .
this paper ( lipsitch et al . , 2000 ) provided a differential equation system model that served as the basis for the undergraduate team 's original work .
this phase began with the student team working through the mathematical analysis conducted by the authors .
then , after some additional analysis , each student selected , for incorporation into this model , one of the control strategies resulting from their literature search .
the four control strategies chosen by the students were antibiotic cycling , bacterial vaccinations , patient isolation practices , and control of bacteria in food .
the students , working individually , combined their own mathematical and biological knowledge , coupled with additional guidance and instruction from dr .
then , using a set of mathematical and computational tools ( that varied from one control strategy to another ) , each student analyzed her own control strategy model , and used the results to draw conclusions regarding the potential for each control strategy to impact the battle against antibiotic - resistant bacterial infections .
over the course of the project period , the students gained first - hand knowledge of one of the important ways that mathematical modeling can contribute to investigating important public health issues .
they also experienced the excitement of seeing their efforts , both individually and as a team , produce results , and the thrill of knowing that they were contributing new understanding and new perspectives on the problem of antibiotic - resistant infections .
the longer - term impact of this project , however , continues to be felt through its influence on the content of the current iqs course .
as summer 2008 unfolded , it became increasingly evident that mathematical modeling , meaningfully applied to a modern , real - life problem , could be learned and used by freshmen with prior exposure to calculus .
the summer experience helped us to identify the key topics from mathematics , biology , and chemistry that are necessary to set the proper scientific and technical context for this modeling topic .
moreover , it became clear that the field of antibiotic- resistance research is a rich area for connections not only with biology and mathematics but also with chemistry and physics ( through the study of antibiotics and their molecular activities ) , as well as computer science ( through simulation studies of infection dynamics ) . recognizing this potential , the designers of iqs selected antibiotic resistance as the theme for the first semester of this two - semester sequence .
this theme , in turn , gave rise to several interesting and relevant student activities , including the following :
a student - developed java program simulating infection spread in a hospital warda set of wet labs for bioprospecting in marine sponge tissue for new antimicrobial compounds active against pathogenic bacteriaa computational chemistry study of stable conformations of simple antibiotic molecules a student - developed java program simulating infection spread in a hospital ward a set of wet labs for bioprospecting in marine sponge tissue for new antimicrobial compounds active against pathogenic bacteria a computational chemistry study of stable conformations of simple antibiotic molecules the final two examples contributed to the signaling theme of the second semester of the iqs course .
laura runyen - janecky ( biology ) specially designed a hybrid wet ( biology ) and dry ( mathematics and physics ) laboratory .
the main laboratory focus is in experimental and theoretical studies of the heat - shock response in mammalian cells .
cells develop a family of proteins that become active after an environmental stress , such as a high rise in temperature .
these heat - shock proteins ( e.g. , hsp70 ) ensure the survival of the cell . the experimental data collected and analyzed by lipan et al .
( 2007 ) show that the mean value response of a cell population to one heat shock can be described by a mathematical expression that is valid for a large range of heat - shock conditions .
students working in this lab are trained to use mathematical models in the early stages of designing their experiments as well as to use basic molecular biology assays to generate experimental data .
they developed a nonlinear stochastic theoretical model that explains the empirical law for the mean response .
moreover , the theoretical model predicts a specific biological probability distribution of responses for a cell population .
they studied the response to one pulse of temperature t1 and duration d1 ; the next step was to find the response to more complex patterns of input functions .
the response to these complex input functions is the basis of an ongoing project funded by the nsf through the ubm program .
specifically , the first cohort of students participating in the ubm focused on measuring the dynamics of the heat - shock promoter hsp70 under the action of two consecutive pulses : one pulse of temperature t1 and duration d1 and the other of t2 and d2 .
this first cohort consisted of four students : two first - year students from biology , a first - year mathematics student , and a third - year mathematics / computer science student . because of their varied backgrounds , the spring semester before their first summer of data gathering and analyses was spent teaching all of the students what they needed to know for the project .
the mathematics students needed to learn wet lab techniques and the biology students had to become comfortable with the mathematical models .
on the biological side , they compared eukaryotic and prokaryotic biochemical strategies to cope with stress .
the students learned the effects of diverse transcription factors on the stress response and the feedback loops that regulate the stress system .
as a parallel activity to the chalkboard meetings , they learned mammalian cell culture and fluorescent activated flow cytometer techniques .
runyen - janecky to insert a construct in the chromosome of the bacteria escherichia coli .
the construct responds to heat shocks and is a basis of the comparative studies between mammalian and bacterial response to heat shocks . for the summer data collection phase
, they designed five different combinations of pairs of heat shocks that were delivered to mammalian cell cultures under a variety of temperature and duration parameters .
the measurements were taken at single - cell level ; by the end of summer , they had accumulated 14 million data points . during the fall data analysis phase
, the data were transferred from the fluorescent activated flow cytometer standard file to a text file and subsequently loaded into matlab ( the mathworks , www.mathworks.com ) .
the analysis produced a list of response parameters available for use in developing mathematical models .
as the iqs course was developed , this experience contributed to our understanding of how a research question amenable to multiple discipline perspectives can motivate both lab - adverse and math - phobic students .
it also helped to gauge the appropriateness of the level of topics to a course for first - year students .
lipan is now teaching the mathematical modeling of cell signaling pathway in the second semester of the iqs course .
this switch appears in many genetic networks , and it is believed to switch on and off the main pathway of the stress response in mammalian cells ( rieger et al . , 2005 ) through a series of phosphorylations .
the ubm group of four students , two with no mathematics background beyond calculus , studied this switch in detail , giving us some indication of how to teach it at a level appropriate for a class of 20 first - year students .
the biology students were skeptical of the power of theoretical thinking , because their previous experience was tightly connected with truth discovered only through experimental techniques .
at the end of the data analysis and modeling period , after they went back and forth many times between experimental facts and theoretical predictions , the biology students understood better the power of the method .
the mathematics students had difficulties with the fluid way of thinking in constructing the theoretical models .
constructing a model based on observations and revising it based on the results of experimentation is not as simple and well organized as analyzing a model in a mathematics classroom .
the frustrations of both the mathematics and biology students better prepared us to accommodate the many different science and mathematics backgrounds of the students in the iqs course .
the ubm faculty found during the first year that it is very effective to have a mature ( junior or senior ) mathematically oriented person in the group who could grasp the mathematical problem faster and help the rest of the group to understand it . based on this experience
, we incorporated several upper - level mathematics and computer science students into the iqs course as teaching assistants .
their duties ranged from trying some of the problems out before they were given to the class to holding problem sessions for some of the assignments . whereas the previous project described contributed to the choice of signaling as the unifying theme for the second semester of the iqs course , the final project we describe grew out of questions raised much later in the process of developing the second - semester topics for iqs .
what began as simply an idea for a project for a student with strong mathematical skills and an interest in applications subsequently made a significant contribution to the course .
this summer project was funded by richmond 's science , technology , engineering and mathematics ( stem ) summer undergraduate research ( ssur ) program , an undergraduate research program funded as part of a science education grant from the hhmi that has as one of its objectives to promote interdisciplinary interactions and collaborations .
the project focused on the connection between diffusion and functionality of the g protein - coupled receptors ( gpcrs ) .
for the cell - signaling theme in the second semester , the iqs designers decided that gpcrs are an important class of receptors and needed to be taught . on a different note ,
brownian motion and diffusion were on the list of essential topics to be included in order to satisfy discipline - specific requirements .
fortunately , it turned out that these phenomena are connected in a nontrivial way : for rhodopsin , a gpcr receptor in retina , diffusion is an important phenomenon ( ramanathan et al . , 2005 ) .
diffusion master equation to the gpcr switch in retinal rods and found the effector protein response to different input signals on the light - activated receptor .
the response of the gpcr switch depends essentially on both reaction and diffusion and displays the interesting phenomenon of stochastic resonance .
the student who worked on this summer project was a highly motivated first - year student , well trained in mathematical approaches to science . however , probabilistic thinking and differential equations were new for him , and only at the end of the 10 wk of research was he comfortable with the stochastic approach .
this experience helped us to design lectures that are understandable by the iqs first - year students . to aid in understanding , we represented the computed results with suggestive contour plots , and we created powerpoint animation of the reaction diffusion mathematical model for the gpcr pathway .
we did not require them to solve these equations ; rather we required them to translate from the qualitative biological description of the pathway to a quantitative model .
the creation of the quantitative model using differential equations presented no problem for the iqs students .
they later studied the solution to the model with the help of the mathematica software tool .
table 1 relates the main ideas from the original ssur project to the second semester of iqs .
relationship between ssur project and iqs course the impact of novel interdisciplinary undergraduate research programs such as those funded by nsf and hhmi extends beyond the gains of the students involved to major contributions to the kind of faculty development that leads to significant curriculum changes connecting the life sciences with mathematics , computer science , and the physical sciences .
it is too early to see the full impact of our experiment integrating the first course of the major of five stem disciplines at university of richmond , but it is clear that externally funded interdisciplinary research projects were key in our ability to conceive the integration and in laying the groundwork for the significant faculty collaboration required to implement the idea .
the first research project described inspired the semester - long experiment used in the laboratory component of the first semester of iqs .
the conception of this experiment was a defining moment early in the iqs course development seminar that set the tone of integrating the discipline - specific material by applying it to solve a modern , real - life problem .
the experiment uses a novel animal system developed with undergraduates in the lab of the biology member of the first - semester team , dr .
the nsf - funded project is an interdisciplinary endeavor between an evolutionary ecologist , a biochemist , a microbiologist , and a developmental geneticist .
one aim of the grant is to develop marine sponges and their microflora as model systems to study host
symbiont associations . over the past few years , one student ( who was originally an hhmi - funded prefreshman in dr .
hill 's lab ) piloted methods to use sponge stem cell cultures to study sponge - associated bacterial symbionts . she found that changing the class of antibiotics used in the experiment led to shifts in the bacterial community , often enriching for different bacterial species .
several of the bacterial species that increased in representation after antibiotic treatment belonged to known marine symbiont families .
building on the data from this project , dr . hill and her research student developed a hypothesis that some of the novel antibiotic - resistant bacteria also might be producers of antimicrobial compounds themselves .
thus , they developed protocols for the iqs students to perform investigations on potential new species of antibiotic - resistant bacteria isolated from marine sponge cultures .
the iqs students used cell culture , dna isolation , polymerase chain reaction , and molecular cloning techniques as well as electron microscopy and antimicrobial activity assays to analyze antibiotic - selected marine sponge bacteria .
students used molecular modeling methods based on chemical and physical principles to study the structures and properties of antibiotics used for selection of resistant strains .
they used mathematical regression analyses to calculate mutation rates for selected bacteria , and they coded computer programs in java to compare dna sequences from newly isolated antibiotic - resistant bacteria .
moreover , this experiment led to further research projects for students in the iqs course .
hill and barry lawson , a computer science faculty member , continued portions of this interdisciplinary work in summer 2010 .
two of the students will pursue genes involved in host / symbiont integration , will further analyze the antibiotic - resistant bacterial species , and will isolate and characterize more species of bacteria from each of the antibiotic selected pools .
two other students will use bioinformatics and other computer science methods to perform analysis on sponge genome sequences as well as symbiont genomes .
these two students will also develop and modify bioinformatic software programs to enhance the analysis of the data .
the next example occurred during summer 2008 ( the summer before the iqs course development seminar began ) and contributed to the development of the first semester of the iqs course .
lester caudill , biomathematician , directed a team of four undergraduates ( three freshmen and one sophomore ) in a research project that integrated microbiology , epidemiology , and mathematics .
three of the students were funded by the nsf 's enhancing the mathematical sciences workforce in the 21st century program , through a grant entitled , long - term undergraduate research experiences ( lure ) , and the fourth student was funded by a grant from the hhmi .
models of antibiotic resistance and hospital - acquired infections and focused on the development and analysis of a mathematical model of bacterial infection dynamics within a hospital ward , including the rise and spread of antibiotic - resistant pathogens .
the overall goal was to use this model to test the potential effectiveness of several different strategies ( control strategies ) proposed in the medical literature for preventing and controlling the rise of antibiotic - resistant bacteria .
like the ubm project , because the students involved were first- and second - year students , dr .
caudill provided background instruction on key aspects of development and analysis of differential equation models , bacterial growth dynamics , and principles of epidemiology and pharmacokinetics , and training on several salient topics , including the following :
principles of mathematical modelingpotential benefits and limitationsbasics of model developmentthe biology and chemistry of antibiotic - resistant hospital - acquired infectionsprimer on bacterial infectionsmechanisms of antibiotic resistanceways to combat resistanceprinciples of antibiotic pharmacokineticsbuilding differential equation systems models via compartment diagramsanalysis of differential equation systems modelsanalytical solution formulasqualitative analysis of long - term behavior via eigen - analysisnumerical analysis with mathematica principles of mathematical modeling potential benefits and limitations basics of model development the biology and chemistry of antibiotic - resistant hospital - acquired infections primer on bacterial infections mechanisms of antibiotic resistance ways to combat resistance principles of antibiotic pharmacokinetics building differential equation systems models via compartment diagrams analysis of differential equation systems models analytical solution formulas qualitative analysis of long - term behavior via eigen - analysis numerical analysis with mathematica simultaneously , the students conducted a search of the medical research literature to produce a list of proposed control strategies .
the students then read and analyzed a research paper from the proceedings of the national academy of sciences .
this paper ( lipsitch et al . , 2000 ) provided a differential equation system model that served as the basis for the undergraduate team 's original work .
this phase began with the student team working through the mathematical analysis conducted by the authors .
then , after some additional analysis , each student selected , for incorporation into this model , one of the control strategies resulting from their literature search .
the four control strategies chosen by the students were antibiotic cycling , bacterial vaccinations , patient isolation practices , and control of bacteria in food .
the students , working individually , combined their own mathematical and biological knowledge , coupled with additional guidance and instruction from dr .
( 2000 ) to allow for the modeling of their chosen control . then , using a set of mathematical and computational tools ( that varied from one control strategy to another ) , each student analyzed her own control strategy model , and used the results to draw conclusions regarding the potential for each control strategy to impact the battle against antibiotic - resistant bacterial infections .
over the course of the project period , the students gained first - hand knowledge of one of the important ways that mathematical modeling can contribute to investigating important public health issues .
they also experienced the excitement of seeing their efforts , both individually and as a team , produce results , and the thrill of knowing that they were contributing new understanding and new perspectives on the problem of antibiotic - resistant infections .
the longer - term impact of this project , however , continues to be felt through its influence on the content of the current iqs course .
as summer 2008 unfolded , it became increasingly evident that mathematical modeling , meaningfully applied to a modern , real - life problem , could be learned and used by freshmen with prior exposure to calculus .
the summer experience helped us to identify the key topics from mathematics , biology , and chemistry that are necessary to set the proper scientific and technical context for this modeling topic .
moreover , it became clear that the field of antibiotic- resistance research is a rich area for connections not only with biology and mathematics but also with chemistry and physics ( through the study of antibiotics and their molecular activities ) , as well as computer science ( through simulation studies of infection dynamics ) . recognizing this potential , the designers of iqs selected antibiotic resistance as the theme for the first semester of this two - semester sequence .
this theme , in turn , gave rise to several interesting and relevant student activities , including the following :
a student - developed java program simulating infection spread in a hospital warda set of wet labs for bioprospecting in marine sponge tissue for new antimicrobial compounds active against pathogenic bacteriaa computational chemistry study of stable conformations of simple antibiotic molecules a student - developed java program simulating infection spread in a hospital ward a set of wet labs for bioprospecting in marine sponge tissue for new antimicrobial compounds active against pathogenic bacteria a computational chemistry study of stable conformations of simple antibiotic molecules
the final two examples contributed to the signaling theme of the second semester of the iqs course .
laura runyen - janecky ( biology ) specially designed a hybrid wet ( biology ) and dry ( mathematics and physics ) laboratory .
the main laboratory focus is in experimental and theoretical studies of the heat - shock response in mammalian cells .
cells develop a family of proteins that become active after an environmental stress , such as a high rise in temperature .
these heat - shock proteins ( e.g. , hsp70 ) ensure the survival of the cell . the experimental data collected and analyzed by lipan et al .
( 2007 ) show that the mean value response of a cell population to one heat shock can be described by a mathematical expression that is valid for a large range of heat - shock conditions .
students working in this lab are trained to use mathematical models in the early stages of designing their experiments as well as to use basic molecular biology assays to generate experimental data .
they developed a nonlinear stochastic theoretical model that explains the empirical law for the mean response .
moreover , the theoretical model predicts a specific biological probability distribution of responses for a cell population .
they studied the response to one pulse of temperature t1 and duration d1 ; the next step was to find the response to more complex patterns of input functions .
the response to these complex input functions is the basis of an ongoing project funded by the nsf through the ubm program .
specifically , the first cohort of students participating in the ubm focused on measuring the dynamics of the heat - shock promoter hsp70 under the action of two consecutive pulses : one pulse of temperature t1 and duration d1 and the other of t2 and d2 .
this first cohort consisted of four students : two first - year students from biology , a first - year mathematics student , and a third - year mathematics / computer science student . because of their varied backgrounds , the spring semester before their first summer of data gathering and analyses was spent teaching all of the students what they needed to know for the project .
the mathematics students needed to learn wet lab techniques and the biology students had to become comfortable with the mathematical models .
on the biological side , they compared eukaryotic and prokaryotic biochemical strategies to cope with stress .
the students learned the effects of diverse transcription factors on the stress response and the feedback loops that regulate the stress system .
as a parallel activity to the chalkboard meetings , they learned mammalian cell culture and fluorescent activated flow cytometer techniques .
runyen - janecky to insert a construct in the chromosome of the bacteria escherichia coli .
the construct responds to heat shocks and is a basis of the comparative studies between mammalian and bacterial response to heat shocks . for the summer data collection phase
, they designed five different combinations of pairs of heat shocks that were delivered to mammalian cell cultures under a variety of temperature and duration parameters .
the measurements were taken at single - cell level ; by the end of summer , they had accumulated 14 million data points . during the fall data analysis phase ,
the data were transferred from the fluorescent activated flow cytometer standard file to a text file and subsequently loaded into matlab ( the mathworks , www.mathworks.com ) .
the analysis produced a list of response parameters available for use in developing mathematical models .
as the iqs course was developed , this experience contributed to our understanding of how a research question amenable to multiple discipline perspectives can motivate both lab - adverse and math - phobic students .
it also helped to gauge the appropriateness of the level of topics to a course for first - year students .
lipan is now teaching the mathematical modeling of cell signaling pathway in the second semester of the iqs course .
this switch appears in many genetic networks , and it is believed to switch on and off the main pathway of the stress response in mammalian cells ( rieger et al . , 2005 ) through a series of phosphorylations .
the ubm group of four students , two with no mathematics background beyond calculus , studied this switch in detail , giving us some indication of how to teach it at a level appropriate for a class of 20 first - year students .
the biology students were skeptical of the power of theoretical thinking , because their previous experience was tightly connected with truth discovered only through experimental techniques . at the end of the data analysis and modeling period , after they went back and forth many times between experimental facts and theoretical predictions , the biology students understood better the power of the method .
the mathematics students had difficulties with the fluid way of thinking in constructing the theoretical models .
constructing a model based on observations and revising it based on the results of experimentation is not as simple and well organized as analyzing a model in a mathematics classroom .
the frustrations of both the mathematics and biology students better prepared us to accommodate the many different science and mathematics backgrounds of the students in the iqs course .
the ubm faculty found during the first year that it is very effective to have a mature ( junior or senior ) mathematically oriented person in the group who could grasp the mathematical problem faster and help the rest of the group to understand it . based on this experience
, we incorporated several upper - level mathematics and computer science students into the iqs course as teaching assistants .
their duties ranged from trying some of the problems out before they were given to the class to holding problem sessions for some of the assignments .
whereas the previous project described contributed to the choice of signaling as the unifying theme for the second semester of the iqs course , the final project we describe grew out of questions raised much later in the process of developing the second - semester topics for iqs .
what began as simply an idea for a project for a student with strong mathematical skills and an interest in applications subsequently made a significant contribution to the course .
this summer project was funded by richmond 's science , technology , engineering and mathematics ( stem ) summer undergraduate research ( ssur ) program , an undergraduate research program funded as part of a science education grant from the hhmi that has as one of its objectives to promote interdisciplinary interactions and collaborations .
the project focused on the connection between diffusion and functionality of the g protein - coupled receptors ( gpcrs ) .
for the cell - signaling theme in the second semester , the iqs designers decided that gpcrs are an important class of receptors and needed to be taught . on a different note ,
brownian motion and diffusion were on the list of essential topics to be included in order to satisfy discipline - specific requirements .
fortunately , it turned out that these phenomena are connected in a nontrivial way : for rhodopsin , a gpcr receptor in retina , diffusion is an important phenomenon ( ramanathan et al . , 2005 ) .
they applied the reaction diffusion master equation to the gpcr switch in retinal rods and found the effector protein response to different input signals on the light - activated receptor .
the response of the gpcr switch depends essentially on both reaction and diffusion and displays the interesting phenomenon of stochastic resonance .
the student who worked on this summer project was a highly motivated first - year student , well trained in mathematical approaches to science . however , probabilistic thinking and differential equations were new for him , and only at the end of the 10 wk of research was he comfortable with the stochastic approach .
this experience helped us to design lectures that are understandable by the iqs first - year students . to aid in understanding , we represented the computed results with suggestive contour plots , and we created powerpoint animation of the reaction diffusion mathematical model for the gpcr pathway .
we did not require them to solve these equations ; rather we required them to translate from the qualitative biological description of the pathway to a quantitative model .
the creation of the quantitative model using differential equations presented no problem for the iqs students .
they later studied the solution to the model with the help of the mathematica software tool .
table 1 relates the main ideas from the original ssur project to the second semester of iqs .
relationship between ssur project and iqs course the impact of novel interdisciplinary undergraduate research programs such as those funded by nsf and hhmi extends beyond the gains of the students involved to major contributions to the kind of faculty development that leads to significant curriculum changes connecting the life sciences with mathematics , computer science , and the physical sciences .
it is too early to see the full impact of our experiment integrating the first course of the major of five stem disciplines at university of richmond , but it is clear that externally funded interdisciplinary research projects were key in our ability to conceive the integration and in laying the groundwork for the significant faculty collaboration required to implement the idea .
| funded by innovative programs at the national science foundation and the howard hughes medical institute , university of richmond faculty in biology , chemistry , mathematics , physics , and computer science teamed up to offer first- and second - year students the opportunity to contribute to vibrant , interdisciplinary research projects .
the result was not only good science but also good science that motivated and informed course development . here , we describe four recent undergraduate research projects involving students and faculty in biology , physics , mathematics , and computer science and how each contributed in significant ways to the conception and implementation of our new integrated quantitative science course , a course for first - year students that integrates the material in the first course of the major in each of biology , chemistry , mathematics , computer science , and physics . |
in a recent genome - wide association study of generalized vitiligo ( gv ) we identified 10 confirmed susceptibility loci . by testing additional loci that showed suggestive association in the genome - wide study , using two replication cohorts of european descent ,
we observed replicated association of gv with variants at 3p13 encompassing foxp1 ( rs17008723 , combined p = 1.04 10 - 8 ) and with variants at 6q27 encompassing ccr6 ( rs6902119 , combined p = 3.94 10 - 7 ) . | |
the osteomalacia is a metabolic bone disease due to the mineralization disturbance of the organic matrix among the adults and its representing causes include the insufficient vitamin d or phosphate or metabolic disorder . among the causes , osteomalacia
due to hypophosphatemia induced by the impaired absorption in the gastrointestinal tract , impaired re - absorption in the renal tubules , tumor and genetic diseases .
the adefovir is used as the therapeutic agent for the wild - type hepatitis b virus ( hbv ) and lamivudine - resistant hbv in the chronic hepatitis b patients . when adefovir is used with high dose for the long time ( 60 - 120 mg once daily ) to treat the human immunodeficiency virus ( hiv )
, it may cause the nephrotoxicity due to the damages of the proximal tubule with hypophosphatemia , hypouricemia , proteinuria and glycosuria .
recently , several papers report the renal diseases in the renal tubules by using the adefovir with the low does ( 10 mg once daily ) and the following hypophosphatemic osteomalacia and the author experiences 2 cases . here
patient : male with the age of 43 chief complain : backache and pain on both knees current history : the patient was diagnosed as the chronic hepatitis b 20 years ago before he came to the hospital , had taken the lamivudine for 13 years .
then , the resistance appeared and has taken 10 mg of adefovir for 5 years .
the backache has appeared for 6 years with the pain on both knees and myalgia and came to the hospital due to abnormal features in gait .
past medical history and family history : no specific observation . physical examination : the vital sign was normal when the patient came to the hospital , the bone tenderness was found on the spine and both knees and the waddling gate appeared while walking .
lab finding : the blood examination shows 8,990/mm of the leukocyte , 15.3 g / dl of the hemoglobin , 429,000/mm3 of the platelet , 2.425 mmol / l of the serum calcium ( normal : 2.05 - 2.55
mmol / l ) , 0.51 mmol / l of the inorganic phosphorus ( normal : 0.74 - 1.52
mmol / l ) , 3.93 mmol / l of the blood urea nitrogen ( bun ; normal : 2.9 - 8.2 mmol / l ) , 114.92 mol / l of the serum creatinine ( normal : 53 - 106
mol / l ) , 21 iu / l of the aspartate transaminase ( ast ; normal : 10 - 30
iu / l ) , 29 iu / l of the alanine transaminase ( alt ; normal : 10 - 40
iu / l ) and 1,218 u / l of the alkaline phosphatase ( alp ; normal : 30 - 120 u / l ) . during the urine examination , 1,000 mg / dl ( 3 + ) of the glycosuria and 0.6 g / day of proteinuria were observed .
pmol / l ( normal : 60 - 108 pmol / l ) but the parathyroid hormone ( pth ) was in the normal range of 42.95 pg / ml ( normal : 10 - 65 pg / ml ) .
radiographic finding : bone density was decreased on the spine and pelvis on simple x - ray .
1 ) . in the bone mineral density ( bmd ) , the t - score of the spine marked -5.1 ( average value of l1-l4 ) and the osteoporosis was observed ( fig .
2a ) and the strong multiple hot uptakes were found in the bone scan ( fig .
the computed tomography ( ct ) test which was performed to find out the primary lesion considering the possibility for the carcinogenic bone metastasis observed the fracture on the 7th spinous process ( fig .
bone biopsy : the bone biopsy was performed on the 7th spinous process with the observation from the ct and a lot of osteoid tissues without mineralization including a lot of osteoblasts were found ( fig .
: it was diagnosed that the osteroporosis appeared due to the hypophosphatemia based on the hypophosphatemia , alp increase , images and biopsy and estimated that it was caused by the adefovir use .
the alendronate ( 70 mg , once a week ) , the bisphosphonate medicine was administered as the treatment , the patient was instructed to take food with a lot of phosphate , to stop using the adefovir and to use the entecavir . since then
, the myalgia was mitigated and the spinal t - score was improved to -1.8 based on the bmd .
patient : male with the age of 42 chief complain : backache and pain on both chests current history : the patient was diagnosed as the chronic hepatitis b 11 years ago before he came to the hospital , had taken the lamivudine .
then , the resistance appeared and has taken 10 mg of adefovir for 7 years .
then the patient came to the hospital due to the backache and pain on both chests suffered from 3 months before he came to the hospital .
physical examination : the vital signs were normal when the patient came to the hospital .
lab finding : the blood examination shows 6,760/mm of the leukocyte , 14.9 g / dl of the hemoglobin , 272,000/mm of the platelet , 2.40 mmol / l of the serum calcium , 0.52 mmol / l of the inorganic phosphorus , 7.5 mmol / l of the bun , 141.4 mol / l of the serum creatinine , 18 iu / l of the ast , 21 iu / l of the alt and 702 u / l of the alp . during the urine examination , 300 mg / dl ( 2 + ) of the glycosuria and 1.1 g / day of the proteinuria were observed .
however , hematuria was not found correlated . the serum 1,25(oh)2d3 recorded 80.0 pmol / l but the pth was in the normal range of 21.1 pg / ml .
radiographic finding : in the bmd , the t - score of the spine marked -2.1 ( average value of l1-l4 ) and the osteoporosis was observed ( fig .
2b ) and the strong multiple hot uptakes were found in the bone scan ( fig .
treatment and progress : as shown in the case 1 , the patient in the case 2 was instructed to stop using the adefovir under the diagnosis of the hypophosphatemic osteomalacia due to hypophosphatemia , alp increase and images , changed to use the entecarvir and the bisphosphonate medicine ( alendronate ) was administered with the among of 70 mg ( once a week ) .
currently , the pain on both chests was improved and the spinal t - score was improved to 0.75 based on the bmd .
the adefovir inhibits the reverse transcriptase and the dna synthetase to prohibit the hbv proliferation .
that is why the adefovir is used for the initial treatment of the chronic hepatitis b or the tolerance against the lamivudine .
the nephrotoxicity appears due to the damages of the proximal tubule when the adefovir is administered with high doses for a long time .
adefovir inhibits the mitochondria dna growth in the epithelial cell of the proximal tubule and prohibits the oxdative phosphorylation , causing the cell necrosis . and
then , absorption of phosphate in the proximal tubule is impaired and lead to hypophosphatemic osteomalacia .
nephrotoxicity of the adefovir was reported depending on the quantity for the high dose over 60 mg to mainly treat the hiv .
reported that comparision with 10 mg and 30 mg of adefovir to treat chronic hepatitis b. the group with 30 mg shows increase the serum creatinine , decrease in the serum phosphate and high portion of nephrotoxicity with proteinuria and glycosuria .
however , the group with 10 mg of the adefovir shows similar result to the control group .
they suggest that the use of the adefovir with the low dose of 10 mg is safe against the nephrotoxicity to treat the chronic hepatitis b. however , local and foreign studies find out that not only the nephrotoxicity but also the osteomalacia appear even after using a small dose of the adefovir of 10 mg .
the osteomalacia is due to the inhibition in the mineralization of newly formed osteoids by the deficiency in the vitamin d or phosphate or metabolic disorder , causes pain on the bone and the muscle weakness .
it may be diagnosed by biochemical examinations including serum calcium , phosphorous , alp , pth , bun and creatinine , image examinations like simple radiography and bone scan and osseous tissue examination .
the osteomalacia due to insufficient vitamin d shows the decrease in the serum calcium , phosphorous and 1,25(oh)2d and increase in the alp and pth .
however , the hypophosphatemic osteomalacia due to damaged proximal tubule may show decrease in the serum phosphorous , alp increase and normal serum calcium , pth and 1,25(oh)2d . in the radiogrphic aspect , the decrease in the bone shadows and pseudofracture
may be found from the simple radiographic image and the hot uptake may be found in the bone scan .
the bmd is not required in the diagnosis but most osteomalacia patients show decrease in the bone density and the osteomalacia is questioned from the osseous tissue examination .
it may be rarely performed if the diagnosis is difficult from the noninvasive examination with the features of increasing the sutured gap between the osteoids , time delay to the mineralization and increase in the osteoid tissues .
the patient in the case 1 of the study has symptoms of bone pain and body weakness shows the opinions of the normal serum calcium and decrease in the serum phosphate from the hematological examination , alp increase and normal pth and the observation of decreasing the bone density and suspicious fracture due to the cracked cortical bone was found in the simple x - ray .
also , the multiple hot uptake was found in the bone scan and a lot of osteoid tissues without the mineralization including the osteoblast in the osseous tissue examination , being diagnosed as the osteomalacia accompanied with the hypophosphatemia .
no family history of the genetic diseases is found and no evidence of the tumors are discovered from the ct . also , there is no intake history of special medicines other than the adefovir and it is diagnosed as the hypophosphatemic osteomalacia due to using the adefovir for a long period of time . similar to the case 2 of the study , the patient shows decrease in the serum phosphate , alp increase and normal pth , as well as the multiple hot uptake from the bone scan and is diagnosed as the hypophosphatemic osteomalacia due to using the adefovir .
the medicine of cause is suspended to treat the hypophosphatemic osteomalacia and it is effective to inject the vitamin d and phosphorous .
two third of the daily recommended dose of the phosphorous for a normal person a day is absorbed to the intestines and most of the phosphate filtered in the proximal tubule is absorbed again .
the oral intake of the phosphorous is effective in treating the hypophosphatemic osteomalacia due to degrading re - absorption of the phosphate from the kidneys by assisting the deposition of the inorganic materials from the bones .
the case also shows the improvement by suspending the use of the adefovir and injecting the phosphate .
about 18 cases of the hypophosphatemic osteomalacia due to using the adefovir with low dose have been reported since 2008 and the study collects the characteristics and various examination results ( table 1 ) .
it is found out that 6 cases in korea , 6 cases in china , 3 cases in japan and 1 cases in france , australia and italy each , showing that most cases are reported in east asian countries .
the gender ratio of male and female patients shows the male domination of 15:3 and the average age is 52.9 ( 22 - 74 ) years old .
half of the patients accompany the liver cirrhosis and its main symptoms include the muscle weakness and bone pain .
the median value of using the adefovir until the symptom appears is 33.7 months ( 8 - 68 months ) and the median value of using the adefovir until the hypophosphatemia appears is 38.3 months ( 6 - 72 months ) .
the phosphate level in the blood is lower than the normal range in most cases , 0.3 - 0.77 mmol / l and most cases with the low serum phosphorous level , glycosuria and proteinuria show that the impaired absorption in the proximal tubule .
after adefovir is discontinued in most cases and phosphate is supplied , the clinical symptoms and hypophosphatemia were improved .
the adefovir is the nucleotide analogue and excreted by the active transportation through the translocator from the renal tubule , as well as the glomerular filtration .
the adefovir causes the nephrotoxicity due to the cell necrosis by inhibiting the mytocondria dna synthesis and the nephrotoxicity increases more due to improper excretion by directly affeting the translocator .
a total of 13 patients stop using the adefovir as the treatment and 6 of them change the medicine to the entecavir . it mainly performs the glomerular excretion with the nucleoside analogue and there are a lot of reports that it shows less nephrotoxicity compared to the adefovir . the 2 cases in the study change to the entecavir and the symptoms are improved .
it is required to periodically measure the serum phosphorous and renal function considering the possibility for the hypophosphatemic osteomalacia due to damaged proximal tubule in the patients who use the adefovir due to the chronic hepatitis b. as well , it is important to conduct the health questionnaire to the patients with the bone pains and muscle weakness on the history of drugs . | adefovir dipivoxil ( adv ) is a nucleotide used as long - term therapy of chronic hepatitis b. many published reports have shown that long - term high - dose therapy with adefovir can be associated with proximal renal tubular dysfunction resulting in significant hypophosphatemia , renal insufficiency and osteomalacia .
we have encountered two patients who developed evidence of hypophosphatemic osteomalacia while on long - term low - dose adefovir therapy for chronic hepatitis b. we report on its clinical features and its potential resolution with cessation of the drug and supplementation with phosphate .
we also reviewed the other published cases associated with hypophosphatemic osteomalacia after low - dose adefovir therapy .
the symptoms and the hypophosphatemia improved after cessation of the drug and supplementation with phosphate in most cases .
patients taking adefovir long - term should receive regular investigation of the phosphate level and renal function . |
sickle cell disease ( scd ) is a form of haemoglobinopathy caused by the abnormal genetic substitution of glutamine by valine in the haem portion of the haemoglobin molecule .
sickle haemoglobin ( hbs ) tends to form polymers and aggregates when deoxygenated and can lead to vasoocclusion and organ ischaemia ( 1 ) .
cardiopulmonary bypass ( cpb ) is commonly associated with hypothermia , hypoxia , hypoperfusion , and acidosis , which are pre - disposing factors that can trigger a profound sickle cell crisis .
when such patients undergo cardiac surgery with cpb , they need special precautions and management to prevent fatal vaso - occlusive episodes .
there are no guidelines for management of sickle cell patients during cardiac surgery , but only case reports are mentioned in the literature .
an 11-year - old child ( weight - 23 kg , height - 122 cm , body surface area - 0.89 m ) presented with a history of shortness of breath while playing , jaundice , and joint pain .
she was a known case of sickle cell anaemia with a history suggestive of sickling crisis in the past , which was managed by blood transfusion .
patient was started on hydroxyurea 250 mg / day ( oral ) . on admission ,
haemoglobin assessment by electrophoresis revealed hbs of 70.80% and adult haemoglobin ( hba ) of 30.60% .
one unit whole blood was transfused and before induction , hb electrophoresis values were obtained , which showed hbs 30.3% , foetal haemoglobin ( hbf ) 12.1% , and hba 49.9% .
cardiopulmonary bypass circuit was primed with 1300 ml of ringers solution , 3 units of fresh donor red cells ( collected within 24 h ) , 1 unit fresh frozen plasma , 60 ml albumin 20% , 100 ml mannitol and 50 ml 7.5% sodium bicarbonate .
after the circuit was primed , the fluid was circulated through oxygenator to achieve adequate oxygenation and warming of blood .
analysis of prime fluid showed pao2 of 310 mm hg , haematocrit of 24% and potassium , 3.0 mmol / dl .
after systemic heparinisation with 3 mg / kg of heparin , aortic and bicaval cannulation was performed . before the start of cpb , exchange transfusion was planned to reduce hbs level and raise hba level [ figure 1 ] .
as 500 ml autologous blood was drained , systolic arterial pressure dropped to 50 mm hg .
venous drainage was stopped temporarily , and prime fluid was infused until haemodynamics got stabilised .
the drainage was slowly restarted , and volume of 1400 ml of autologous blood was collected with simultaneous infusion of priming fluid .
after exchange transfusion , hb electrophoresis showed hbs 6.5% , hbf 3.2% , and hba 78.2% .
cpb was initiated with flows at 2.3 - 2.5 l / min at normothermia [ table 1 ] .
venous reservoir level was maintained by adding fresh blood to keep haematocrit level near 25% .
patient was weaned from bypass by inotropic support of adrenaline 0.02 g / kg / min . as the patient was off the bypass , remaining blood in the reservoir
post - cpb , patient was maintained normothermic using warm fluid , warm air mattress , and at room temperature .
after heparin reversal , hb was 8.4 g / dl and one unit fresh blood was transfused .
post - operative hb was 10.2 g / dl and hbs 10% [ table 2 ] .
cardiopulmonary bypass circuit showing arrangements for exchange transfusion laboratory parameters in the peri - operative period
sickle cell disease is an autosomal recessive condition that results from a single nucleotide polymorphism in the -globin gene . under the right conditions
haemoglobin ss molecules with this point mutation can polymerise , stiffening the erythrocyte membrane and distorting the cellular structure to the characteristic sickle shape .
certain circumstances such as hypoxia , hypothermia , acidosis or diminished blood flow can lead to aggregation , vascular occlusion and thrombosis .
chronically , scd can give rise to multiorgan damage secondary to haemolysis and vascular obstruction .
reduced life expectancy and a tendency for complications in sickle cell trait or disease can negatively affect the likelihood of survival after open heart surgery .
have reported heart valve surgery and surgery for congenital heart diseases performed safely in patients with scd or sickle cell trait with acceptable outcome and survival rates .
described a method of complete intraoperative exchange transfusion in the operating room that reduces the haemoglobin s ( hbs ) level to < 5% , without pre - operative exchange transfusions .
plasma and platelet fractions separated intra - operatively from the patient 's native red cell mass were used , in addition to hemoconcentration while on cpb , to effectively reduce the red cell and clotting factor transfusion requirements after the procedure .
suggested minimally invasive , warm , beating heart approach to try and minimise the risk of sickling due to cpb , low - flow states , cold cardioplegia and aortic cross - clamping .
maddali et al . have reported case of elective coronary artery bypass graft surgery in a scd patient using cpb techniques following pre - operative transfusions to increase the haemoglobin a levels to above 60% . in this case , we used pre - operative blood transfusion and hydroxyurea . pre - operative blood transfusion is indicated in scd patients to raise the haematocrit level and lower hbs levels .
hydroxyurea is the main treatment in symptomatic sickle cell patients because of its beneficial effects .
hydroxyurea increases hbf , which prevents sickling and also has beneficial effects on red blood cells hydration and vascular wall adhesion .
there is no consensus on safe values of hbs in patients undergoing surgery , but some reports suggest hbs should be decreased to < 30% for major surgeries or < 5% for cardiac surgeries prior or at the time of operation . in our case , even though hbs was 30.3% before surgery , we decided to do pre - operative exchange transfusion to promote perfusion , lower the incidence of peri - operative sickling crisis and perioperative multiorgan failure .
exchange transfusion is known to decrease hbs , increases pre - operative hba , haematocrit , and cellular oxygen delivery .
the classic pre - disposing factors for sickling include exposure to cold stress , dehydration , infections , hypoxia , inflammatory cascades , and acidosis .
hypothermia is prevented by employing normothermic cpb , use of warm air blankets and maintaining operating theatre temperature .
we avoided hypoxia by hyperoxygenating pump prime , maintaining haematocrit above 24% by using fresh donor blood ( collected within 24 h ) and keeping venous oxygenation saturation above 80% .
frequent monitoring of arterial blood gas is needed to maintain ph in normal range . in our case , warm induction of cardioplegia with oxygenated crystalloid solution
advantages of this technique are delivery of high pao2 to myocardium , improved distribution of cardioplegic solution due to decreased viscosity and flushing of hbs from the myocardium , thereby reducing the risk of localised anoxia .
use of continuous haemofiltration and modified ultrafiltration reduces the inflammatory response and improves lung compliance .
we did not use cell saver and autotransfusion was not done during or after the surgery .
cardiac surgery constitutes major stress , especially in paediatric patients , so it is strongly recommended to give the patients adequate analgesia and sedation .
thorough pre operative stabilisation of the patient with respect to hb and use of initial exchange transfusion , warm cardioplegia and maintenance of blood gases enable cpb to be conducted safely in sickle cell patients . | an 11-year - old child , a known case of sickle cell anaemia with a history suggestive of sickling crisis in the past was scheduled for surgical pulmonary valvotomy .
pre - operative blood transfusion and hydroxyurea were administered .
pre - operative blood transfusion is indicated in sickle cell disease patients to raise the haematocrit level and lower sickle haemoglobin ( hbs ) levels . before the start of cardiopulmonary bypass ( cpb ) ,
exchange transfusion was performed to reduce hbs level and raise adult haemoglobin level .
hypothermia was prevented by employing normothermic cpb . |
the principal physiological function of the prostate gland is the synthesis , accumulation and secretion of the anion citrate .
citrate may be used as an important energy source for spermatozoa , or involved as a buffer or chelator of cations in seminal fluid .
nadependent uptake of aspartate from plasma is achieved by two kinetically distinct na - dependent transport systems to create a high cytosolic aspartate concentration .
aspartate provides the intra - mitochondrial source of oxaloacetate while glucose provides the source of acetyl - coa for citrate biosynthesis .
ouabain - sensitive na , k - atpase - mediated transport is critical for aspartate uptake , citrate production and prostatic fluid formation since the inward nagradients generated by na , k - atpase are utilized for the nadependent uptake of aspartate .
naand kalso represent a large component of prostatic fluid osmolarity and their levels are finely regulated by plasma membrane transport systems that have yet to be identified .
furthermore , androgen activation of na , k - atpase serves as a metabolic pacemaker in the prostate exerting transcriptional control over the expression of na , k - atpase subunits , which play a critical role in the biogenesis of na , k - atpase in prostate cancer . in normal prostate ,
however , citrate production levels are significantly reduced as a result of altered cellular metabolism and bioenergetics .
na , k - atpase is an important regulator of intracellular electrolyte levels in almost all mammalian cells .
it is a mg - dependent p - type transport pump responsible for maintaining the low intracellular na : kratio that is essential for cell homeostasis and physiological function .
it catalyzes the active uptake of kand extrusion of naat the expense of hydrolyzing atp with a stoichiometry of 3nafor 2k .
the active form of na , k - atpase is an integral membrane protein complex primarily composed of two non - covalently attached subunits ; a 110-kda catalytic subunit and a 4555-kda glycosylated subunit .
the subunit has binding sites for na , k , atp and cardiac glycosides ( digitalis and ouabain ) .
four isoforms encoded by different genes have been identified which are ~85% identical at the protein level .
the subunit is a complex type ii glycoprotein with a short cytoplasmic nh2 terminus , a single transmembrane domain and a large globular cooh ectodomain containing three disulfide bridges and sites for n - linked glycosylation .
renal na , k - atpase consists of an additional small component known as the subunit .
the subunit is a member of the fxyd family of small ion transport regulators and is believed to be responsible for fine regulation of natransport in the nephron by modulating the transport function of renal na , k - atpase .
we have previously shown that human and rat prostatic epithelial cells express the 1 , 1 and 2 isoforms of na , k - atpase . despite the importance of na ,
k - atpase function for citrate production there is no information about its expression patterns in hyperplastic or neoplastic prostate .
the objective of this study was to determine the localization of na , k - atpase and to compare expression of na , k - atpase isoforms in normal canine prostate , benign prostatic hyperplasia ( bph ) and prostatic adenocarcinoma ( pca ) in order to determine whether reduced citrate levels in pca are also accompanied by changes in na , k - atpase expression .
1 , panel a ) glands are lined with a relatively continuous sheet of simple cuboidal to low columnar / pseudostratified epithelial cells which are separated by stromal connective tissue consisting of smooth muscle , blood vessels and neuroendocrine cells . normal glands exhibit large lumens without evidence of tufting or micropapillae .
the tissue in panel b exhibits all the classic morphological features of bph ; the tubuloalveoli vary in size and form with many of the individual glands appearing cystically dilated or collapsed .
panel c shows sheets of neoplastic epithelial cells that are morphologically typical of high - grade adenocarcinoma .
there is marked cellular and nuclear pleomorphism and numerous mitotic figures are observed particularly in the epithelial cells layer . enlarged hyperchromatic nuclei display marked anisokaryosis .
micro - glands exhibit ill - defined lumens significantly constricted by the geometric progression of proliferating cells .
histological appearance of normal ( a ) , bph ( b ) and pca canine prostate tissues incorporated in this study . in the normal prostate continuous sheets of epithelial cells ( ec ) are separated by connective tissue stroma ( s ) consisting of blood capillaries ( indicated by closed arrows ) , smooth muscle and neuroendocrine cells .
bph glands display smaller lumens ( l ) compared to normal tissue with evidence of tufting and micropapillae .
pca glands are lined with neoplastic cells exhibiting cellular and nuclear pleomorphisms and numerous mitotic figures ( open arrows ) .
the basal cells ( bc ) were irregular in contour but the basement membrane appears confluent .
expression of na , k - atpase 1 and 1 isoforms was observed in the lateral and basolateral plasma membrane domains of prostatic epithelial cells in normal and bph prostates ( fig .
na , k - atpase expression was significantly lower compared to normal and bph glands ( fig .
2 ) . the expression observed in high grade ( poorly differentiated ) adenocarcinomas was significantly lower than low grade ( well differentiated tumours ) ; the abundant basolateral immunostaining observed in normal and bph tissue was significantly reduced in high grade pca as determined by immunohistochemistry using a monoclonal pan antibody ( mab9a7 ) that has been shown to recognize the 1 isoform in addition to all other known isoforms ( fig .
identical results were obtained using the 5 monoclonal pan antibody ( results not shown ) .
image analysis confirmed the down - regulation of na , k - atpase ( fig .
immunohistochemical staining of na , k - atpase 1 and 1 subunits in canine normal , bph and pca tissues . sites of na , k - atpase immunoreactivity are stained red ( using fast - red tr / naphthol as - mx as precipitating substrate for the alkaline phosphatase conjugated secondary antibody ) . in normal and bph sections na , k - atpase immunostaining is clearly visible in the basolateral membrane domain of epithelial cells ( arrows ) . in three different pca specimens na , k - atpase expression levels appear to be significantly lower than in normal or bph tissues and na , k - atpase is diffusely spread within focal areas of the neoplasm .
nuclei were counterstained with haematoxylin . comparing the immunohistochemical localization of na , k - atpase " " subunits in normal , bph and pca tissues using a pan monoclonal antibody ( mab9a7 ) that recognizes all known isoforms .
the pan monoclonal antibody produces a staining pattern which is identical to the 1 specific antibodies providing further evidence for the predominance of 1 expression in the prostate and the downregulation of na , k - atpase in pca .
quantitative analysis of na , k - atpase immunoreactivity in normal , bph and pca canine prostate .
panel a ; results of image analysis experiments performed in triplicate comparing na , k - atpase expression levels in tissue sections immunoassayed with a polyclonal antibody to the 1 subunit of na , k - atpase under identical experimental conditions .
panels d and e ; plot profiles of na , k - atpase abundance in the bph and pca sections .
the 1 isoform was detected in abundance but there was no evidence of 2 or 3 isoform expression ( fig .
canine kidney was used as a positive control to confirm that the polyclonal antibodies raised against the rodent isoform recognize the canine isoform expressed in the nephron ( fig .
although the isoform was not detected in all nephron segments , it was found in low levels in basolateral membranes of proximal convoluted tubules and in very high levels in the medullary thick ascending limbs , distal convoluted tubules and connecting tubules .
panel a : absence of na , k - atpase 2 , 3 and isoform expression and comparison with 1 expression in canine prostate .
panel b : evidence to confirm that the c33 polyclonal raised against the rodent isoform recognizes the canine isoform protein in the canine kidney .
the expression of the isoform is not detected in all nephron segments ( i.e. medullary and papillary collecting ducts ) but it is present in low levels in proximal convoluted tubules and in high levels in the medullary thick ascending limbs , and the distal nephron .
the results of this study reveal that the 1 and 1 subunits are the dominant na , k - atpase isoforms expressed in the basolateral membranes of epithelial cells in normal and bph canine prostate .
the 2 , 3 and subunits of na , k - atpase are not expressed in this tissue .
immunohistochemical and image analyses performed in this study suggest that na , k - atpase expression is significantly reduced in canine pca
. the cause of this down - regulation is not known at present but it may be associated with the loss of epithelial polarity and function in prostate cancer .
it may also be related to the reduced citrate production and secretion that accompanies neoplastic development in the prostate .
there is , however , an argument against the latter scenario : unlike the human prostate , the canine counterpart does not produce huge quantities of citrate ( l.c .
therefore , the down - regulation of na , k - atpase could be intricately involved in a series of other metabolic changes that occur during the progression of prostate malignancy , or it could merely be a consequence of such changes , which has little effect in the process of neoplastic transformation .
androgen ablation therapy is often used to treat advanced prostate cancer a treatment that is successful until the malignant growth evolves resistance to this and becomes androgen - independent .
previous studies have indicated that the -subunit of na , k - atpase is down - regulated in the prolonged presence of a synthetic androgen at a transcriptional level , resulting in a reduction of functional na , k - atpase in androgen - dependant prostate cell - lines .
studies have also shown that voltage activated sodium channel ( vasc ) activity and expression is altered in prostate cancer cell lines and vasc protein expression has been shown to enhance the invasive , metastatic properties of rat and human prostate cancer cells . taken together , these results suggest that neoplastic development in the prostate is accompanied by changes in cell homeostasis and expression levels of ion transporters including na , k - atpase and vasc . whether na , k - atpase expression is also reduced in human pca remains to be determined .
the metabolic transformation of zinc - accumulating citrate - producing normal prostate epithelial cells to citrate - oxidizing malignant neoplastic epithelial cells has important implications for cell metabolism and appears to be accompanied by a down regulation of na , k - atpase .
given the importance of the altered intermediary metabolism of prostatic cells in the pathogenesis of malignant prostatic adenocarcinoma and the progression of malignancy , the focus of future studies will be to address the role of na , k - atpase and other ion and metabolite transporters as potential epigenetic factors which may contribute to the metabolic transformation of sane cells to neoplastic cells .
sodium and potassium flux pathways ( including na , k - atpase , na / k/2cl co - transport and voltage - gated nachannels ) have already been studied in prostate cancer cells lines in terms of their potential value as targets for cytotoxic anti - neoplastic therapy [ 26 - 28 ] .
our results suggest that down - regulation of na , k - atpase in prostate cancer may accompany the up - regulation of voltage - gated sodium channels that has been observed by other investigators , contributing to the hyper - excitability of prostate cancer cells .
these phenotypic changes will influence citrate biosynthesis , zinc uptake and energy metabolism ( fig .
6 ) and may promote neoplastic development , galvanotaxis and metastasis . collectively these observations have important implications for cellular homeostasis , bioenergetics and metabolism and justify further functional ( i.e. naand kflux measurements ) and molecular studies to evaluate the role of ion transport pathways and the regulation of their expression during neoplastic development and metastasis in the prostate .
proposed scheme for the role of na , k - atpase in maintaining prostatic epithelial cell polarity and citrate - related energy metabolism ( adapted from a concept first proposed by costello and franklin , 2000 ) .
normal prostate cells abundantly express basolateral na , k - atpase to maintain cell homeostasis and epithelial polarity .
these cells contain specific transport mechanism for the accumulation of high levels of zinc and aspartate for optimal citrate biosynthesis .
aspartate , is transported into prostate epithelial cells on a high - affinity na - dependent carrier which resides in the basal cell membrane and is dependant on na , k - atpase activity .
the citrate transporter itself has yet to be identified but has been proposed to be localized in the apical membrane where it contributes to the accumulation of a citrate rich prostatic fluid .
the genetic transformation of a normal prostate cell to a neoplastic cell has been proposed to be accompanied by an impaired ability to accumulate high levels of citrate .
the neoplastic cell begins a metabolic transformation to a citrate - oxidizing cell , losing the ability to accumulate important factors such as zinc .
thus begins a metabolic conversion to a pre - malignant or malignant citrate - oxidizing cell .
the loss of basolateral na , k - atpase appears to be one of a number of phenotypic changes that accompany this transformation . this alteration will no doubt influence the membrane potential , homeostatic responses to ionic perturbations mediated by ion channels and secretory functions .
normal ( 2 prostates ) , bph ( 2 prostates ) and pca ( 3 prostates ) were dissected from the cadaver of canines following euthanasia . normal and bph
canine kidneys were used as positive controls for expression of na , k - atpase subunit .
tissues were fixed for 48 hrs in neutral buffered formalin before being embedded in paraffin wax .
the sections of prostate tissue were histologically and morphologically analyzed using established histpathological criteria ( i.e. cellular and nuclear pleomorphisms as evidence of dysplastic and neoplastic alterations ) by two independent veterinary pathologists and were categorized into normal , bph , well differentiated ( low grade ) and poorly differentiated ( high grade ) adenocarcinomas . all chemicals and secondary antibodies used were purchased from sigma biosciences ( poole , dorset , uk ) .
fast - red alkaline phosphatase precipitating agent was purchased from sigma / aldrich ( poole , dorset , uk ) .
immunohistochemical studies were carried out on 8m paraffin sections mounted on 3-aminopropyl - triethoxysilane ( apes ) treated slides .
normal , bph and pca tissues were incubated for identical periods of time with antibodies and the precipitating agent throughout the studies .
sections were dewaxed in 100% xylene for 5 minutes and taken through a graded series ( 100% , 70 % and 50% ) of alcohol baths for 1 min each before antigen retrieval in the microwave oven in the presence of 10 mm citrate buffer ( ph 6.0 ) for 12 minutes .
the sections were allowed to cool before washing in phosphate buffered saline solution ( pbs ) .
endogenous alkaline phosphatase was blocked for one hour at room temperature ( rt ) by treatment with 1.25 mm levamisole solution ( vector laboratories , peterborough , uk ) .
non - specific protein binding sites were blocked by addition of 10% normal goat serum in pbs ( 1 hour rt ) .
a panel of monoclonal and polyclonal antibodies to the and subunits of na , k - atpase was used ( table 1 ) .
sections were incubated with primary antibodies ( various dilutions in pbs ) for 24 hrs at 4c and subsequently washed in pbs before a two - hour incubation with goat anti - rabbit igg or goat anti - mouse igg conjugated to alkaline phosphatase .
tissue sections were washed in pbs and alkaline phosphatase active sites were developed using fast - red tr / naphthol as - mx as precipitating agent for 1015 minutes .
sections were counterstained with haematoxylin for 1 min and washed in distilled water before mounting in aqueous medium and visualization under a light microscope .
photographs were taken using a nikon microphot - fx microscope fitted with a nikon dxm1200 digital camera or goat anti - mouse igg .
isoform specific antibodies used to detect the isoforms of na , k - atpase in canine prostatic tissue by immunohistochemical analysis .
the 5 monoclonal broadly recognizes na , k - atpase subunits of avian , mammalian and insect species .
there are 4 known isoforms and the designation " 5 " does not indicate the existence of a fifth na , k - atpase isoform . the 5 antibody developed by d. fambrough
was also obtained from the developmental studies hybridoma bank developed under the auspices of the nichd and maintained by the university of iowa , department of biological sciences , iowa city , ia 52242 , u.s.a . the monoclonal antibody xvif9g10 developed by k.p .
image analysis was carried out using scion image for windows ( version 4.0.2 ) based on nih image for macintosh .
sections used for image analysis were only exposed to the fast - red precipitating agent and not counterstained with hematoxylin .
the results are expressed as the means + /-sd of a representative experiment performed in triplicate .
normal ( 2 prostates ) , bph ( 2 prostates ) and pca ( 3 prostates ) were dissected from the cadaver of canines following euthanasia . normal and bph
canine kidneys were used as positive controls for expression of na , k - atpase subunit .
tissues were fixed for 48 hrs in neutral buffered formalin before being embedded in paraffin wax .
the sections of prostate tissue were histologically and morphologically analyzed using established histpathological criteria ( i.e. cellular and nuclear pleomorphisms as evidence of dysplastic and neoplastic alterations ) by two independent veterinary pathologists and were categorized into normal , bph , well differentiated ( low grade ) and poorly differentiated ( high grade ) adenocarcinomas .
all chemicals and secondary antibodies used were purchased from sigma biosciences ( poole , dorset , uk ) .
fast - red alkaline phosphatase precipitating agent was purchased from sigma / aldrich ( poole , dorset , uk ) .
immunohistochemical studies were carried out on 8m paraffin sections mounted on 3-aminopropyl - triethoxysilane ( apes ) treated slides .
normal , bph and pca tissues were incubated for identical periods of time with antibodies and the precipitating agent throughout the studies .
sections were dewaxed in 100% xylene for 5 minutes and taken through a graded series ( 100% , 70 % and 50% ) of alcohol baths for 1 min each before antigen retrieval in the microwave oven in the presence of 10 mm citrate buffer ( ph 6.0 ) for 12 minutes . the sections were allowed to cool before washing in phosphate buffered saline solution ( pbs ) .
endogenous alkaline phosphatase was blocked for one hour at room temperature ( rt ) by treatment with 1.25 mm levamisole solution ( vector laboratories , peterborough , uk ) .
non - specific protein binding sites were blocked by addition of 10% normal goat serum in pbs ( 1 hour rt ) .
a panel of monoclonal and polyclonal antibodies to the and subunits of na , k - atpase was used ( table 1 ) .
sections were incubated with primary antibodies ( various dilutions in pbs ) for 24 hrs at 4c and subsequently washed in pbs before a two - hour incubation with goat anti - rabbit igg or goat anti - mouse igg conjugated to alkaline phosphatase .
tissue sections were washed in pbs and alkaline phosphatase active sites were developed using fast - red tr / naphthol as - mx as precipitating agent for 1015 minutes .
sections were counterstained with haematoxylin for 1 min and washed in distilled water before mounting in aqueous medium and visualization under a light microscope .
photographs were taken using a nikon microphot - fx microscope fitted with a nikon dxm1200 digital camera or goat anti - mouse igg .
isoform specific antibodies used to detect the isoforms of na , k - atpase in canine prostatic tissue by immunohistochemical analysis .
the 5 monoclonal broadly recognizes na , k - atpase subunits of avian , mammalian and insect species .
there are 4 known isoforms and the designation " 5 " does not indicate the existence of a fifth na , k - atpase isoform .
the 5 antibody developed by d. fambrough was also obtained from the developmental studies hybridoma bank developed under the auspices of the nichd and maintained by the university of iowa , department of biological sciences , iowa city , ia 52242 , u.s.a . the monoclonal antibody xvif9g10 developed by k.p .
image analysis was carried out using scion image for windows ( version 4.0.2 ) based on nih image for macintosh .
sections used for image analysis were only exposed to the fast - red precipitating agent and not counterstained with hematoxylin .
the results are expressed as the means + /-sd of a representative experiment performed in triplicate .
in a very recent study of na , k - atpase and subunit expression in bladder tumours , human urothelial cancer tissue microarrays have been successfully used to demonstrate that the mean protein expression for both and subunits of na , k - atpase is reduced in invasive bladder tumours compared to benign and dysplastic tissue .
the authors have suggested that na , k - atpase and subunit expression levels may be useful predictors of clinical outcomes .
research grants from the pet plan charitable trust ( grant no . 0211 ) and the wellcome trust ( u.k . ) supported this work . we wish to thank the pathologists of the department of veterinary pathology at the university of liverpool for supplying tissues and original histopathological interpretations .
caplan ( yale university school of medicine ) for their continued generosity in provision of antibodies .
d. alvarez de la rosa ( yale university school of medicine ) for critical comments on the manuscript and dr . | backgroundan important physiological function of the normal prostate gland is the synthesis and secretion of a citrate rich prostatic fluid . in prostate cancer , citrate production levels are reduced as a result of altered cellular metabolism and bioenergetics .
na , k - atpase is essential for citrate production since the inward na+ gradients it generates are utilized for the na+ dependent uptake of aspartate , a major substrate for citrate synthesis .
the objective of this study was to compare the expression of previously identified na , k - atpase isoforms in normal canine prostate , benign prostatic hyperplasia ( bph ) and prostatic adenocarcinoma ( pca ) using immunohistochemistry in order to determine whether reduced citrate levels in pca are also accompanied by changes in na , k - atpase expression.resultsexpression of na , k - atpase 1 and 1 isoforms was observed in the lateral and basolateral plasma membrane domains of prostatic epithelial cells in normal and bph prostates .
canine kidney was used as positive control for expression of na , k - atpase 1 and isoforms .
the 1 isoform was detected in abundance in prostatic epithelial cells but there was no evidence of 2 , 3 or subunit expression . in advanced pca ,
na , k - atpase 1 isoform expression was significantly lower compared to normal and bph glands .
the abundant basolateral immunostaining observed in normal and bph tissue was significantly attenuated in pca.conclusionthe loss of epithelial structure and function and the transformation of normal epithelial cells to malignant cells in the canine prostate have important implications for cellular metabolism and are accompanied by a down regulation of na , k - atpase . |
acute abdomen due to spontaneous rupture of an intra - abdominal vessel is an extremely rare clinical condition particularly in otherwise healthy subjects .
we report a case of acute abdomen in a young man due to spontaneous rupture of the posterior gastric artery .
a 33-year - old man came to emergency department with severe upper abdominal pain of 45 h duration .
he had self - induced vomiting by using his finger to trigger his gag reflex . following this
there was no history of trauma or any medical disease , and he was not on any medication . his general condition was fair with stable vital signs .
his pulse was 77 beats per minute with a blood pressure of 110/70 mm hg .
he was afebrile with a respiratory rate of 22 breaths per minute . on abdominal examination
, there was no gross distension but there was generalized guarding , tenderness and rebound tenderness consistent with acute peritonitis .
his chest x - ray showed no free gas under the domes of diaphragm and the abdominal x - ray was unremarkable with a non - specific bowel gas pattern .
12.6 g / dl , platelet 239 10 l ,
glucose routine bloods were normal , apart from a low potassium ( 3.1 mmol / dl ) .
an ultrasound showed free fluid in his peritoneal cavity and computerized tomography ( ct ) scan of abdomen with oral and intravenous contrast reported a large well - defined soft tissue mass of about 10 cm 12 cm posterior to the stomach , anterior and above the body and tail of pancreas with loss of fat plane from upper surface of body without enhancement or any blush ( fig .
repeat laboratory investigations were unremarkable without any rising trend of amylase . in view of the clinical findings strongly suggestive of acute peritonitis and the suspicion of ruptured pancreatic mass after ct
his peritoneal cavity was full of free and clotted blood in the paracolic gutter , pelvis , perihepatic and perisplenic areas .
on opening the lesser sac a large blood clot was found in the same region as suggested by ct abdomen .
after clearing the clot a spurting vessel was seen which was found to be a bleeding posterior gastric artery ( fig .
2 ) , controlled by ligation . there was no visible or palpable abnormality in his pancreas , spleen or posterior part of stomach .
after control of the bleeding , the entire peritoneal cavity explored which appeared to be normal .
hemorrhage within the peritoneal cavity in itself is not uncommon , but intra - abdominal hemorrhage from a non - traumatic and non - iatrogenic cause is extremely rare .
this condition has been named abdominal apoplexy or more recently , idiopathic spontaneous intraperitoneal hemorrhage ( isih ) .
frederick p. ross in a case report published in 1950 has stressed that this condition is a rare occurrence and that reporting of cases is warranted .
common causes of spontaneous abdominal hemorrhage are visceral ( rupture of spleen , liver or kidney ) , gynecologic ( rupture of ectopic pregnancy , or ovarian follicle ) , coagulopathy related conditions and vascular conditions .
examples of spontaneously ruptured vessels which have been reported include branch of middle colic artery , short gastric artery , and hepatic artery .
the etiology of spontaneous rupture of vessels may be related to hypertension , arteriosclerosis , splanchnic artery aneurysm mainly of splenic and hepatic artery and the erosion of a vessel by an adjacent neoplastic or inflammatory process . in our patient ,
this led to the development of acute abdomen with abrupt onset of severe abdominal pain progressing to peritonitis , which finally turned out to be due to rupture of posterior gastric artery .
reports are available in the literature in which violent vomiting led to rupture of arteries supplying the stomach .
there is no pathognomonic sign or symptom of abdominal apoplexy , however , cushman and kilgore have described the syndrome and believe that it can help in diagnosis .
signs and symptoms of spontaneous abdominal hemorrhage depend upon the site , volume and rate of bleeding .
usually it presents with sudden onset of severe abdominal pain , distension , signs of generalized peritoneal irritation and an acute drop of hematocrit level .
the most important aid in the diagnosis is to maintain a high index of suspicion in ambiguous cases of acute abdomen .
ultrasound abdomen can detect free fluid in the abdominal cavity and is particularly helpful in unstable cases .
the availability of computerized tomography can be used to confirm the diagnosis with more accuracy in equivocal cases .
the primary aim of the treatment in cases of internal bleeding is to control the hemorrhage by ligating or repairing the involved vessel . in some clinically suspected cases of abdominal apoplexy
if the patient is stable with more gradual onset , ct and visceral angiography can diagnose and localize bleeding vessel preoperatively . in such clinical setting , embolization may be a useful alternative to open operation .
the most important strategy in management of equivocal cases is early surgical exploration to establish the diagnosis and treat accordingly .
mohammad ali al qarni : management decision , responsibilities , share in making the article as reviewed and history & examination of management details ; sarwer jawaid : wrote the references and the article design . | introductionspontaneous hemoperitoneum in healthy males is an extremely rare , life threatening emergency condition with high mortality and morbidity , if not diagnosed and managed early.presentation of casewe present a 33-year - old male who presented with hemoperitoneum following self - induced vomiting.discussionto suspect and diagnose the condition is a challenge for clinician if ambiguity in presentation prevails.conclusionthe most important strategy in management of equivocal cases is early surgical exploration to establish the diagnosis and treat accordingly . |
fungal infection has rarely been reported in patients with primary biliary cirrhosis ( pbc ) ; however it may occur following live transplant ( lt ) in patients with pbc , predisposing factors for the development of fungal infection following lt include diabetes mellitus , cholestasis , hypertransfusion , acute rejection , treatment with high - dose of steroids and immunosuppressive agents , renal failure and bacterial infection ( 1 ) . the present study aimed to analyze the imaging , clinical and pathological features of fungal infection involvement in pbc by retrospectively analyzing and reviewing the features of two patients with fungal infection involvement in pbc who were admitted to our department .
the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction
lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 .
liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . in 2007 , facial and yellow sclera and dark urine gradually developed . following the administration of compound glycyrrhizin tablets ( 50 mg , 3/d ; minophagen pharmaceutical co. , ltd . ,
tokyo , japan ) and ademrtionine ( 500 mg , 3/d ; hospira uk ltd . , hurley , uk ) ,
the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved .
abdominal distension , loss of appetite , dysfunction of liver , pancytopenia and ascites followed .
a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) .
the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) .
the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following progressive aggravation of her symptoms , the patient was admitted to our hospital .
physical examination demonstrated the following : moon face ; symmetry congestive erythema on the face and hands ; petechia and ecchymosis on the upper limbs ( predominantly ) and the nape of the neck ; no jaundice of the skin ; the superficial lymph node was not large and the lungs were normal ; and hepatosplenomegaly , shifting dullness was negative .
laboratory tests results were conducted and the results were as follows ( normal reference range ) : routine blood tests : white blood cell count ( wbc ) , 0.59 ( 3.510)x10/l ; red blood cell count ( rbc ) , 3.12 ( 3.55.5)x10/l ; hemoglobin ( hb ) , 94 ( 110176 )
routine stool tests : helicobacter pylori , weakly positive ; erythrocyte sedimentation rate ( esr ) , 11 mm / h ; immunoglobin g ( igg ) , 1730 ( 7001600 ) mg / dl ; iga 35.7 ( 70400 ) mg / dl , c3 , 43.2 ( 90180 ) mg / dl ; c4 , 7.63 ( 1040 ) mg / dl ; and c - reactive protein ( crp ) , < 0.308 ( 00.8 ) mg / dl .
autoantibody tests : anti - nuclear antibody ( ana ) ( 1:1,000 particles ) , positive , anti - ssa antibody , positive ; anti - ssb antibody , positive ; and anti - mitochondrial antibody ( ama ) , positive . blood biochemistry tests : albumin ( alb ) , 27.1 ( 3550 )
g / l ; total bilirubin ( tbil ) , 66.8 ( 021 ) mol / l ; direct bilirubin ( dbil ) , 48.9 ( 08.6)/mol / l ; alanine aminotransferase ( alt ) , 296.7 ( 040 ) u / l ; aspartate aminotransferase ( ast ) , 109.5 ( 040 )
u / l ; alkaline phosphatase ( alp ) , 244.9 ( 0130 ) u /
u / l ; lactate dehydrogenase , 346.6 ( 0200 ) u / l ; glucose ( glu ) 9.07 mmol / l ; blood urea nitrogen ( bun ) , 7.35 ( 1.87.5 ) mmol / l ; creatinine ( cr ) , 76 ( 30110 ) mol / l ; cancer antigen ( ca ) 125 , 421.3 u / ml ; ca19 - 9 , 455.7 u / ml ; and blood ammonia , 92.1 mol / l . purified protein derivative ( ppd ) test demonstrated and tuberculosis antibody were negative ( 1 ) .
magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig .
following admission to our hospital , the patient suffered from a cough producing white sputum .
lung ct tests showed that the upper lung was shadowed , and the possibility of tuberculosis was not excluded .
moxifloxacin hydrochloride ( 0.4 g ; 1/d ; bayer ag , leverkusen , germany ) , streptomycin ( 0.75 g ; i m ; 1/d ; merro pharmaceutical co. , ltd . , dalian , china ) and ethambutol ( 0.75 g ; 1/d ; china resources double - crane pharmaceutical co. , ltd . ,
serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp .
the patient was treated with caspofungin acetate ( 70 mg ; i.v . ; 1/d ; msd ; merck millipore , darmstadt , germany ) ; two slices of cotrimoxazole ( 3/d ; southwest synthetic pharmaceutical co. , ltd . , chongqing , china ) and bedside hemofiltration .
the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig .
the final diagnosis was pbc and pneumonia caused by pneumocystis carinii , which led to respiratory and circulatory failure . a 49-year - old female presented with intermittent fever and abdominal distension for more than a year .
the patient suffered from an intermittent fever without any known cause or regularity a year prior to admission ( body temperature , 38c ) .
fever was accompanied by fatigue , anorexia , cough , yellow sputum , night sweats and abdominal distension .
ana was positive , anti - dsdna was confirmed as weakly positive and liver transaminase levels were mildly elevated .
the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . ; 1/d ; beijing tide pharmaceutical co. , ltd . ,
the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon .
taking the autoimmune cirrhosis and pulmonary infection into consideration , hepatic and anti - inflammatory symptomatic treatment consisting of ursodeoxycholic acid and cefuroxime axetil tablets ( 0.5 g , 2/d ; glaxosmithkline , brentford , uk ) was administered , and the symptoms of cough and expectoration eased but were accompanied by an intermittent fever .
one week prior to hospital admission , hypogastrium pain ( persistent dull pain ) appeared with no nausea , vomiting , diarrhea or other symptoms . during the course of the disease
, symptoms of thirst and joint pain were noted ; however no obvious limitations in ingesting dry food were detected .
the patient did not suffer from dry eyes , muscle pain , muscle weakness , facial butterfly spots or photophobia .
the patient suffered from tuberculosis for 12 years , which was subsequently been cured . physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs .
laboratory test results were as follows : ana autoantibodies ( 1:1,000 homogeneous particles ) , positive ; anti - dsdna , positive ; anti - ssa antibody , negative ; anti - ssb antibody , negative ; iga , 853 ( 70400 ) mg / dl ; igg , 2820 ( 7001600 ) mg / dl ; igm , 924 ( 40230 ) mg / dl ; ige , 1400 ( 0100 ) iu / ml ; c3 , 79.3 mg / dl ; c4 , 17.6 mg / dl ; and rf , 5410 ( 020 ) iu / ml .
g / l ; rbc , 3.4610/l ; wbc , 1.010/l ; plt , 8610/l ; esr , 129 ( < 20 ) mm / h ; and crp , < .313 mg / dl .
routine urine tests revealed a normal range and tests for hepatitis b and c and hiv were detected negative .
umol / l ; alt , 35 u / l ; ast , 84.1 u / l ; alp , 184.2 u / l ; ggt , 140.8 u / l ; bun , 2.42 ( 1.87.5 ) mmol / l ; cr , 38.8 ( 30110 ) mol / lu ; k , 3.47 ( 3.55.5 ) mmol / l , ca , 2.20 ( 2.252.75 ) mmol / l ; acid - fast stain ( sputum ) , negative ; tumor marker , negative .
schirmer 's test was 0 cm ; labial gland biopsy showed interstitial multifocal lymphocytic infiltration of the salivary gland tissue ( fig .
4 ) . bone marrow aspiration revealed hyperplasia of marrow activity , megakaryocytes were visible .
abdominal ultrasound indicated cirrhosis , splenauxe , portal hypertension , gallbladder wall thickening , echo nodules next to the spleen , an accessory spleen and small amount of ascites .
plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear .
in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum .
she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . ,
basel , switzerland ) , ursodeoxycholic acid ( 0.25 g ; 3/d ) , and polyene phosphatidylcholine capsules ( 456 mg , 3/d ; sanofi - aventis hong kong ltd . ,
hong kong , china ) and cefuroxime sodium for injection ( 1.5 g , 2/d ) . following treatment ,
the patient 's body temperature dropped to within normal levels . compared to the symptoms exhibited upon admission to our hospital
however , 14 days after she was admitted to hospital , routine urine tests indicated the following : wbc15 , ~18/hp ; pro , negative ; 80% of erythrocytes were abnormally formed and 20% were uniform . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs .
flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot .
the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig .
the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation .
an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared .
the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed .
osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease , a renal biopsy
the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . ,
shenyang , china ) and nutritional support [ human serum albumin ( 10 g , 1/d , shanghai raas , shanghai , china ] . due to the severity of the illness , the patient succumbed to the condition .
the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction
lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 .
liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . in 2007 , facial and yellow sclera and dark urine gradually developed . following the administration of compound glycyrrhizin tablets ( 50 mg , 3/d ; minophagen pharmaceutical co. , ltd . ,
tokyo , japan ) and ademrtionine ( 500 mg , 3/d ; hospira uk ltd . , hurley , uk ) ,
the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved .
abdominal distension , loss of appetite , dysfunction of liver , pancytopenia and ascites followed .
a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) .
the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) .
the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following progressive aggravation of her symptoms , the patient was admitted to our hospital .
physical examination demonstrated the following : moon face ; symmetry congestive erythema on the face and hands ; petechia and ecchymosis on the upper limbs ( predominantly ) and the nape of the neck ; no jaundice of the skin ; the superficial lymph node was not large and the lungs were normal ; and hepatosplenomegaly , shifting dullness was negative .
laboratory tests results were conducted and the results were as follows ( normal reference range ) : routine blood tests : white blood cell count ( wbc ) , 0.59 ( 3.510)x10/l ; red blood cell count ( rbc ) , 3.12 ( 3.55.5)x10/l ; hemoglobin ( hb ) , 94 ( 110176 )
routine stool tests : helicobacter pylori , weakly positive ; erythrocyte sedimentation rate ( esr ) , 11 mm / h ; immunoglobin g ( igg ) , 1730 ( 7001600 ) mg / dl ; iga 35.7 ( 70400 ) mg / dl , c3 , 43.2 ( 90180 ) mg / dl ; c4 , 7.63 ( 1040 ) mg / dl ; and c - reactive protein ( crp ) , < 0.308 ( 00.8 ) mg / dl .
autoantibody tests : anti - nuclear antibody ( ana ) ( 1:1,000 particles ) , positive , anti - ssa antibody , positive ; anti - ssb antibody , positive ; and anti - mitochondrial antibody ( ama ) , positive . blood biochemistry tests : albumin ( alb ) , 27.1 ( 3550 )
g / l ; total bilirubin ( tbil ) , 66.8 ( 021 ) mol / l ; direct bilirubin ( dbil ) , 48.9 ( 08.6)/mol / l ; alanine aminotransferase ( alt ) , 296.7 ( 040 ) u / l ; aspartate aminotransferase ( ast ) , 109.5 ( 040 )
u / l ; alkaline phosphatase ( alp ) , 244.9 ( 0130 ) u /
u / l ; lactate dehydrogenase , 346.6 ( 0200 ) u / l ; glucose ( glu ) 9.07 mmol / l ; blood urea nitrogen ( bun ) , 7.35 ( 1.87.5 ) mmol / l ; creatinine ( cr ) , 76 ( 30110 ) mol / l ; cancer antigen ( ca ) 125 , 421.3 u / ml ; ca19 - 9 , 455.7 u / ml ; and blood ammonia , 92.1 mol / l . purified protein derivative ( ppd ) test demonstrated and tuberculosis antibody were negative ( 1 ) .
magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig .
following admission to our hospital , the patient suffered from a cough producing white sputum .
lung ct tests showed that the upper lung was shadowed , and the possibility of tuberculosis was not excluded .
moxifloxacin hydrochloride ( 0.4 g ; 1/d ; bayer ag , leverkusen , germany ) , streptomycin ( 0.75 g ; i m ; 1/d ; merro pharmaceutical co. , ltd . , dalian , china ) and ethambutol ( 0.75 g ; 1/d ; china resources double - crane pharmaceutical co. , ltd . ,
serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp .
the patient was treated with caspofungin acetate ( 70 mg ; i.v . ; 1/d ; msd ; merck millipore , darmstadt , germany ) ; two slices of cotrimoxazole ( 3/d ; southwest synthetic pharmaceutical co. , ltd . , chongqing , china ) and bedside hemofiltration .
the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig .
the final diagnosis was pbc and pneumonia caused by pneumocystis carinii , which led to respiratory and circulatory failure . a 49-year - old female presented with intermittent fever and abdominal distension for more than a year .
the patient suffered from an intermittent fever without any known cause or regularity a year prior to admission ( body temperature , 38c ) .
fever was accompanied by fatigue , anorexia , cough , yellow sputum , night sweats and abdominal distension .
ana was positive , anti - dsdna was confirmed as weakly positive and liver transaminase levels were mildly elevated .
the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . ; 1/d ; beijing tide pharmaceutical co. , ltd . ,
the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon .
taking the autoimmune cirrhosis and pulmonary infection into consideration , hepatic and anti - inflammatory symptomatic treatment consisting of ursodeoxycholic acid and cefuroxime axetil tablets ( 0.5 g , 2/d ; glaxosmithkline , brentford , uk ) was administered , and the symptoms of cough and expectoration eased but were accompanied by an intermittent fever .
one week prior to hospital admission , hypogastrium pain ( persistent dull pain ) appeared with no nausea , vomiting , diarrhea or other symptoms . during the course of the disease
, symptoms of thirst and joint pain were noted ; however no obvious limitations in ingesting dry food were detected .
the patient did not suffer from dry eyes , muscle pain , muscle weakness , facial butterfly spots or photophobia .
the patient suffered from tuberculosis for 12 years , which was subsequently been cured . physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs .
laboratory test results were as follows : ana autoantibodies ( 1:1,000 homogeneous particles ) , positive ; anti - dsdna , positive ; anti - ssa antibody , negative ; anti - ssb antibody , negative ; iga , 853 ( 70400 ) mg / dl ; igg , 2820 ( 7001600 ) mg / dl ; igm , 924 ( 40230 ) mg / dl ; ige , 1400 ( 0100 ) iu / ml ; c3 , 79.3 mg / dl ; c4 , 17.6 mg / dl ; and rf , 5410 ( 020 ) iu / ml .
g / l ; rbc , 3.4610/l ; wbc , 1.010/l ; plt , 8610/l ; esr , 129 ( < 20 ) mm / h ; and crp , < .313 mg / dl .
routine urine tests revealed a normal range and tests for hepatitis b and c and hiv were detected negative .
umol / l ; alt , 35 u / l ; ast , 84.1 u / l ; alp , 184.2 u / l ; ggt , 140.8 u / l ; bun , 2.42 ( 1.87.5 ) mmol / l ; cr , 38.8 ( 30110 ) mol / lu ; k , 3.47 ( 3.55.5 ) mmol / l , ca , 2.20 ( 2.252.75 ) mmol / l ; acid - fast stain ( sputum ) , negative ; tumor marker , negative .
schirmer 's test was 0 cm ; labial gland biopsy showed interstitial multifocal lymphocytic infiltration of the salivary gland tissue ( fig .
4 ) . bone marrow aspiration revealed hyperplasia of marrow activity , megakaryocytes were visible .
abdominal ultrasound indicated cirrhosis , splenauxe , portal hypertension , gallbladder wall thickening , echo nodules next to the spleen , an accessory spleen and small amount of ascites .
plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear .
in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum .
she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . ,
basel , switzerland ) , ursodeoxycholic acid ( 0.25 g ; 3/d ) , and polyene phosphatidylcholine capsules ( 456 mg , 3/d ; sanofi - aventis hong kong ltd . ,
hong kong , china ) and cefuroxime sodium for injection ( 1.5 g , 2/d ) . following treatment ,
the patient 's body temperature dropped to within normal levels . compared to the symptoms exhibited upon admission to our hospital
however , 14 days after she was admitted to hospital , routine urine tests indicated the following : wbc15 , ~18/hp ; pro , negative ; 80% of erythrocytes were abnormally formed and 20% were uniform . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs .
flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot .
the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig .
the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation .
an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared .
the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed .
osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease , a renal biopsy
the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . ,
shenyang , china ) and nutritional support [ human serum albumin ( 10 g , 1/d , shanghai raas , shanghai , china ] . due to the severity of the illness , the patient succumbed to the condition .
the present study was approved by the ethics committee of the chinese pla general hospital and written informed consent was obtained from all patients . a 20-year - old female presented with the following : liver dysfunction
lasting > 2 years ; xanthochromia lasting three months ; and edema of the lower limb lasting one week , and was hospitalized in 2008 .
liver dysdunction was confirmed in the patient following a physical examination in 2005 ; however , the patient refused standard treatment . in 2007 , facial and yellow sclera and dark urine gradually developed . following the administration of compound glycyrrhizin tablets ( 50 mg , 3/d ; minophagen pharmaceutical co. , ltd . ,
tokyo , japan ) and ademrtionine ( 500 mg , 3/d ; hospira uk ltd . , hurley , uk ) ,
the patient suffered from a fever for five days ; however , after terminating ingestion of the drugs , the fever was relieved .
abdominal distension , loss of appetite , dysfunction of liver , pancytopenia and ascites followed .
a local hospital diagnosed the patient with sicca syndrome , cirrhosis and hypersplenism , and she was prescribed intravenous methylprednisolone ( 40 mg ; 8 d ; pfizer manufacturing belgium , puurs , belgium ) , followed by implosive therapy of intravenous methylprednisolone ( 1 g ; 1 d ) .
the dosage of intravenous methylprednisolone was maintained at 40 mg / d , and the patient was treated with ursodeoxycholic acid ( 500 mg , 3/d ; losan pharma gmbh , neuenburg am rhein , germany ) and glutathione ( 0.6 g ; 3/d , pharmainvest spa , boumedian , algeria ) .
the liver function of the patient was not greatly improved , and she presented with the following : petechia on both upper limbs ; congestive rash on the palms ; malaise ; progressive decline of blood corpuscle ; and edema below the lower limbs . following progressive aggravation of her symptoms , the patient was admitted to our hospital .
physical examination demonstrated the following : moon face ; symmetry congestive erythema on the face and hands ; petechia and ecchymosis on the upper limbs ( predominantly ) and the nape of the neck ; no jaundice of the skin ; the superficial lymph node was not large and the lungs were normal ; and hepatosplenomegaly , shifting dullness was negative .
laboratory tests results were conducted and the results were as follows ( normal reference range ) : routine blood tests : white blood cell count ( wbc ) , 0.59 ( 3.510)x10/l ; red blood cell count ( rbc ) , 3.12 ( 3.55.5)x10/l ; hemoglobin ( hb ) , 94 ( 110176 )
routine stool tests : helicobacter pylori , weakly positive ; erythrocyte sedimentation rate ( esr ) , 11 mm / h ; immunoglobin g ( igg ) , 1730 ( 7001600 ) mg / dl ; iga 35.7 ( 70400 ) mg / dl , c3 , 43.2 ( 90180 ) mg / dl ; c4 , 7.63 ( 1040 ) mg / dl ; and c - reactive protein ( crp ) , < 0.308 ( 00.8 ) mg / dl .
autoantibody tests : anti - nuclear antibody ( ana ) ( 1:1,000 particles ) , positive , anti - ssa antibody , positive ; anti - ssb antibody , positive ; and anti - mitochondrial antibody ( ama ) , positive . blood biochemistry tests : albumin ( alb ) , 27.1 ( 3550 )
g / l ; total bilirubin ( tbil ) , 66.8 ( 021 ) mol / l ; direct bilirubin ( dbil ) , 48.9 ( 08.6)/mol / l ; alanine aminotransferase ( alt ) , 296.7 ( 040 ) u / l ; aspartate aminotransferase ( ast ) , 109.5 ( 040 )
u / l ; alkaline phosphatase ( alp ) , 244.9 ( 0130 ) u /
u / l ; lactate dehydrogenase , 346.6 ( 0200 ) u / l ; glucose ( glu ) 9.07 mmol / l ; blood urea nitrogen ( bun ) , 7.35 ( 1.87.5 ) mmol / l ; creatinine ( cr ) , 76 ( 30110 ) mol / l ; cancer antigen ( ca ) 125 , 421.3 u / ml ; ca19 - 9 , 455.7 u / ml ; and blood ammonia , 92.1 mol / l . purified protein derivative ( ppd ) test demonstrated and tuberculosis antibody were negative ( 1 ) .
magnetic resonance ( mr ) imaging of the abdomen also indicated liver cirrhosis , splenomegaly , a small amount of ascites ( fig .
following admission to our hospital , the patient suffered from a cough producing white sputum .
lung ct tests showed that the upper lung was shadowed , and the possibility of tuberculosis was not excluded .
moxifloxacin hydrochloride ( 0.4 g ; 1/d ; bayer ag , leverkusen , germany ) , streptomycin ( 0.75 g ; i m ; 1/d ; merro pharmaceutical co. , ltd . , dalian , china ) and ethambutol ( 0.75 g ; 1/d ; china resources double - crane pharmaceutical co. , ltd . ,
serum -glucan test was positive and polymerase chain reaction analysis of the sputum detected inclusion bodies of pneumocystis spp .
the patient was treated with caspofungin acetate ( 70 mg ; i.v . ; 1/d ; msd ; merck millipore , darmstadt , germany ) ; two slices of cotrimoxazole ( 3/d ; southwest synthetic pharmaceutical co. , ltd . , chongqing , china ) and bedside hemofiltration .
the patient 's condition became progressively aggravated and a chest radiograph showed pneumonia of both upper lungs ( fig .
the final diagnosis was pbc and pneumonia caused by pneumocystis carinii , which led to respiratory and circulatory failure .
a 49-year - old female presented with intermittent fever and abdominal distension for more than a year .
the patient suffered from an intermittent fever without any known cause or regularity a year prior to admission ( body temperature , 38c ) .
fever was accompanied by fatigue , anorexia , cough , yellow sputum , night sweats and abdominal distension .
ana was positive , anti - dsdna was confirmed as weakly positive and liver transaminase levels were mildly elevated .
the patient was diagnosed with autoimmune hepatitis and was treated using compound glycyrrhizin tablets ( 50 mg ; 3/d ) and alprostadil ( 20 mg ; i.v . ; 1/d ; beijing tide pharmaceutical co. , ltd . ,
the patient 's condition did not improve , rather it gradually worsened as her temperature reached up to 40c , peaking in the afternoon .
anti - smooth muscle antibody and ama were positive . taking the autoimmune cirrhosis and pulmonary infection into consideration , hepatic and anti - inflammatory symptomatic treatment consisting of ursodeoxycholic acid and cefuroxime axetil tablets ( 0.5 g , 2/d ; glaxosmithkline , brentford , uk ) was administered , and the symptoms of cough and expectoration eased but were accompanied by an intermittent fever .
one week prior to hospital admission , hypogastrium pain ( persistent dull pain ) appeared with no nausea , vomiting , diarrhea or other symptoms . during the course of the disease ,
symptoms of thirst and joint pain were noted ; however no obvious limitations in ingesting dry food were detected .
the patient did not suffer from dry eyes , muscle pain , muscle weakness , facial butterfly spots or photophobia .
physical examination revealed scattered spider angioma on the anterior part of the neck and the left palm and mild pitting edema of the lower limbs .
laboratory test results were as follows : ana autoantibodies ( 1:1,000 homogeneous particles ) , positive ; anti - dsdna , positive ; anti - ssa antibody , negative ; anti - ssb antibody , negative ; iga , 853 ( 70400 ) mg / dl ; igg , 2820 ( 7001600 ) mg / dl ; igm , 924 ( 40230 ) mg / dl ; ige , 1400 ( 0100 ) iu / ml ; c3 , 79.3 mg / dl ; c4 , 17.6 mg / dl ; and rf , 5410 ( 020 ) iu / ml .
g / l ; rbc , 3.4610/l ; wbc , 1.010/l ; plt , 8610/l ; esr , 129 ( < 20 ) mm / h ; and crp , < .313 mg / dl .
routine urine tests revealed a normal range and tests for hepatitis b and c and hiv were detected negative .
g / l ; tbil , 33.9 umol / l ; dbil , 19.4 umol / l ; alt , 35 u / l ; ast , 84.1 u / l ; alp , 184.2 u / l ; ggt , 140.8 u / l ; bun , 2.42 ( 1.87.5 ) mmol / l ; cr , 38.8 ( 30110 ) mol / lu ; k , 3.47 ( 3.55.5 ) mmol / l , ca , 2.20 ( 2.252.75 ) mmol / l ; acid - fast stain ( sputum ) , negative ; tumor marker , negative .
schirmer 's test was 0 cm ; labial gland biopsy showed interstitial multifocal lymphocytic infiltration of the salivary gland tissue ( fig .
4 ) . bone marrow aspiration revealed hyperplasia of marrow activity , megakaryocytes were visible .
abdominal ultrasound indicated cirrhosis , splenauxe , portal hypertension , gallbladder wall thickening , echo nodules next to the spleen , an accessory spleen and small amount of ascites .
plain ct scanning of the lung revealed bilateral patchy high - density shadows , the boundary was unclear .
in addition to changes in the lungs that were indicative of inflammation , several lymph glands were seen in the bilateral oxters and mediastinum .
she was treated with hormones ( methylprednisolone ; 36 mg / d ) immunosuppressants ( mycophenolate ; 1 g ; 2/d ; roche pharma inc . ,
basel , switzerland ) , ursodeoxycholic acid ( 0.25 g ; 3/d ) , and polyene phosphatidylcholine capsules ( 456 mg , 3/d ; sanofi - aventis hong kong ltd . ,
hong kong , china ) and cefuroxime sodium for injection ( 1.5 g , 2/d ) .
following treatment , the patient 's body temperature dropped to within normal levels . compared to the symptoms exhibited upon admission to our hospital
however , 14 days after she was admitted to hospital , routine urine tests indicated the following : wbc15 , ~18/hp ; pro , negative ; 80% of erythrocytes were abnormally formed and 20% were uniform . following this , gross hematuria appeared , the offside costalpinal angle exhibited tenderness and percussion pain , and pitting edema was confirmed in the patient 's lower limbs .
flocculent echo was detected in the right renal pelvis , which was indicative of a blood clot .
the kidney mri and dynamic contrast - enhanced scan suggested cirrhosis , splenomegaly , a little ascites with abnormality of the right kidney ( fig .
the patient subsequently suffered from sudden impaired vision of the left eye , hyperemia of bulbar conjunctiva , photophobia and lacrimation .
an eye examination indicated hypopyon of the left eye , the liquid was at the height of ~1/5 of the anterior chamber , cellulose before cystals of floating liquid material of aqueous humour ( + + + ) exuded and vitreous opacities appeared .
the patient was diagnosed with hypopyon of the left eye and uveitis of the left eye was confirmed .
osaka , japan ) was administered into the left eye and a dexamethasone and gentamicin mixture was inserted next to the left eye ball ; however , the patient 's vision did not markedly improved . to identify the nature of the disease ,
the patient was provided with active anti - fungal amphotericin b therapy ( 2 mg , iv , 1/d ; northeast pharmaceutical group co. , ltd . ,
shenyang , china ) and nutritional support [ human serum albumin ( 10 g , 1/d , shanghai raas , shanghai , china ] . due to the severity of the illness , the patient succumbed to the condition .
this condition predominantly occurs in middle - aged women and is characterized by progressive bile duct destruction in the liver , inflammation and fibrosis in the portal area and around the periportal , and eventual portal hypertension , liver cirrhosis and liver failure ( 2 ) . with advances in diagnostic techniques and knowledge of the disease in recent years , the incidence rates of this disease have increased in china ( 3 ) .
previous studies have confirmed that pbc and other autoimmune diseases , such as sjogren 's syndrome and rheumatoid arthritis , have a common immunopathologic basis ( 4,5 ) .
there is currently a lack of satisfactory therapies for the disease , as symptomatic treatment remains the main therapy option .
ursodeoxycholic acid has been successful in some patients for the relief of symptoms and has been shown to improve the indicators of liver function , delay disease progression and improve quality of life ( 6 ) . if the patient has an additional connective tissue diseases , the patient is required to take hormones and immunosuppressive therapy simultaneously .
long - term use of hormones and immunosuppressive agents may increase the patient 's risk of developing a variety of concurrent infections , particularly opportunistic infections ( 7,8 ) . in the present study ,
two cases of fungal infection involvement in pbc , which was diagnosed in line with american association for the study of liver disease 's pbc diagnosis recommendations published in 2000 ( 9 ) . in case 1 , the patient suffered from subsequent respiratory and cardiac failure ; in case 2 , the patient 's condition was complicated by sjogren 's syndrome .
both of the patients were receiving hormonal and immunosuppressive therapy due to pbc prior to the development of concurrent p. carinii and mucormycosis infection .
individuals of any age can be infected with pcp , and people with normal immune function do not typically exhibit symptoms after infection . however , when the host 's immune system is weakened , pneumocystis bacteria begin to multiply , and spread in the lungs , resulting in interstitial plasma cell pneumonia .
patients with connective tissue disease are at an increased risk of infection with pcp as they receive large doses of hormonal and immunosuppressive agents for long periods of time ( 10 ) . in recent years , with the extensive use of biological agents , the incidence of fungal infections has significantly increased ( 1114 ) . in japan , the rate of rheumatoid arthritis ( ra ) involvement in pcp infection after applying infliximab treatment is 0.4% , at an average of 8.5 weeks after the application of infliximab ( 15 ) .
rituximab monoclonal antibody treatment of ra and wegener 's granulomatosis ( wg ) has also been demonstrated to increase involvement in pcp ( 16 ) .
numerous clinical studies have reported systemic lupus erythematosus and wg involvement pcp infection ( 1618 ) .
reports of wg involvement in pcp infection are most common . in a meta - analysis of 11,905 cases of patients with connective tissue disease ,
12% of the 578 cases of wg were complicated by pcp infection , whereas dermatomyositis / polymyositis involvement in pcp infection was only 6% , sle was 5% and ra was 1% ( 19 ) .
connective tissue disease patients ' infection with fungi and pcp is not only related to immune disorders of connective tissue disease but is also associated with the long - term use of hormonal and immunosuppressive therapy ( 20 ) .
however , it is difficult to elucidate which factor specifically causes the disease . in the present study ,
the two cases exhibited leukopenia upon admission to our hospital , as leukopenia may increase the chance of opportunistic infection .
connective tissue disease involvement in pcp infection is related to treatment with cyclophosphamide , methotrexate , hormones , azathioprine , cyclosporine and other biological agents , such as anti - tnf inhibitors and anti - cd20 inhibitors ( 21,22 ) .
hormone therapy is the uppermost risk factor ; > 90% of patients who present with fungal infection involvement pcp have received long - term hormone therapy ( 23 ) . if elderly patients , patients with nutritional deficiency or patients with low - lymphocytes lipoproteinaemia receive > 16 mg / d equivalent hormone ( > 2 months ) or > 20 mg / d ( > 1-month ) hormone therapy , the risk of pcp infection markedly increases ( 24 ) .
cyclophosphamide treatment involvement in pcp infection may be related to significant inhibition of the lymphocyte count ; therefore , patients regularly treated with cyclophosphamide should consider monitoring their cd4 lymphocyte count . to the best of our knowledge , there are no previous reports of pbc involvement in renal mucormycosis infection . in a clinical setting , there have been a small number of reports about liver involvement in mucormycosis infection after transplantation ; the majority involve lung mucormycosis infection and reports of renal mucormycosis infection are rare ( 25 ) .
patients with the disease are predominantly complicated by diabetic acidosis , malnutrition , severe burns , trauma , leukemia , lymphoma , acquired immune deficiency syndrome or other serious wasting diseases , or have received long - term immunosuppressive agents , cytotoxic drugs or adrenocortical hormone ( 27 ) .
most of the mucormycosis diseases are dangerous and the mortality rates are high ( 28 ) .
the main feature of mucormycosis is sexual propagation producing zygosperm and asexual reproduction forming sporangia .
features include : i ) hyphae are relatively thick , unseparated or rarely separated ; ii ) the mycelium wall is relatively thick , the side shoot is at right angle with the base shoot ; and iii ) tend to violate the vessel wall and lumen , thus forming a vessel clot that leads to ischemia , hemorrhage infarction and necrotizing inflammation of adjacent tissues ( 29 ) .
the clinical manifestations of mucor infections are cerebral , lung , gastrointestinal , cutaneous and systemically - spreading infections .
the symptoms of patients with acute disease progress quickly and patients often succumb to the disease within several days or several weeks ( 30 ) .
chronic infection can be manifested as simple granuloma or pyogenic inflammation and granulomatous mixed inflammation ( 31,32 ) .
clinical diagnosis is often based on clinical symptoms , susceptible factors , mycological examination and pathological biopsy , of which , locating hypha in pathological tissue is of most diagnostic significance .
its initial performance is unilateral or bilateral bronchial pneumonia integrating into a large consolidation rapidly and often forming porosis . single or multiple nodules can also be detected .
if the pulmonary embolism is relatively large , the wedge - shaped shadow close to the pleura at the bottom can be seen , which has diagnostic significance .
high - resolution ct can detect symptoms that can not be seen on an x - ray , such as lumen blocking , vignette and pulmonary artery pseudoaneurysm caused by mucormycosis within the bronchus .
mucormycosis progresses rapidly , therefore , early diagnosis and effective treatment are of decisive significance for the prognosis of patients with mucormycosis .
renal involvement of systemically disseminated mucormycosis is rare , and simple renal mucormycosis is scarcer .
case 2 received hormonal and immunosuppressive therapy due to sjogren 's syndrome involvement in pbc . following therapy , the patient was infected with renal mucormycosis . despite active amphotericin b anti - infective therapy ,
the present study provided a retrospective analysis of two cases of pbc involvement in opportunistic fungal infections .
the patients in these cases were infected with fungi after taking hormones and immunosuppressive agents for a long period of time .
this suggests that in patients with rheumatic autoimmune diseases , who receive long - term hormonal and immunosuppressive therapy , the probability of opportunistic pathogen infections increases .
when confronted with an infection whose response to antibiotic therapy is not ideal , a sputum smear and tests for fungi should be conducted .
when necessary , an early histopathological biopsy should be performed to make time for the treatment of the patient . at the same time , actively seeking pathological diagnosis will improve our understanding of these difficult diseases . | the present study aimed to analyze the imaging , clinical and pathological features of fungal infection involvement in primary biliary cirrhosis ( pbc ) by retrospectively analyzing and reviewing the features of two patients with fungal infection involvement in pbc .
both patients were female .
one patient had a confirmed diagnosis of pbc .
the other patient had confirmed sjogren syndrome and pbc .
the two cases of pbc were infected with fungal infection after treatment with hormonal and immunosuppressive agents .
rcr of sputum confirmed pneumocystis spp .
infection in the patient with pbc alone .
the mucormycosis infection was confirmed in the other patient after pathological examination of a renal biopsy .
the state of the illnesses progressed quickly and both patients ultimately succumbed to their conditions .
the patient prognosis of fungal infection involvement pbc is poor .
patients treated with long - term hormone and immunosuppressive agents should be monitored . |
improvements in composite mechanical properties have made them more reliable for use in posterior teeth than they were some years ago14,17 .
the development of shrinkage during polymerization is the major disadvantage of light cured composites and compomers13,16
.the conversion of the monomer molecules into a polymer network is accompanied by a closer packing of the molecules , causing contraction in the composite9,10 .
this contraction creates mechanical stresses in the resin composite , which can disrupt the marginal seal between the composite restoration and dentin or enamel9 .
polymerization shrinkage leads to several clinical problems , such as marginal discoloration , restoration fractures , solubility of the bonding system and marginal leakage12 .
there are three phases in polymerization shrinkage : the pre - gel , gel point and post gel phase , but the only one capable of disrupting the marginal seal between the composite restoration and dentin / enamel is the post - gel phase10,12 .
this occurs because , in the phase before the gel point , the monomers can still move or slip into new positions within the resin matrix , without causing stress at the interface12 . as the number of monomers converted into polymers increases , the flow gradually decreases , while the resin composite becomes stiffer and the material becomes strong enough to exert forces or stress in the bond system9,10 .
post gel polymerization results in clinically significant stress in the composite - tooth bond and surrounding tooth structure24 , leading to adhesion failure12 .
adhesive bond strength , restorative material elastic modulus , cavity design , light intensity and curing time are some of the factors influencing the marginal quality of restorations10,23 .
a number of polymerization techniques have been developed in an attempt to reduce the stress caused by polymerization shrinkage .
initial polymerization with low intensity light followed by final cure with high intensity light may result in improved marginal integrity without losing the achievable material properties19 .
the aim of the technique using low light curing intensity is to prolong the time span before gel point is reached and to increase the material flow capability .
afterwards , high light intensity is necessary to achieve complete polymerization and optimal mechanical properties10 .
another technique that prolongs the time span before reaching gel point , similar to the soft start method , is the pulse activation technique16 .
it differs from the soft start , as there is a waiting interval between the initial low intensity and the final exposure to the high intensity light .
several studies1,21 have investigated the pulse delay technique and the general consensus is that it reduces polymerization shrinkage stress without decreasing mechanical properties . however , these two techniques have long cure times , which are inconvenient for the patient , impractical with children , uncomfortable for the dentist and make the treatment more expensive because of the extra time spent in the chair20 . a different method of providing extremely high irradiance levels is to use curing units that emit high intensity light , as they allow resin composite polymerization to occur very quickly and are recommended because of the curing depth and mechanical properties achieved .
high light curing , which is compensated by the intensity , can polymerize the resin composite in a shorter time .
however , these high light intensities do not allow enough flow for reducing internal stress , thus contribute to high polymerization shrinkage3,19 , and increased leakage3 .
different polymerization modes can lead to the resulting polymer having different structures , even though the degree of monomer conversion is the same6 .
thus , some physical properties , like hardness , diametral tensile strength and wear resistance show differences15,18 .
the differences among the physical properties of composite resin cured with different polymerization systems has been shown to be more evident when this material was immersed in ethanol6,18 .
ethanol causes the resin composite surface to soften , by removing the polymer structure , such as unreacted monomer , oligomers and linear polymers6 or imparting an opener structure to the polymer , facilitating the sorption of pigment agents and increasing wear5,18,22 .
asmussen and hansen5 ( 1986 ) studied the relation between composite resin surface softening and discoloration , and concluded that the softening effect on a composite resin surface can increase its susceptibility to staining .
material color stability over the time is an important factor in the success of an esthetic restoration19 .
thus , this study investigated the effect of different polymerization modes and ethanol immersion on the composite resin susceptibility to staining .
the null hypotheses to be tested were : ( 1 ) there is no difference in the susceptibility to staining of composite resin light - cured by different polymerization modes ; ( 2 ) specimens immersed in water or ethanol present no difference in the susceptibility to staining .
forty cylindrical specimens were prepared using teflon ring moulds ( 5.0 mm in internal diameter and 2.0 mm depth ) held between two glass slabs separated by milar matrix strips , and then pressed with a 500 g load .
the cavity was filled in one increment and was randomly polymerized according to the four experimental light curing mode groups ( table 1 ) .
the initial intensity of soft start group was obtained by polymerization with the light tip at a distance of 0.9 cm from the composite resin top surface ( controlled by an electronic digital caliper ) .
specimens were stored in distilled water at 37c for 24 hours and the top surface of each specimen was polished with flexible aluminum oxide disks ( sof - lex pop - on - 3 m espe ) under water spray .
all specimens were randomly assigned to one of the two media ( distilled water or absolute ethanol ) at 37c for 24 hours . after this period
, the specimens were washed in tap water , immersed separately in 4 ml of 2% methylene blue solution for 12 hours at 37c and thereafter , rinsed in tap water and air - dried .
each specimen was weighed and ground into powder in a hard tissue mill ( marconi equip .
the resulting powder was weighed , and , if the difference between the initial and final weight was greater than 10% , the specimen should be discarded . in this study
the powder from each specimen was immersed for 24 hours in a glass tube containing 4 ml of absolute alcohol pa in order to dilute the methylene blue .
the supernatant ( floating solution ) was analyzed with a spectrophotometer ( beckman du-65 - instruments , inc . ,
fullerton , ca , usa ) adjusted to a measurement wavelength of 668 nm . to calibrate the spectrophotometer , the absorbance of the standard solutions ( 0.1 ; 0.2 ; 0.3 ; 0.5 ; 1 ; 2 ; 4 ; 6 g / ml ) was determined at wavelengths ranging from 400 to 700 nm , and the maximum value was obtained at 668 nm . at this wavelength ,
a coefficient of linear correlation ( r=0.9998 ) and a straight - line equation ( y = a + bx ) were determined .
the following relation was obtained : absorbance = 0.2716 x ( dye concentration ) - 0.0075 . to calculate the quantity of the dye concentration ( mg / ml ) in the experimental samples , the " y " was changed fo the absorbency value of each specimen . for statistical analyses ,
two - way anova and tukey 's test were performed on the data at 0.05 confidence level .
the shapiro - wilk 's test ( test for normality ) ( p>0.05 ) confirmed the normality and homoscedasticity of the groups .
anova ( table 3 ) showed no statistically significant differences ( p>0.05 ) between the two immersion media .
the tukey 's test was applied to individual comparisons and showed that the soft start polymerization mode had the lowest staining means , significantly different from the high intensity and pulse - delay curing modes ( p<0.05 ) .
the conventional polymerization mode did not present statistically significant differences from any other polymerization mode ( p>0.05 ) ( table 2 ) .
degree of freedom ; sq sum of squares ; mq mean square ; - statistically significant difference ; ns - no statistically significant difference .
an important factor in the success of an esthetic restoration is the material color stability over time8 .
the susceptibility of a resin composite to staining may , subject to several factors , be associated with the polymerization mode . under the experimental conditions of this study , the results showed that different polymerization methods can alter composite resin susceptibility to staining .
resin discoloration may be associated with its affinity for stains and water sorption due to its lower monomer conversion degree7 .
water sorption is associated with polymer degradation , and can also be associated with the type of polymer11 .
asmussen and petutzfeldt6 ( 2001 ) reported that light intensity and polymerization time can modify polymer structure formation .
polymerization with pulse delay causes little growth at the centre of the polymer and , consequently , propagation will predominantly add one molecule of monomer after another to a growing polymer chain .
this results in a more linear oligomer or polymer structure with relatively few cross - links .
the final cure will activate a large part of the remaining camphoroquinone and thus give rise to a multitude of growth centers that will increase the tendency to form a branched polymer .
the authors of the above - mentioned study credit the polymer structures formed with the pulse delay technique to the softness of the surface after being immersed in ethanol .
this polymer structure may be associated with a greater sorption of liquid , resulting in more susceptibility to staining .
the high intensity polymerization mode did not show significant differences from pulse delay and conventional polymerization .
initial high intensity develops higher polymerization shrinkage stress due the rapid and immediate polymerization reaction .
a high initial intensity can result in a polymer structure with higher cross - link density , due the large amount of monomer that reacts , generating several growth centers .
however , a supposition for the high susceptibility to staining of this polymerization mode is that 10 seconds of polymerization are not sufficient for an effective cure at a deeper layer of the sample .
aguiar , et al.1 ( 2005 ) showed that reduced polymerization times at high intensity provide an unsatisfactory cure at the deepest layer of the composite resin , and this under - cured layer is more susceptible to staining .
the soft start technique showed less susceptibility to staining and it did not show statistically significant differences from conventional technique .
asmussen and petutzfeldt6 ( 2001 ) reported that conventional polymerization with a continuous intensity will initiate a multitude of growth centers , resulting in a polymer structure with higher crosslink density .
it may be compared with a ladder in which each original monomer molecule represents a step . in the above - mentioned study ,
the intensity for the conventional technique was 450 mw / cm . in the soft start groups of the present study ,
the initial intensity was 300 mw / cm , which is a value close to the one used in asmussen 's study6 . based on these values
, it can be supposed that both soft start groups and conventional groups ( 550 mw / cm ) had comparable polymer structures and showed an equivalent behavior regarding to the susceptibility to staining . however , the conventional technique presented an intermediate behavior , and showed no statistically significant differences from either pulse delay and high intensity , or the soft start group .
ethanol simulates certain beverages and alcoholic drinks , and distilled water simulates the wet intra - oral environment provided by saliva and water .
it was expected that the ethanol medium could increase the resin composite susceptibility to staining .
ethanol causes resin composite surface to soften , by removing the polymer structure such as unreacted monomer , oligomers and linear polymers6 , or imparting a opener structure to the polymer , thereby facilitating the sorption of pigment agents and increasing wear5,18,22 . in a composite resin with the same degree of monomer conversion
, it may be that dissolution by ethanol is more selective in a relatively linear polymer than in one that is expected to have a more cross - linked structure .
in other studies that associated softening agents with staining , the pigment agent was associated with the softening agent5,18,22 .
aguiar , et al.2 ( 2004 ) used alcoholic and aqueous staining solutions to test susceptibility of colored restorative materials to staining , and concluded that alcoholic solutions had higher staining means than aqueous ones .
in the mentioned studies , the pigment penetrated at the same time as the ethanol altered the polymeric matrix . in the present study ,
ethanol was first applied to the resin composite and then the pigment agent was applied .
it is possible that ethanol removed the organic matrix responsible for absorbing the pigment , thus not interfering in the staining .
in vivo studies
it is difficult to establish a direct relation between in vitro and in vivo outcomes .
however , in vitro studies may guide one to conclusions that can be later confirmed in vivo and then improves the clinical activity .
further studies must also been done with another dyes associated with dietary habits , such as consumption of coffee , soft drinks , alcoholic drinks and others .
within the limits of this study , it may be concluded that : the ability of a composite resin surface to be stained was not related to the two immersion media studied ; the mode of polymerization can affect the staining of composite resin restorations ; the soft start polymerization mode resulted in the lowest staining for the composite resin evaluated , statistically different from high intensity and pulse delay , and similar to conventional light curing mode . | the aim of this in vitro study was to evaluate the susceptibility of a hybrid composite resin ( filtek z250 3 m espe ) to staining , when light cured in four different modes and immersed in two different media .
composite resin specimens were randomly prepared and polymerized according to the experimental groups ( conventional - 550 mw / cm2 / 30 seconds ; soft start - 300mw / cm2/ 10 seconds + 550 mw / cm2 / 20 seconds ; high intensity - 1060 mw / cm2 10 seconds ; pulse delay 550 mw / cm2 1 seconds + 60 seconds of waiting time + 550 mw / cm2 20 seconds ) and immersed in one of two media ( distilled water or absolute ethanol ) for 24h .
next , the specimens were immersed in a 2% methylene blue solution for 12 hours .
afterwards , the specimens were washed and prepared for the spectrophotometric analysis . for statistical analysis ,
two - way anova ( 42 ) and tukey 's test were performed on the data at 0.05 confidence level .
soft start showed the least staining , and was statistically different from the high intensity and pulse delay light curing modes ( p<0.05 ) .
conventional light curing unit did not show statistically significant differences from any other groups ( p>0.05 ) .
there were no significant differences between the two immersion media ( p>0.05 ) .
the soft start polymerization mode showed lower susceptibly of the composite resin to staining than high intensity and pulse delay , irrespective of the immersion medium . |
tuberous sclerosis ( tsc ) is a multi system genetic disorder affecting skin , brain / nervous system , kidneys , heart and lung and leading to growth of non malignant tumors .
a combination of symptoms may include seizures , developmental delay , behavioral problems , skin abnormalities , lung and kidney disease .
tuberous sclerosis lesions infrequently cause symptoms in the endocrine , gastrointestinal ( gi ) , and lymphatic systems .
a 26-year - old female presented with galactorrhea for the past 10 days and menstrual irregularities over the past six months .
she denied any history of substance abuse , drug intake ( antipsychotic , anti emetic , hormonal preparations ) , hypothyroidism , chronic liver or kidney diseases , epilepsy .
she was a well nourished female with mild pallor , tiny nodule on face , subungual fibroma in hands .
serum prolactin was 85 ng / ml with fsh-4.66 and lh- 4.21 miu / ml . in view of the theoretical possibility of involvement of cell groups of other anterior pituitary lineage ,
tests for evaluation of other anterior pituitary hormones were carried out and were found to be normal . abdominal and pelvic ultrasound revealed no abnormality .
computed tomography ( ct ) scan revealed multiple intracerebral calcifications . these calcified lesions / subependymal hamartomas are seen along the lateral surface of the lateral ventricles giving rise to characteristic candle dripping appearance .
magnetic resonance imaging ( mri ) of the brain ruled out the presence of any pituitary mass .
the combined clinical scenario along with the radiologic findings leads to the diagnosis of tsc with hyperprolactinemia .
patient was prescribed cabergoline 0.5 mg twice daily , which resulted in amelioration of galactorrhea and regularization of menses .
there is a growing body of evidence that tsc patients develop hormone secreting tumors involving the neuroendocrine system at higher frequency than the general population .
cushing 's disease , hypoglycemia secondary to insulinomas , precocious puberty , thyrotoxicosis , hypercalcemia secondary to parathyroid adenomas , hyperprolactinemia and acromegaly have all been reported in tsc patients .
the circulating prolactin of our patient may be of pituitary origin or may possibly be secreted ectopically by a hamartoma .
there has been evidence linking neuroendocrine tumors to the akt / mtor / s6 kinase pathway that is regulated by the hamartin / tuberin ( tsc1/tsc2 ) complex .
thus , it is beginning to appear that the occurrence of these neuroendocrine tumors ( net ) in tsc is more than random coincidence . | tuberous sclerosis is a multi system genetic disorder affecting skin , central nervous system , kidney , heart and lungs .
we present a case report of a 26 year old female with tuberous sclerosis who presented with galactorrhea and menstrual irregularities due to hyperprolactinemia . |
left ventricle non - compaction ( lvnc ) or persistence of spongy myocardium is a rare form of congenital cardiomyopathy which presents with cardiac failure , thromboembolic events , arrhythmia and sudden death .
we report a family with two deceased children and two alive offspring diagnosed with left ventricular ( lv ) non - compaction . a 9-year - old boy and
a 16-year - old girl of our reported family suddenly died following exercise and emotional provocation .
both had a history of convulsion and syncope , with the latter taking anticonvulsant medication .
the remaining two offsprings , one boy and one girl were diagnosed as having isolated lvnc .
syncope and convulsion can be first manifestations of lvnc and heralding signs for sudden death in patients with lvnc .
isolated left ventricular non - compaction ( lvnc ) is one of the rare genetic heart diseases recently known , and characterized by heterogeneous defects in endocardium accompanied by increased trabeculation due to abnormal embryogenesis of the normal myocardium . in this disease ,
the myocardium is composed of two layers : spongy or non - compacted and normal or compacted ones .
the clinical manifestations include symptoms and signs of left ventricular failure ( common ) , those of right ventricle ( rare ) , ventricular arrhythmias , sudden death , mural thrombi and embolic events with cardiac origin .
we report a family having the history of two offspring deaths , at the ages of 9 and 16 , with two live ones suffering from lvnc .
we reported sudden death of a 9-year old boy with a history of two episodes of unprovoked convulsion and syncope in school and his 16-year old sister due to sudden death while she was getting back home after a feast . a 12year - old boy and a 7-year - old girl
were referred to pediatric cardiology clinic for cardiac evaluation following occurrence of sudden death in their siblings .
the first pregnancy of the mother ended in abortion at the age of two months of gestation , and the second offspring was a girl who died one week after birth following an operation to repair esophageal atresia .
the first victim of suspected lvnc in this family was a nine year old boy with a history of two episodes of unprovoked convulsion and syncope in school .
the first attack was in break time with spasm of neck and body , ended uneventfully .
the next one was during exercise , with sudden fall but no urinary incontinence , eye deviation or sialorrhea . the electrocardiogram ( ecg ) , electroencephalography and lab tests showed no abnormalities at that time .
the family lost their 16-year old daughter due to sudden death while she was getting back home after a feast .
she also had experienced two seizure episodes associated with loss of consciousness , one at the age of 9 during exercise at school and the other at the age of 10 on vacation .
both episodes had ended uneventfully , and brain ct scan with contrast demonstrated no pathologic finding .
the 12-year - old boy had no history of seizure , syncope , easy fatigability , exertional dyspnea or hospitalization .
on 2d transthorasic echocardiography , the parasternal short axis and four chamber views demonstrated increased left ventricular ( lv ) wall thickness accompanied by trabeculations and deep recesses that were indicative of lv non - compaction .
four chamber and short axis views demonstrating the ratio of non - compacted to normally compacted segments the coronary arteries were normal and no pericardial effusion and intracavitary thrombi were detected
. the 7-year - old girl also had no positive history of hospitalization , seizure , easy fatigue .
her ecg was normal , however , her 2d echocardiograms were compatible with the diagnosis of lv non - compcation , based on the diagnostic criteria already reported by frischknecht et al , with increased lateral , inferior and apical wall thickness and deep recesses and trabeculations .
the coronary arteries were normal and no pericardial effusion and intracavitary thrombi were detected ( fig .
parasternal long axis view and m - mode echocardiography showing abnormal trabeculation and spongy pattern of left ventricle the parents were evaluated clinically and by echocardiography .
this case report highlights the importance of syncope and seizure as heralding signs for sudden death in two deceased children of a family with two offspring having documented lv non - compaction .
although syncope and sudden death have been previously reported in adults with lvnc but to date , as to best of our knowledge , sudden death with prior heralding sign of syncope and seizure has not been reported in children with lv non - compaction . in our reported family
, both of the unexpected deaths occurred after previous episodes of uneventful seizure and syncope . considering the common embryologic origins of central nervous system and myocardium , occurrence of syncope and seizure in lvnc could be explained .
lv non - compaction or persistence of spongy myocardium , first described in 1984 , is a rare form congenital cardiomyopathy with a prevalence of 0.05 - 0.24/year .
the limitation of our report is lack of echocardiographic evidence for confirmation of diagnosis in the two deceased and not timely - referred and timely - diagnosed children .
we suggest careful biannual follow up ( ecg , holter monitoring , echocardiography ) and anticoagulation therapy ( aspirin , dose : 3 - 5mg/ kg / daily ) for alive sibling .
unexplained seizure and syncope in a child , particularly in a familial pattern , may warrant further echocardiographic evaluation for early diagnosis of left ventricular non - compaction to prevent catastrophic events .
although the numbers of our patients are limited , but it seems that seizure and syncope can be considered as warning signs in those patients with lvnc who are at high risk for sudden death . | backgroundleft ventricle non - compaction ( lvnc ) or persistence of spongy myocardium is a rare form of congenital cardiomyopathy which presents with cardiac failure , thromboembolic events , arrhythmia and sudden death.case presentationwe report a family with two deceased children and two alive offspring diagnosed with left ventricular ( lv ) non - compaction .
a 9-year - old boy and a 16-year - old girl of our reported family suddenly died following exercise and emotional provocation .
both had a history of convulsion and syncope , with the latter taking anticonvulsant medication .
following their demise , the other members of the family were evaluated by echocardiography .
the remaining two offsprings , one boy and one girl were diagnosed as having isolated lvnc.conclusionsyncope and convulsion can be first manifestations of lvnc and heralding signs for sudden death in patients with lvnc .
echocardiography can be helpful for early diagnosis . |
pharmacology forms the backbone of rational therapeutics , being both a basic and applied science .
the primary objective of teaching pharmacology is to enable undergraduate medical students to take rational therapeutic decisions in clinical practice.1 it is one of the most evolving branches in medical sciences .
hence , not only concepts but also the teaching methodologies and evaluation methods need to be kept under continuous review .
traditionally , it has focused more on factual information , with little or no emphasis on clinical and applied aspects . dispensing
pharmacy and experimental pharmacology has remained the cornerstone of conventional pharmacology practical exercises . however ,
clinical utility and relevance of these practical exercises have always been questioned and criticised.2 the primary objective of teaching pharmacology to undergraduate students can be fulfilled only if they are well acquainted with the subject with respect to innovations in this field .
kashibai navale medical college and general hospital ( sknmc and gh ) is as per maharashtra university of health sciences which mainly focuses on traditional teaching methodologies like lectures in whole class and practicals based on experimental and clinical pharmacy .
little emphasis has been given to clinical patient - related teaching , and newer teaching methods like problem base learning / microteaching and integrated teaching are not advocated as part of regular teaching .
however , there is strong demand from students for these newer teaching methodologies . keeping this demand in view
it is accepted that the feedback from students serves as an effective tool in developing teaching methodology and evaluation methods in undergraduate teaching.3 the present study is a step towards achieving this tool by assessing the student 's attitude , perception and feedback on teaching
this cross - sectional study involved 140 second year medical students from iii , iv and v terms studying in sknmc and gh , who were surveyed with pre - validated questionnaire designed for them .
the questionnaire was adapted from the previous studies that assessed feedback of second year medical students on teaching
learning methodology and evaluation methods in pharmacology.47 a few modifications were done in the questionnaire to best fit with reference to university syllabus .
questionnaire validation was done in 15 ( 5 - 10% ) students to review the questionnaire to determine whether the questionnaire measured what it was designed to measure .
content validation method was used for the validation procedure.8 answer to each question was reviewed by our experts and the requisite modifications and deletions were done to validate the 22 questions out of total 25 questions in the questionnaire .
the following validation criteria were used :
time requirement for completion of questionnaire ( 5 - 10 min)appropriateness of questionnaire for collecting datarepetition or inappropriate questionslogical order of questionsclear , concise and unambiguous questionseasy and meaningful instructionscomments and suggestions specified to the application guidelines.after getting the protocol approved by the institutional ethics committee ( sknmc no / ethics / app/2010/72 ) , the list of second year medical students studying at sknmc was obtained . after getting the list ,
a total of 140 students were administered a pre - validated questionnaire containing 22 questions at the end of pharmacology class and they were asked to fill up the questionnaire .
suggestions were also asked regarding the quality of good pharmacology teachers and modification needed in pharmacology teaching methods .
the filled questionnaires were collected immediately once they were filled up , on the same day .
time requirement for completion of questionnaire ( 5 - 10 min ) appropriateness of questionnaire for collecting data repetition or inappropriate questions logical order of questions clear , concise and unambiguous questions easy and meaningful instructions comments and suggestions specified to the application guidelines .
out of total 140 students , 64 were females and 76 were males , and the mean age of the students was 19.7 0.26 years . in reply to the questions ,
majority of the students ( 63.62% ) were found to know somewhat about the subject ; only 17.28% students were totally unaware of the subject .
pharmacology was opined to be a very useful , practically important and interesting subject by 47.89% of senior students when asked about their opinion .
about 7% senior students gave variable responses stating that although useful and interesting , it is a very difficult , volatile and confusing subject .
the same response was obtained from second year students , i.e. , majority ( 59.62% ) of them found the subject to be very useful , practically important and interesting .
when the students were asked to grade the subject in comparison with the other subjects , majority ( 51.3% ) of them found no difference from the other subjects , i.e. , they treated all the subjects equally important .
46.06% students mentioned that they would like to have few lectures on newer drugs / and recent advancement on therapy during internship .
39.93% students also suggested that there should be pharmacology orientation course of few days during internship .
when the students were asked about their opinion on pharmacology teachers , 41.7% students found most of the pharmacology teachers good and knowledgeable and 40.68% found most of the teachers good and few of them average .
58.42% students mentioned that pharmacologists are respected as expert therapeuticians undoubtedly , whereas 24.45% answered they had no idea .
significant number ( i.e. , 73.8% ) of students mentioned that the course time of 1 years is adequate for teaching second year mbbs students .
only 14.07% students gave favourable stand on the argument that second year medical course time of 1 year is adequate .
we received varied suggestions and opinions on the qualities of a good pharmacology teacher . according to them ,
the teacher should be knowledgeable , approachable , student interactive , problem solving , should come up with good audiovisual presentations , should have good communication , clarity in speech , expressivity and clear basic knowledge of the subject .
when the students were asked about being a pharmacologist in future , only 4.22% students accepted that they wanted to become a pharmacologist [ figure 1 ] .
comparison of responses to general questions ( % ) of the participants to the questionnaire ( n = 140 ) majority of the students found cardiovascular system ( 49.25% ) , central nervous system ( 39.19% ) and general pharmacology ( 36.85% ) as the most interesting topics , while autonomic nervous system ( 32.09% ) , respiratory system ( 13.91% ) , gastrointestinal system ( 9.08% ) and autacoids ( 7.68% ) were treated as less interesting topics in pharmacology .
cardiovascular system ( 60.10% ) , chemotherapy ( 54.06% ) and central nervous system ( 44.15% ) are going to be the most useful topics in internship , as per their opinion .
59.06% students suggested change in the order in which pharmacology topics are taught , whereas 40.94% accepted the present order to be the best .
48.53% students preferred audiovisual - aided lectures as the best teaching method , and the second highest ( 39.13% ) preference was given to clinical / patient - related pharmacology followed by multiple choice question ( mcq)-based study ( 39.13% ) and interactive classes ( 24.23% ) with strict bilateral communication .
prescription writing ( 64.89% ) , comments on fixed - dose combinations ( 35.88% ) and criticism and rewriting of prescription ( 29.90% ) were found amongst the most interesting and useful topics from pharmacology practicals in future [ figure 2 ] . when the students were asked about the methods to make pharmacology teaching more interesting , about 46.06% students suggested addition of problem - based learning in conventional teaching and 37.33% insisted in microteaching sessions .
furthermore , when they were asked about the topics they want to be discussed in lecture / practicals of pharmacology in addition to the usual topics , about 46.05% mentioned dose schedule and calculation and 44.44% suggested drugs used in special conditions like kidney dysfunction , sexual dysfunction and emergency drugs [ figure 2 ] .
comparison of responses to teaching and evaluation methods ( % ) of the participants to the questionnaire ( n = 140 ) students were also questioned about the learning materials and methods that they use to learn pharmacology .
comparable ( 47.85% ) number of students admitted using the combination of teacher 's class notes , self - prepared notes and textbook .
about 36.79% students mentioned that they study pharmacology regularly for gaining more knowledge with understanding ( 57.80% ) [ figure 2 ] . amongst the evaluation methods for preparing for university examination , preliminary examination ( 51.12% ) was given the highest score , followed by terminal examination ( 48.52% ) and mcq test ( 38.16% ) [ figure 3 ] .
for making pharmacology more interesting / approachable / understandable and practicable , the students suggested incorporation of new drug information along with prototype drugs in a comparative manner , conduction of mcq tests and viva at the end of every topic taught , accompanied by video clipping and student slide show .
comparison of responses to learning methodology ( % ) of the participants to the questionnaire ( n = 140 )
it is very important to emphasize the academic need of reviewing the teaching programs from time to time and making adequate modifications , to keep pace with progress in the subject and to cope with the requirements of the beneficiaries . in the present study ,
many interesting things came into picture and students suggested many ways that could be incorporated in conventional pharmacology teaching so as to make the subject more interesting and understandable .
although majority of the entrant students had somewhat knowledge about pharmacology , there was a class of students who were totally unaware of the subject ; hence , there is a need to conduct subject orientation programme before the students enter into medical education .
majority of the students found cardiovascular system , central nervous system and general pharmacology as the most interesting topics in pharmacology , whereas autonomic nervous system , gastrointestinal system , autocoids and respiratory system were the less interesting topics .
so , these topics need to be emphasized more to draw attention of the students . this could be done by utilizing their suggestions like use of microteaching sessions , problem - based learning , patient - related teaching and interactive teaching with strict bilateral communication in conventional teaching .
involvement of students improves learning , and thereby their performance in university examinations.4 this approach towards teaching pharmacology would be appreciated by students than theoretical conventional teaching .
some studies mentioned about the inability of doctors in writing rational prescription and it is suggested that there should be incorporation of some training of clinical pharmacology in the internship.9 presently , we are conducting few lectures on pharmacovigilance and rational prescribing as a part of internship - orientation programme . in this study , students have additionally asked for conducting few lectures on new drugs and recent advances in the therapies , during internship .
hence , few lectures on recent therapies may be incorporated in this programme for the benefit of the students .
this reflects their lack of knowledge about the subject scope and it may be their mindset to treat the subject as a very difficult one .
hence , they need to be well versed with the future prospective in the subject . by assisting the students in subject learning through simple and understandable illustrations , this mindset may be changed .
students unanimously accepted all pharmacology teachers as good and knowledgeable ; however , they expected free interaction and problem solving with the teachers .
they suggested that teachers should come up with good presentation , clarity in speech and expressivity .
definitely efforts can be taken in this direction through discussion and seeking guidance from senior experienced faculties .
conducting mcq tests and viva sessions at the end of every topic was suggested by many students , as they study pharmacology regularly due to these tests . although not feasible , few mcqs may be provided as home assignment from time to time and viva sessions may be planned at the end of term during revision classes . in light of the results of the present study , we should understand that all these issues must be taken into consideration while updating the undergraduate curriculum .
also , there is a definite need for modification of undergraduate curriculum so as to make pharmacology more interesting and practicable . | background : to assess the student 's attitude , perception and feedback on teaching learning methodology and evaluation methods in pharmacology.materials and methods : one hundred and forty second year medical students studying at smt .
kashibai navale medical college , pune , were selected .
they were administered a pre - validated questionnaire containing 22 questions .
suggestions were also asked regarding the qualities of good pharmacology teachers and modification in pharmacology teaching methods .
descriptive statistics were used and results were expressed as percentage.results:majority of the students found cardiovascular system ( 49.25% ) as the most interesting topic in pharmacology , whereas most of the students opined that cardiovascular system ( 60.10% ) , chemotherapy ( 54.06% ) and central nervous system ( 44.15% ) are going to be the most useful topics in internship .
48.53% students preferred clinical / patient - related pharmacology and 39.13% suggested use of audiovisual - aided lectures .
prescription writing and criticism of prescription were amongst the most useful and interesting in practical pharmacology .
students expressed interest in microteaching and problem - based learning , whereas seminars , demonstrations on manikin and museum studies were mentioned as good adjuvants to routine teaching .
multiple choice question ( mcq ) practice tests and theory viva at the end of a particular system and periodical written tests were mentioned as effective evaluation methods .
students were found to have lot of interest in gathering information on recent advances in pharmacology and suggested to include new drug information along with prototype drugs in a comparative manner.conclusion:there is a need of conducting few microteaching sessions and more of clinical - oriented problem - based learning with mcq - based revisions at the end of each class in the pharmacology teaching at undergraduate level . |
cardiovascular diseases ( cvd ) is the leading cause of death globally , causing nearly 17.3 million deaths in 2008 , with the number expected to increase to 23.3 million by 2030 , . according to world health organization , over 80% of the world
s deaths from cvds occur in low and middle income countries , as they have less access to effective and equitable healthcare services which respond to their needs , including early detection service .
easy to use diagnostic and screening devices , amenable to field deployment for screening and triaging are important in such scenarios .
carotid artery stiffness has been demonstrated to have strong association with increased intima - media thickness and even with severity of plaques in aorta .
hence , non - invasive measurements of carotid artery stiffness , that can estimate cardiovascular health , are an attractive option for screening and early detection .
however , state of the art systems for measuring arterial stiffness are highly expensive , and have a laborious operating procedure , and are hence not suited for field applications . ultrasound echo tracking systems , such as aloka e - tracking , artlab etc . ,
require expert sonologists to examine ultrasound data of the artery to perform stiffness measurements . there exists an unmet need for a practical , affordable , easy - to - use technology to non - invasively measure arterial stiffness in an automated manner , which could be used by general medical practitioners and health workers .
such a non - expert operable device could overcome the skill barrier and also reduce the time taken for test , thereby making it suitable for large scale cardiovascular screening in addition to diagnosis . to address this need ,
we have developed artsens ( arterial stiffness evaluation for non - invasive screening ) , a device that overcomes the limitations of present systems effectively , while performing the measurements with accuracy and precision according to clinically accepted standards , .
we had previously presented the concept of an image - free system for measurement of arterial stiffness and also demonstrated the accuracy and repeatability of measurements in controlled laboratory settings , . in this paper , we start with a technical overview of artsens device , and its operation , and present a detailed validation of the ability of the device to perform accurate measurements of arterial stiffness in - vivo , in clinical settings . the feasibility of using stiffness measurements performed in sitting posture is also investigated . from the list of major clinically accepted estimates of arterial stiffness enlisted in table 1
, it is evident that evaluation of the arterial stiffness requires an accurate measurement of arterial distension ( \documentclass[12pt]{minimal }
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} { } $ \delta d)$
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} { } $ d_{s}= d_{d}+\delta d ) $
\end{document } and the systolic and diastolic blood pressures ( \documentclass[12pt]{minimal }
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} { } $ { p}_{d})$
\end{document}.table 1clinically accepted measures of arterial stiffness.measure of arterial stiffnessequationpressure strain elasticity , \documentclass[12pt]{minimal }
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} { } $ e_{p}$
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} { } $ e_{p}=\frac{d_{d } \times \delta p}{\delta d}$
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} { } $ ac$
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} { } $ ac= \frac{\pi(d_{s}^{2 } - d_{d}^{2})}{4\delta p}$
\end{document}stiffness index , \documentclass[12pt]{minimal }
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} { } $ \beta$
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} { } $ \beta=\frac{{\rm in}({p_{s}}/p_{d})}{(\delta d / d_{d})}$
\end{document } artsens utilizes a single element ultrasound transducer to interrogate the artery and obtain all relevant arterial dimensional measurements in an automated manner .
the principle of image - free measurement and system architecture of artsens is presented in section ii .
this section explains the hardware designed to excite the transducer , receive echo signals from the artery and digitize the signals , and also provides a detailed description of algorithms used to extract required parameters from the echo signals .
section iv presents the results of the study , and illustrates the accuracy of artsens by comparison with state of the art echo tracking system for evaluating carotid artery stiffness .
1 . a single element ultrasound probe , operated in the pulse echo modality is used to investigate arterial dynamics .
this probe , placed on the neck over the carotid artery , sends sharp pulses of high frequency ultrasound into the body that are reflected by the artery walls and other structures in the sound propagation path .
intelligent signal processing and automated measurement algorithms automatically identify the arterial wall echoes , track wall motion and measure arterial distension ( \documentclass[12pt]{minimal }
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} { } $ \delta d)$
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} { } $ { d}_{d})$
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these are used to compute various measures of arterial stiffness such as stiffness index ( \documentclass[12pt]{minimal }
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} { } $ e_{p})$
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figure 1.system architecture of artsens .
the measurement requires no intervention from the operator other than positioning of the probe over the artery as illustrated in fig .
the system utilizes a single element 5 mhz ultrasound transducer ( 10 mm diameter , hengxuannanshi , china ) .
the ultrasound frequency of 5 mhz was selected based on the conflicting requirements of high resolution while ensuring required depth of penetration which is about 60 mm for measurements on carotid artery considering the attenuation coefficient of soft tissue as 0.7db / cm / mhz .
the transducer s narrow half angle beam width of about 1.3 ensures that strong and distinct echoes will be obtained only when the transducer is kept normal to the artery .
custom designed probe housings were developed for the single element transducer , to enable easy operation .
the white probe was used in the current study . the custom analog front end electronics hardware developed for artsens
5 . the board has a low voltage ( + /15 v , 500 ma ) power supply section for the digital logic and analog front end sections , and a high voltage ( 100 v , 50 ma ) section used for ultrasound transducer excitation .
digital pulses generated using a microcontroller are translated to high voltage levels and used to excite the transducer in the pulse echo modality .
the reflected echo signals are then passed to an analog signal conditioning section consisting of a high pass filter of cut off frequency 2.56 mhz and a dual stage amplifier with a total gain of 40 db .
a transmit receive switch is used to protect the amplifier section from the high voltage transmit pulses .
figure 4.single element ultrasound probes designed for artsens .
the conditioned analog signals are digitized at the rate of 100 ms / s using a ni usb 5133 high speed digitizer ( national instruments ) .
the digitized signals , referred to as frames , are then given to subsequent signal processing algorithms for automated measurement .
the digitized signals are transferred in real time into a laptop computer and processed using a signal processing virtual instrument , developed in labview using state machine architecture .
the raw ultrasound data frames ( where each frame is defined as the echo obtained for a transducer excitation pulse ) are pre - processed to improve its signal to noise ratio ( snr ) .
the frames are filtered using a \documentclass[12pt]{minimal }
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} { } $ 4^{\mathrm { th}}$
\end{document } order zero phase butterworth band pass filter with upper cut off frequency of 8 mhz and lower cut off frequency of 1 mhz and then passed through a time gain compensation ( tgc ) block to compensate for the signal attenuation encountered as it passes through the soft tissue , as seen in fig . 7 .
the arterial near ( proximal ) and far ( distal ) walls are identified based on the inherent out of phase motion of the wall echoes , as the artery contracts and relaxes during systole and diastole respectively .
the phase of wall motion is detected by locating artery wall echo peaks in the first frame , and then using the next few ( 5 to 10 ) frames to identify the two consecutive artery wall echoes that move opposite to each other .
figure 7.(a ) raw echo signal ( frame ) received from the artery ( b ) signal after pre - processing .
notice significant improvement in snr , elimination of transducer ringing artefacts and compensation for echo attenuation to improve amplitude of farther echoes .
( a ) raw echo signal ( frame ) received from the artery ( b ) signal after pre - processing .
notice significant improvement in snr , elimination of transducer ringing artefacts and compensation for echo attenuation to improve amplitude of farther echoes .
once the artery wall locations are identified , the system goes to tracking state where the movements of near wall and far wall echoes are tracked for a few cardiac cycles as they move out of phase w.r.t each other in a quasi - periodic manner .
correlation based algorithm is used to find the shift in the echo location from that of the previous frame .
arterial distension \documentclass[12pt]{minimal }
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} { } $ d(t)$
\end{document } is calculated from each frame as the sum of relative near and far wall shifts w.r.t .
the carotid artery lumen diameter is calculated as the distance between the intima layers on both walls , corresponding to the trailing edge of the near wall echo and leading edge of the far wall echo . a smoothened hilbert envelope of the region of interest ( roi )
a wall motion negative correlation check running simultaneously along with tracking ensures that tracking is done on near and far walls without fail , .
this is ensured by checking that the near and far wall echoes are moving opposite to each other , i.e. , the absolute values of the near and far wall motion patterns have a negative correlation .
if the condition is not satisfied , the system goes back to wall identification state .
the distension and diameter values as obtained from the tracking stage are recorded and stored in a buffer of size \documentclass[12pt]{minimal }
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} { } $ b$
\end{document } , calculated as the number of points required to store distension of around two cardiac cycles .
once the buffer is filled , the valley locations are found out to obtain distension cycles , from which \documentclass[12pt]{minimal }
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} { } $ \delta d$
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the diameter values corresponding to these valley points in the distension array are identified as end diastolic diameters \documentclass[12pt]{minimal }
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.
gaussian error elimination is performed to eliminate any random data which could have been included in the measurement .
the arterial stiffness index ( \documentclass[12pt]{minimal }
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} { } $ d_{d}$
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} { } $ { p}_{d})$
\end{document } , separately measured and entered by the operator prior to artsens measurement .
a snapshot of the various versions of the artsens device that were developed is illustrated in fig .
the hardware for data acquisition and subsequent signal processing modules were embedded together in the device , making it easier for the operator to use .
figure 8.technology evolution of artsens ( a ) desktop prototype ( b ) tablet version ( c ) rugged version for field deployment ( d ) handheld version under development .
technology evolution of artsens ( a ) desktop prototype ( b ) tablet version ( c ) rugged version for field deployment ( d ) handheld version under development .
the performance of the signal processing algorithms , the repeatability and reproducibility of the instrument and artsens usability in controlled laboratory settings have already been validated and reported . here we present an extensive validation of artsens in clinical setting . the objectives of this study are the following .
( a)to establish feasibility of artsens measurements in clinical setting on large number of subjects(b)to verify the ability of artsens to provide accurate estimates of stiffness with the subject in sitting posture.(c)to establish the measurement accuracy of artsens in comparison with state of art image based ultrasound echo tracking system.the aloka \documentclass[12pt]{minimal }
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} { } $ \alpha 10 $
\end{document } prosound e - tracking system was taken as the reference equipment for evaluation of carotid artery stiffness . to establish feasibility of artsens measurements in clinical setting on large number of subjects to verify the ability of artsens to provide accurate estimates of stiffness with the subject in sitting posture . to establish the measurement accuracy of artsens in comparison with state of art image based ultrasound echo tracking system .
the procedure of arterial stiffness measurements and the overall study protocol approved by the institutional review board are explained below and are shown in fig .
figure 9.artsens validation study protocol ( a ) informed consent ( b ) blood pressure ( c ) blood sampling ( d ) anthropometry ( e ) artsens measurement ( f ) aloka e - tracking measurement .
artsens validation study protocol ( a ) informed consent ( b ) blood pressure ( c ) blood sampling ( d ) anthropometry ( e ) artsens measurement ( f ) aloka e - tracking measurement .
male and female subjects , above the age of 18 years , with no documented history of cardiovascular or peripheral vascular disease were included .
all subjects were informed of the study objectives , backgrounds and protocol before data collection and informed consents were obtained from them .
each subject was registered to the study with a unique identifier , and the personal details were filled in .
subjects were given a light breakfast and allowed to relax for at least 15 minutes before proceeding with measurements .
blood pressure at the brachial artery was measured using a sphygmomanometer in both sitting and supine position .
the stiffness of the carotid artery of each subject was evaluated both using artsens , and also using an ultrasound echo tracking system ( aloka \documentclass[12pt]{minimal }
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} { } $ \alpha 10 $
\end{document } prosound e - tracking system ) .
the left carotid artery location was identified by palpation and the ultrasound probe was placed nearly 2.5 cm below the carotid bifurcation .
the angle of the probe was adjusted to get strong echoes from the near and far wall of the artery .
artsens was configured to give the average of five measurement results where each measurement comprised of five continuous cycles of distension .
10 shows a screenshot of artsens graphical user interface ( gui ) indicating all measurements .
screenshot of artsens gui indicating measurements . for measurement using the e - tracking system , 3-lead electrocardiogram ( ecg ) was placed on the subject s body .
the near and far walls of the artery were manually identified on the b - mode image and the wall motion was tracked by the aloka \documentclass[12pt]{minimal }
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} { } $ \alpha 10 $
\end{document } prosound e - tracking system . when a steady waveform was obtained for 45 cycles ,
image is frozen to acquire the data which were accumulated over the period to start arterial stiffness analysis . in the analysis window ,
measurements were taken in both sitting and supine postures with corresponding systolic and diastolic pressures entered .
10 shows a screenshot of aloka measurement and post analysis screen . the agreement between the arterial stiffness readings given by artsens to those given by the ultrasound imaging system
least square regression models , performing linear fit for stiffness estimates from imaging systems and those from artsens were obtained under the assumption that random error is associated with only artsens results and that the results from imaging system are true values without random errors .
further , a bland altman analysis was performed to examine the degree of variation between the two readings .
the limits of agreement were defined as \documentclass[12pt]{minimal }
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. two artsens measurements were performed on a few ( \documentclass[12pt]{minimal }
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} { } $ \text{n}=33 $
\end{document } ) randomly selected subjects . in such cases ,
the repeatability of measurements performed by artsens was evaluated by computing coefficient of variability ( repeatability ) as the ratio of the standard deviation of the differences between the two artsens measurements to the average of the means .
a trend analysis was also performed to investigate the ability of artsens to detect age - related changes in arterial stiffness .
artsens was used to measure the carotid artery stiffness of all these subjects . typical time taken for a measurement was less than 5 minutes .
this demonstrated the ability of artsens to perform in - vivo measurements of arterial stiffness .
in very few cases , the measurement took more time , as it was difficult to position the probe at the correct location to get stable distension waveforms .
of the total 125 subjects , a few subjects on whom it was difficult to perform the measurements on the exact same locations due to change in posture or probe dimensions illustrated less repeatability in measurements .
such suspected cases of measurement error , in which there was significant variation in the stiffness values recorded by the two instruments ( artsens and aloka e - tracking ) were eliminated as outliers . after eliminating a few suspected cases of data entry error and cases with missing data , a total of 111 subjects were selected for analysis .
a summary of the linear regression analysis is provided in table 2 and table 3 .
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} { } $ \delta d , d_{d},d_{s},\beta , ac$
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\end{document } were found to have a strong positive correlation with the corresponding values obtained from the imaging system , measured in sitting posture , as indicated in table 2 .
arterial distension measured using artsens was found to have a correlation coefficient ( \documentclass[12pt]{minimal }
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} { } $ r$
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the correlation of both end - diastolic diameter and systolic diameter were observed to be 0.5 .
this slightly lower values of correlation coefficient are expected in direct comparison of dimensional measurements , as these measurements were performed sequentially , using two different instruments and hence perfect matching of the site of measurement is not possible due to practical limitation of positioning the larger imaging probe on the exact same location as that of the smaller artsens probe . moreover
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} { } $ \beta $
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the pressure strain elastic modulus ( \documentclass[12pt]{minimal }
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} { } $ e_{p})$
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the correlation of arterial compliance ( \documentclass[12pt]{minimal }
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} { } $ ac)$
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figure 11.screenshot of aloka e - tracking ( a ) b - mode indicating cursor locations ( b ) post analysis screen .
figure 12.comparison of arterial distension ( \documentclass[12pt]{minimal }
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} { } $ \delta d)$
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figure 13.comparison of end - diastolic diameter ( \documentclass[12pt]{minimal }
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} { } $ { d}_{d})$
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figure 14.comparison of systolic diameter ( \documentclass[12pt]{minimal }
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} { } $ { d}_{s})$
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figure 15.comparison of stiffness index ( \documentclass[12pt]{minimal }
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} { } $ \beta ) $
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screenshot of aloka e - tracking ( a ) b - mode indicating cursor locations ( b ) post analysis screen .
comparison of arterial distension ( \documentclass[12pt]{minimal }
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comparison of end - diastolic diameter ( \documentclass[12pt]{minimal }
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comparison of systolic diameter ( \documentclass[12pt]{minimal }
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comparison of stiffness index ( \documentclass[12pt]{minimal }
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} { } $ \beta ) $
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strong correlation was observed even when the artsens stiffness estimates measured on subjects in sitting posture were compared with those made using the imaging system on subjects in supine posture .
it may be observed from table 3 , that the arterial stiffness estimates ( both \documentclass[12pt]{minimal }
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} { } $ \beta $
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} { } $ e_{p})$
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} { } $ r$
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the \documentclass[12pt]{minimal }
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} { } $ r$
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it may be remembered that there is a difference in the arterial pressures , both systolic and diastolic , when the subject moves from sitting to supine posture , and this will cause corresponding differences in the arterial distension and end - diastolic diameter values .
however , the strong observed correlation of arterial stiffness estimates validates the use of artsens to evaluate stiffness in the sitting posture .
this is relevant , as a supine measurement may not be often possible in field settings , when artsens is deployed for screening . to evaluate the degree of agreement between stiffness measurements provided by artsens and the imaging system , bland - altman plots with limits of agreement defined as \documentclass[12pt]{minimal }
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} { } $ \pm 2sd$
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figure 16.comparison of pressure strain elastic modulus ( \documentclass[12pt]{minimal }
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figure 17.comparison of arterial compliance ( \documentclass[12pt]{minimal }
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} { } $ ac)$
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figure 18.bland altman plot of end diastolic diameter ( \documentclass[12pt]{minimal }
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} { } $ _ { \mathrm { artsens}})$
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figure 19.bland altman plot of arterial distension ( \documentclass[12pt]{minimal }
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figure 21.bland altman plot of pressure strain elastic modulus ( \documentclass[12pt]{minimal }
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} { } $ _ { \mathrm { artsens}})$
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figure 22.bland altman plot of arterial compliance ( \documentclass[12pt]{minimal }
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figure 23.distribution of the differences between beta measurements given by artsens and imaging system in sitting posture
.
figure 24.distribution of the differences between \documentclass[12pt]{minimal }
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figure 25.distribution of the differences between \documentclass[12pt]{minimal }
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comparison of pressure strain elastic modulus ( \documentclass[12pt]{minimal }
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} { } $ e_{p}$
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comparison of arterial compliance ( \documentclass[12pt]{minimal }
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bland altman plot of end diastolic diameter ( \documentclass[12pt]{minimal }
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distribution of the differences between \documentclass[12pt]{minimal }
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distribution of the differences between \documentclass[12pt]{minimal }
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it may be observed that there is a high degree of agreement between artsens readings and stiffness values provided by the imaging system .
the mean of differences , known as the bias , for directly measured artery dimensional parameters is slightly higher than those reported in literature using image based systems , owing to reasons explained earlier in section iv.c1 .
the bias is close to zero for the arterial stiffness results \documentclass[12pt]{minimal }
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} { } $ e_{p}$
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it can be also be inferred from the plots that the bias and the limits of agreement are very low when compared to the absolute values of the parameters measured .
the variability in measurements exhibits even distribution in all plots , irrespective of the magnitude of the value measured .
it may be noted that the analysis also takes into account the minor differences introduced owing to minor changes in probe location and subject physiology as the measurements are performed sequentially.table 4results of bland altman analysis of arterial stiffness measurements by artsens with those given by imaging system in sitting posture.parameter measuredmean ( bias)standard deviation\documentclass[12pt]{minimal }
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} { } $ \delta_{d}$
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} { } $ d_{d}$
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} { } $ e_{{\rm p}}$
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} { } $ ac ( { \mathrm { mm}}^{2}/{\mathrm { kpa}})$
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} { } $ 0.06 \pm 1.04 $
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bland altman plot indicates the difference to be randomly distributed , with 99% of the measurements within 2sd as shown in fig .
the difference between the stiffness index measurements , \documentclass[12pt]{minimal }
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} { } $ \beta $
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the noticeable bias in the lumen diameter measurement may be attributed to the fact that the automated algorithm in artsens measures the inner lumen diameter , whereas it is normally difficult to locate lumen interface on ultrasound imaging system and hence the measurement by imaging system is expected to overestimate the diameter .
figure 20.bland altman plot of arterial stiffness index ( \documentclass[12pt]{minimal }
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} { } $ \beta _ { \mathrm { image(sit)}})$
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} { } $ ac$
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also , as already mentioned , the correlation of \documentclass[12pt]{minimal }
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hence it may be concluded that \documentclass[12pt]{minimal }
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} { } $ ac$
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} { } $ { e}_{p } $
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two artsens measurements were performed on a few randomly selected subjects ( \documentclass[12pt]{minimal }
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} { } $ \text{n}=33 $
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the repeatability of the arterial stiffness index value ( \documentclass[12pt]{minimal }
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the coefficient of variability ( repeatability ) computed as the ratio of the standard deviation of the differences between the two artsens measurements to the average of the means was calculated from the data .
the calculated coefficients of variability shown in table 5 are comparable to the previously reported results on carotid stiffness measurements performed using artsens and other ultrasound imaging systems , , , , .
this indicates the ability of the instrument to give precise measurements of stiffness irrespective of the field conditions.table 5coefficients of repeatability for artsens measurements.parameter measuredcoefficient of repeatability ( variability ) in % \documentclass[12pt]{minimal }
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\end{document}16.59 to investigate the ability of artsens to detect subtle changes in arterial stiffness , the data was analyzed to examine if artsens could pick up trends in arterial stiffness associated with age . to eliminate the influence of other factors that may affect stiffness ,
a total of 102 subjects were included in this age - trend analysis . to study the trend in variation of each parameter with age , the mean and standard deviation of stiffness index (
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was slightly skewed towards the younger age group , a classification based on age quartiles will not properly reflect the age related changes in arterial stiffness . hence age bins selected for analysis were chosen to ensure nearly uniform distribution of the overall subject pool among the various age bins .
a strong positive trend between arterial stiffness measures and age could be inferred from these plots .
this illustrates the ability of artsens to detect subtle changes in arterial stiffness that occurs due to ageing , thereby indicating potential use in vascular screening and diagnosis .
figure 26.age related increase in stiffness index ( \documentclass[12pt]{minimal }
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figure 27.age related increase in pressure strain elastic modules ( \documentclass[12pt]{minimal }
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figure 28.age related decrease in arterial compliance ( \documentclass[12pt]{minimal }
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age related decrease in arterial compliance ( \documentclass[12pt]{minimal }
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} { } $ ac)$
\end{document } measured using artsens . unlike the commercially available image - based systems , artsens has completely automated measurement and is the only image free system available for direct measurement of arterial dimensions to evaluate stiffness .
because of its image free technology , an expert ultrasonographer is not required for performing the measurement .
the time taken for measurement ( typically 5 minutes ) is much less than that required for image - based systems , and the measurement procedure is simple and less laborious .
the portable nature of the instrument and the ability of the intelligent algorithms to quickly perform the measurement without any operator input make it very amenable for field deployment .
artsens measurements could not be performed on few volunteers , where the artery was very superficial due to which the near wall could not be identified clearly .
the hardware of the device is being improved to reduce the apparent blind spot close to the probe surface , by increasing the transducer damping . in all subjects
whose data were eliminated as outliers in this study , the quality of the echo signal was found to be very low , as the artery walls could not be distinguished clearly .
the enforcement of a stricter protocol of probe positioning and angulation to ensure high quality echo signals during measurement will eliminate such cases .
an automated signal quality evaluation algorithm is being developed to avoid this in screening scenarios .
a novel image free instrument for non - invasive evaluation of arterial stiffness , called artsens was presented .
artsens uses a single element ultrasound probe , and is an affordable , compact , user friendly device for measurement of arterial stiffness . the hardware and software architecture of the system was explained .
the ability of artsens to easily perform in - vivo measurement of carotid artery stiffness was verified by the study conducted on 125 subjects .
the accuracy of the measurements provided by artsens was verified by comparing with a reference , state of art ultrasound imaging system .
the stiffness estimates provided by artsens strongly correlated with those obtained using the imaging system .
bland altman analysis demonstrated significant degree of agreement between artsens measurements and imaging system measurements .
the results show that artsens is at par with state of art technology for measurement of arterial stiffness .
measurements of \documentclass[12pt]{minimal }
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\end{document } stiffness index , considered to be better estimate , as it characterizes stiffness independent on distending pressure , was found to be repeatable and accurate in artsens .
artsens device demonstrated enough sensitivity to detect age related trends in arterial stiffness , thereby indicating potential for use in large scale screening studies .
artsens allows quick and easy measurement of arterial stiffness even in sitting posture , and its automated measurement algorithms allow it to be used by any operator with minimal training .
artsens can thus be deployed in primary or secondary care facilities , and even in field scenarios , thereby making vascular stiffness measurements feasible in large scale epidemiological studies as well as for early detection and screening .
the relative significance of various measures of stiffness , and normative values in indian population would be explored in future epidemiological studies using artsens . | vascular stiffness is an indicator of cardiovascular health , with carotid artery stiffness having established correlation to coronary heart disease and utility in cardiovascular diagnosis and screening .
state of art equipment for stiffness evaluation are expensive , require expertise to operate and not amenable for field deployment . in this context , we developed arterial stiffness evaluation for noninvasive screening ( artsens ) , a device for image free , noninvasive , automated evaluation of vascular stiffness amenable for field use .
artsens has a frugal hardware design , utilizing a single ultrasound transducer to interrogate the carotid artery , integrated with robust algorithms that extract arterial dimensions and compute clinically accepted measures of arterial stiffness .
the ability of artsens to measure vascular stiffness in vivo was validated by performing measurements on 125 subjects .
the accuracy of results was verified with the state - of - the - art ultrasound imaging - based echo - tracking system .
the relation between arterial stiffness measurements performed in sitting posture for artsens measurement and sitting / supine postures for imaging system was also investigated to examine feasibility of performing artsens measurements in the sitting posture for field deployment .
this paper verified the feasibility of the novel artsens device in performing accurate in vivo measurements of arterial stiffness . as a portable device that performs automated measurement of carotid artery stiffness with minimal operator input , artsens has strong potential for use in large - scale screening . |
experimental details of the synthesis and characterisation , in vitro assays and cell - based studies , as well as supplementary figures , are given in the supporting information available via http://dx.doi.org/10.1002/cmdc.201300428 .
as a service to our authors and readers , this journal provides supporting information supplied by the authors .
such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors | the 2-oxoglutarate ( 2og)-dependent jumonji c domain ( jmjc ) family is the largest family of histone lysine demethylases .
there is interest in developing small - molecule probes that modulate jmjc activity to investigate their biological roles .
5-carboxy-8-hydroxyquinoline ( iox1 ) is the most potent broad - spectrum inhibitor of 2og oxygenases , including the jmjc demethylases , reported to date ; however , it suffers from low cell permeability . here
, we describe structure activity relationship studies leading to the discovery of an n - octyl ester form of iox1 with improved cellular potency ( ec50 value of 100 to 4 m ) .
these findings are supported by in vitro inhibition and selectivity studies , docking studies , activity versus toxicity analysis in cell cultures , and intracellular uptake measurements .
the n - octyl ester was found to have improved cell permeability ; it was found to inhibit some jmjc demethylases in its intact ester form and to be more selective than iox1 .
the n - octyl ester of iox1 should find utility as a starting point for the development of jmjc inhibitors and as a use as a cell - permeable tool compound for studies investigating the roles of 2og oxygenases in epigenetic regulation . |
it is estimated that over a million such medical procedures occur annually world - wide . however , despite the minimal rate of early complications , up to 30% of all surgeries are revised within 1014 years of initial surgery ( 1 ) .
these particles are identified as one of the main causal reasons for total hip arthroplasty .
the wear between primary binding surfaces of the femoral head and acetabular components in total hip replacements is considered to be the most significant source of prosthetic particles ( 2 ) .
one estimation , from a study utilizing a metal on polyethylene joint , suggested the generation of hundreds of thousands of polyethylene particles during each gait cycle ( 3 ) .
another study found that metal - onmetal or ceramic - on - ceramic pairings had significantly less wear on the prosthesis ( 4 ) .
however , despite the variances in particle generation of different materials , osteolysis is indicated in all types of hip replacements .
particle debris generated from the mechanical wear of prosthetic devices is known to alter the function of multiple cell types in the periprosthetic area , including macrophages , fibroblasts , osteoblasts , and osteoclasts ( 57 ) .
the generated biologically active particles appear to provoke the formation of lytic areas through the inhibition of bone formation and increased fluid production ( 810 ) .
bones are highly dynamic tissues undergoing a constant remodeling process that is regulated by a tightly - controlled balance of osteoblast and osteoclast activity .
the major challenge of revision hip arthroplasty is related to osteolysis and bone loss decreasing stability of the implant .
the loss of bone can surmount due to surgical bone loss from the primary operation , migration of the cup , and as mentioned previously , particle induced osteolysis ( 11 ) .
consequently , the goal of revision hip surgery should be anatomical placement of the acetabular component , but this can be difficult to achieve in cases where contact between host bone and the implant is diminished .
this bone loss can be managed by either filling the absent area with cement , the use of allograft bone , or by using special implants such as those constructed with trabecular metal ( 1216 ) .
allograft incorporation can be implemented to reinforce the lytic area , thereby stabilizing the implant .
allografts do not suffer from osteoclastic invasion and it has been shown that osteoblasts deposit osteoid across the host bone and the dead graft ( 17 ) .
this union results in an increased mechanical resistance that is indicated by increased radiological density throughout the graft ( 18 ) .
the structure of trabecular metal resembles bone and approximates its physical and mechanical properties more closely than other prosthetic materials .
the highly porous trabecular configuration is conducive to bone ingrowth , facilitating rapid and extensive tissue infiltration resulting in strong implant attachment ( 19 ) .
trabecular metal consists of interconnecting pores resulting in a crystalline micro - textured biomaterial that is 80% porous .
this allows for greater tissue ingrowth ( 23 fold higher compared to conventional porous coatings ) with approximately double the interface shear strength ( 19 ) . in hip arthroplasties , porous trabecular metal facilitates bone ingrowth and fixation of the device ;
multiple studies have shown that most replacements can be reconstructed with an uncemented trabecular hemispherical cup with screws with or without morselized bone graft ( 2022 ) . because of their porous surface and mechanical properties trabecular metal cups provide a promising approach for offering a better environment for bone graft remodeling ( 23 ) . in this case study
we detail the use of a trabecular metal cup to halt the migration of the component into the acetabulum and promote greater fixation and bone ingrowth .
additionally , we show the use of a tibial allograft coupled with bone chips and cement to cover and support a lytic cyst in the proximal femur , distal to the greater trochanter .
a 73 year old man presented with instability in the left hip , 17 years after bilateral hip arthroplasty was completed .
the original replacement was a ceramic head on a polyethylene lined acetabular component . still working full time and an active individual , instability and
sharp pain presented down the lateral aspect of his left leg during jarring actions . during physical examination ,
good range of motion and gait was displayed in both hips , although pain was produced during forced external rotation of the left hip .
x - rays showed significant osteolysis of the proximal femur , distal to the greater trochanter of his left hip ( figure 1 ) . despite the bone loss , the stem appeared to still be solid .
it was anticipated that the progressive wear of the polyethylene cup would induce small particle disease involving the greater and lesser trochanter areas .
comparison to the current x - rays ( figure 1 ) revealed that the particle disease had expanded and significantly increased the endosteal lytic zone , it was also evident that the ceramic head had penetrated deeper into the acetabulum .
to avoid impending femoral fracture or avulsion of the greater trochanter from lytic bone loss , revision of the left hip was scheduled .
since the stem had good fixation , the choice was made to leave it in place and use a bone graft to support the lucent area distal to the greater trochanter .
a tibial allograft was used to secure the femoral component and stabilize the lytic area .
the allograft was irradiated with 2.5 mrad ( 25,000 gy ) in the hospital bone bank , accrediated by the american association of tissue banks , and consequently stored at 70 degrees celsius .
the approximate allograft size was templated preoperatively and a longer graft ordered to account for any intraoperative adjustments .
the allograft was only brought into the operating room after possible infection of the to - be - revised hip was ruled out . in order to reduce operative time , the allograft was prepared on a separate table by members of the surgical team while the revision was initiated .
the femoral component had good fixation so additional bone ingrowth was not a major concern .
thus , cement was used to fill in the lytic area and provide additional structural support .
polymethylmethacrylate bone cement has been known to strengthen allograft bone , impair resorbtion , and allow for the delivery of antibiotics ( 24 ) .
cement was not used between the allograft and the host bone , only in the lytic area between the allograft and the implant , so as not to impair healing at the host - donor interface .
additionally , morselized bone from the allograft was used between contact sites of host and donor bone in an effort to increase bone ingrowth .
operative visualization confirmed the cup had protruded into the acetabulum and wear on the polyethylene lining was evident , necessitating its replacement .
a zimmer trilogy acetabular system was selected to replace the acetabular component , consisting of a metal shell , polyethylene liner , and screws .
the shell , made from tivanium ti-6al-4v alloy , is pourous to allow for fixation with tivanium alloy cortical screws . in this case ,
three 6.5 mm screws of lengths 25 , 30 , and 35 mm were used to secure the cup into the acetabulum .
the original cup was uncemented , 56 mm in size and was replaced with a similar 56 mm trabecular metal cup .
the porous trabecular metal serves to facilitate bone ingrowth and increased fixation of the arthrography .
bone chips from the excess allograft were also used to fill in the protruded acetabulum from the original implant .
immediately following the surgery the patient was taken to radiology for post - operative x - rays ( figure 2 ) .
revision of the acetabular component with a trabecular metal cup was then visualized and correct placement within the acetabulum was confirmed .
the original femoral component was also visualized , shown reinforced with a well placed tibial allograft .
revision cases are usually rehabilitated more conservatively than primary replacements , as was the case here .
this patient was treated with additional care due to the use of allograft bone to stabilize the femoral component . during the first week post - op ,
our patient was encouraged to begin range - of - motion ankle exercises and moving with a walker / assistance 5 to 10 feet the first day after surgery which was done with success . throughout the first week ,
aided walking was extended to 2545 feet and stair - climbing with crutches was introduced after day 5 .
stair climbing and other motions that bend the knee must be watched carefully as for the first 3 months the patient is advised not to bend their knee past 90 degrees .
additional x - rays were taken after 3 months to confirm fixation of the aetabular component and evaluate the success of the tibial allograft over the osteolytic area in the proximal femur ( figure 3 ) .
the acetabular component appeared to be well placed and the observed migration into the acetabulum was halted .
the tibial allograft , marked with a white arrow , remains in place supporting the area of osteolysis .
the level of transparency had reduced significantly after successful cleaning of the cyst and filling the lytic zone with biocompatible polymethylmethacrylate bone cement .
physical examination was completed at the same time as x - rays to ensure the patient had continued with the rehabilitation program and was continuing to make significant progress .
ambulation , range of motion , and functionality were all observed and were progressing well .
the patient is now fully recovered , ambulating and functioning without pain or discomfort . for reasons including
particle disease , revision of total hip replacements generally occur upon indication of a painful loose prosthesis .
davis et al . ( 25 ) used the western ontario and mcmaster universities osteoarthritis ( womac ) questionnaire to grade pain and function pre- and 2-year - postoperatively .
the study reported that a higher pain level and number of comorbidities before the surgery predicted poorer outcomes at 24 month post - surgical follow - up .
patients with better preoperative womac pain and function scores had better scores postoperatively , suggesting the benefit of performing total hip revision not only to relieve current symptoms , but to reduce the chance that pain and function will worsen while waiting for surgery . in this case , loosening was first noticed by the patient through instability and loss of gait control and later confirmed through radiography .
if surgery had waited until higher levels of pain were expressed to maximize the life of the original device , more serious complications may have occurred . by performing the revision more complicated surgeries with worse outcomes , potentially for failure due to fracture ,
the use of allograft is shown as an effective way to support components threatened with osteolysis . donor bone in combination with biocompatible cement can greatly reduce the potential for injury and pain due to lytic bone loss . | total hip replacement is considered to be a highly successful and routine surgery ; however , the internal components produce particles through friction and wear in the device .
these particles are identified as one of the main reasons for total hip revisions .
the generated , biologically active , particles provoke the formation of osteolytic areas through the inhibition of bone formation and increased fluid production .
the resulting bone loss can be managed through the use of allograft bone in combination with bone chips and cement .
in addition , implants constructed with highly porous trabecular metal can be used to further facilitate rapid and extensive tissue infiltration resulting in strong implant attachment . in this case study
we show the use of a tibial allograft coupled with bone chips and cement to cover and support a lytic cyst in the proximal femur , distal to the greater trochanter . additionally , we detail the use of a trabecular metal cup to halt the migration of the component into the acetabulum and promote greater fixation and bone ingrowth . |
the " super scan " of bone scintigraphy is defined as a bone image that " looks too good . "
its characteristics include intense activity in the bones and diminished renal parenchymal activity ( 1 ) .
( 2 ) had reported the case of f - fluorodeoxyglucose positron emission tomography ( fdg pet ) super scan involving skeletons , similar to the super scan of bone scintigraphy .
a " hepatic super scan " by fdg pet has been reported in a patient with hodgkin disease who had unusually elevated hepatic radioactivity , in addition to the multiple areas of focal uptake in the chest , mediastinal , left axillary , and abdominal lymph nodes , and the adrenal glands .
here we report a case of f - fdg pet / ct super scan showing extensive hypermetabolic lesions throughout the skeletons and liver .
contrary to the intense hypermetabolism of skeletons and liver , the skeletal muscles of limbs , mediastinum , bowel , and especially brain showed very low fdg uptake .
subsequent diagnostic ct imaging of chest and abdomen revealed 3.8 cm sized mass in left lower lung with multiple lymph nodes and hepatic metastasis .
excision biopsy of supraclavicular lymph node was performed and histopathologic diagnosis was small cell lung cancer .
the patient 's mental status was clear and he underwent f - fdg pet / ct ( biograph sensation 16 , siemens medical systems , usa ) for staging of lung malignancy , 1 hr after injection of 444 mbq ( 12 mci ) f - fdg .
f - fdg pet / ct demonstrated focal hypermetabolic lung mass in left lower lobe with diffuse and intense hypermetabolism throughout the skeleton and liver .
interestingly , skeletal muscles of limbs , mediastinum , bowel , and especially brain showed very low fdg uptake ( fig .
1 ) . this patient had no medications could disturb cerebral glucose metabolisms , such as corticosteroids and sedatives .
a super scan is well - known phenomenon which is characterized by a strikingly high bone to soft tissue ratio on skeletal scintigraphy , with a uniform symmetrical increase in bone uptake and absent renal visualization ( 4 ) .
the super scan is associated with various malignancies , and most commonly in carcinomas of the prostate , stomach and breast ( 5 ) .
metastasis from lung cancer rarely causes a super scan , although multiple bone metastases or bone marrow involvement frequently occur in patients with lung cancer ( 6 ) .
sy et al . ( 7 ) hypothesized that the increased uptake of radiopharmaceutical by diseased bone results in reduced phosphate excretion , thereby producing faint renal images in the bone scintigraphy .
first , even though this is not uncommon feature in patients with extensive metastasis , intense and diffuse hypermetabolism throughout the skeleton and liver was demonstrated .
the similar findings had reported in other cases of fdg pet super scan , however , this case had shown the best contrast between metastatic organs and non - metastatic organs ( 2 , 3 , 8) .
second , fdg uptakes in skeletal muscles of limbs , mediastinum , bowel , and especially brain were remarkably low and this is the unique characteristic of the present case .
the faint visualization of the brain , renal cortex , and soft tissue might be the result of extraordinarily high uptake of fdg by bony and hepatic metastatic lesions .
low brain uptake of fdg has been reported when using corticosteroids and sedatives , and these conditions should be taken into consideration ( 9 , 10 ) .
the present patient had no medications could disturb cerebral glucose metabolisms , such as corticosteroids and sedatives .
because of some characteristics in common with super scan on skeletal scintigraphy , this case could be considered as ' metabolic super scan ' . | a 50-yr - old man presented with intermittent hemoptysis and was diagnosed small cell lung cancer .
18f - fdg pet / ct for staging demonstrated extensive hypermetabolic lesions throughout the skeleton and liver .
interestingly , skeletal muscles of limbs , mediastinum , bowel , and especially brain showed very low fdg uptake .
because of some characteristics in common with super scan on skeletal scintigraphy , this case could be considered as ' metabolic super scan ' . |
annually , falls occur in about one third of community - dwelling adults aged 65 and older living in the us.1 the physical and functional impacts ( fractures , soft - tissue injury , premature institutionalization , and deaths ) of falls have been well documented , but little is known about the long - term emotional and cognitive impact of falls.26 also , falls , depression , and cognitive impairments frequently coexist in the same patient , raising the possibility of shared risk factors and shared etiology . preventing falls - related
disability requires a good understanding of how experience of falls affects subsequent emotional and cognitive function .
such understanding can inform development of interventions to stem the onset of not only physical disability but any fall - related cognitive disability .
falls , depression , and cognitive impairment shared common risk factors.3,5,712 past studies showed a relationship between cognitive impairment and falls,3,8,9 cognitive impairment and depression,10 and falls and depression.6,11,12 however , the synergistic relationship among these three syndromes has not been explored in depth .
raji et al found an association between lower scores on the mini mental state examination ( mmse ) and high depressive symptomatology in older mexican americans.10 a later study of the effects of cognitive and emotional status on physical functioning found that having high depression and low cognition was associated with poor physical performance.13 in another study of 283 adults aged 60 and older , biderman et al determined that the common risk factors for falls and depression are poor self - rated health , poor cognitive status , impaired ability to perform activities of daily living , two or more clinic visits in the past month , and slow walking speed.14 another explanation for the complex relationships among falls , depression , and cognitive decline may be due to subsequent events following development of one of the conditions .
. falls in older persons have been associated with development of fear of falls,15 with a subsequent decrease in levels of physical activity , walking , participation in social activities , and an increase in loneliness and social isolation.1519 over time , reduced social and physical activity may lead to onset of depression and future cognitive decline.2022 we wanted to explore this hypothesis further by testing whether , in a group of cognitively intact elders , recurrent falls would be independently associated with faster cognitive decline , and whether presence of depression would at least in part mediate this association . because of the high prevalence of depressive symptoms and cognitive impairments in older mexican american adults,11,23,24 a fast growing segment of the us population , we wanted to test whether having a fall would trigger social isolation , depression , and subsequent worsening of cognition . to avoid the floor effects of the mmse
, we chose to include only those with normal baseline cognition ( mmse 21 ) , a cut - off point recommended in populations with low educational attainment.25 mean education of our sample was 6.3 years . understanding the fall - cognition pathway is key to designing interventions that address all the potential pathways to cognitive and motor
first , we examined the association of falls versus no falls on subsequent change in cognitive function ( using the mmse ) over a 6-year period in a community sample of older mexican americans who were initially cognitively normal .
second , because past research has shown an association between depression and cognitive decline,10 we tested whether depressive symptoms would have a mediating effect on the association between falls and cognitive change .
data are from the h - epese ( hispanic established populations for epidemiologic studies of the elderly ) , an ongoing longitudinal study of mexican americans 65 years and older who reside in texas , colorado , california , new mexico , and arizona , usa.23 in - home interviews were conducted of 3,050 participants or their proxy at the beginning of the study ( 19931994 ) .
the sampling procedure assured a sample that is generalizable to approximately 500,000 older mexicans americans living in the southwest and has been described elsewhere.23 data were collected during home visits by trained interviewers in the language of the subject s choice ( spanish or english ) .
the face - to - face interviews included questionnaires , interviews , and physical assessments . the present study used participants interviewed in 20042005 ( wave 5 ) , 20072008 ( wave 6 ) , and 20102011 ( wave 7 ) .
wave 5 in this study is considered as baseline with two follow - ups ( wave 6 and wave 7 ) .
we excluded those with a mmse score < 21 ( n=681)25 because we were interested in examining how falls and high depressive symptoms influenced changes in cognitive function in those with normal or high cognition . also , we excluded those with missing information on the mmse ( n=199 ) .
participants with missing information on the mmse and those with mmse < 21 were significantly more likely to be older , have low level of education , perform lower in hand grip strength , report less hypertension , and report more stroke , hip fracture , high depressive symptoms , and falls compared with those with mmse 21 . after excluding participants with an mmse score < 21 at baseline and those with missing data on mmse , fall status , and all covariates , the final sample consisted of 1,119 participants .
subjects excluded were significantly more likely to be older , unmarried , and to have reported more hypertension , stroke , hip fractures , and high depressive symptoms . they were also more likely to have less education , a lower mmse score , and have had two or more falls .
fall status was established by asking the participants at each interview the following question : during the last 12 months , how many times did you fall and land on the floor or ground ? fall status was categorized as having no falls , having one fall , or having two or more falls .
scored on a 30-point scale , the mmse screens for difficulties in episodic memory , orientation , working memory , naming , language , and image copying.26,27 higher scores indicate better cognition .
interviewers were thoroughly trained in administering and scoring the mmse , through both workshops and videotaped instruction .
the english and spanish versions of the mmse were adopted from the diagnostic interview scale used in prior community surveys.28 this spanish version of the mmse has met standard criteria for development of translated tests , including formal translation , back - translation , and consensus by committee for final item content .
additionally , the spanish mmse has been successfully used in community surveys of mexican americans.29 baseline sociodemographic variables included age , sex , marital status , and years of education .
the presence of various medical conditions was determined with a series of questions asking participants if a doctor had ever told them that they had diabetes , heart attack , stroke , or hypertension .
depressive symptomatology was measured with the center for epidemiologic studies depression scale.30 this scale consists of 20 items that ask how often specific symptoms were experienced during the past week ; responses were scored on a 4-point scale ( ranging from rarely or none of the time to most or all of the time : 0 , 1 , 2 , 3 ) with potential total scores ranging 060 .
as is common in the literature , we consider persons scoring 16 or over to experience high depressive symptomatology.10,11,30
data are from the h - epese ( hispanic established populations for epidemiologic studies of the elderly ) , an ongoing longitudinal study of mexican americans 65 years and older who reside in texas , colorado , california , new mexico , and arizona , usa.23 in - home interviews were conducted of 3,050 participants or their proxy at the beginning of the study ( 19931994 ) .
the sampling procedure assured a sample that is generalizable to approximately 500,000 older mexicans americans living in the southwest and has been described elsewhere.23 data were collected during home visits by trained interviewers in the language of the subject s choice ( spanish or english ) .
the face - to - face interviews included questionnaires , interviews , and physical assessments . the present study used participants interviewed in 20042005 ( wave 5 ) , 20072008 ( wave 6 ) , and 20102011 ( wave 7 ) .
wave 5 in this study is considered as baseline with two follow - ups ( wave 6 and wave 7 ) .
we excluded those with a mmse score < 21 ( n=681)25 because we were interested in examining how falls and high depressive symptoms influenced changes in cognitive function in those with normal or high cognition . also , we excluded those with missing information on the mmse ( n=199 ) .
participants with missing information on the mmse and those with mmse < 21 were significantly more likely to be older , have low level of education , perform lower in hand grip strength , report less hypertension , and report more stroke , hip fracture , high depressive symptoms , and falls compared with those with mmse 21 . after excluding participants with an mmse score < 21 at baseline and those with missing data on mmse , fall status , and all covariates , the final sample consisted of 1,119 participants .
subjects excluded were significantly more likely to be older , unmarried , and to have reported more hypertension , stroke , hip fractures , and high depressive symptoms . they were also more likely to have less education , a lower mmse score , and have had two or more falls .
fall status was established by asking the participants at each interview the following question : during the last 12 months , how many times did you fall and land on the floor or ground ? fall status was categorized as having no falls , having one fall , or having two or more falls .
scored on a 30-point scale , the mmse screens for difficulties in episodic memory , orientation , working memory , naming , language , and image copying.26,27 higher scores indicate better cognition .
interviewers were thoroughly trained in administering and scoring the mmse , through both workshops and videotaped instruction .
the english and spanish versions of the mmse were adopted from the diagnostic interview scale used in prior community surveys.28 this spanish version of the mmse has met standard criteria for development of translated tests , including formal translation , back - translation , and consensus by committee for final item content .
additionally , the spanish mmse has been successfully used in community surveys of mexican americans.29 baseline sociodemographic variables included age , sex , marital status , and years of education .
the presence of various medical conditions was determined with a series of questions asking participants if a doctor had ever told them that they had diabetes , heart attack , stroke , or hypertension .
depressive symptomatology was measured with the center for epidemiologic studies depression scale.30 this scale consists of 20 items that ask how often specific symptoms were experienced during the past week ; responses were scored on a 4-point scale ( ranging from rarely or none of the time to most or all of the time : 0 , 1 , 2 , 3 ) with potential total scores ranging 060 .
as is common in the literature , we consider persons scoring 16 or over to experience high depressive symptomatology.10,11,30
fall status was established by asking the participants at each interview the following question : during the last 12 months , how many times did you fall and land on the floor or ground ? fall status was categorized as having no falls , having one fall , or having two or more falls .
scored on a 30-point scale , the mmse screens for difficulties in episodic memory , orientation , working memory , naming , language , and image copying.26,27 higher scores indicate better cognition .
interviewers were thoroughly trained in administering and scoring the mmse , through both workshops and videotaped instruction .
the english and spanish versions of the mmse were adopted from the diagnostic interview scale used in prior community surveys.28 this spanish version of the mmse has met standard criteria for development of translated tests , including formal translation , back - translation , and consensus by committee for final item content .
baseline sociodemographic variables included age , sex , marital status , and years of education .
the presence of various medical conditions was determined with a series of questions asking participants if a doctor had ever told them that they had diabetes , heart attack , stroke , or hypertension .
depressive symptomatology was measured with the center for epidemiologic studies depression scale.30 this scale consists of 20 items that ask how often specific symptoms were experienced during the past week ; responses were scored on a 4-point scale ( ranging from rarely or none of the time to most or all of the time : 0 , 1 , 2 , 3 ) with potential total scores ranging 060 .
as is common in the literature , we consider persons scoring 16 or over to experience high depressive symptomatology.10,11,30
chi - square , analysis of variance , and post hoc tukey s tests were used to examine the distribution of covariates for participants by fall status at baseline .
general linear mixed models using the mixed procedure in sas ( sas institute , inc .
, cary , nc , usa ) were used to examine the factors associated with decline in cognitive function over a 6-year period as a function of fall status .
all variables were analyzed as time - dependent covariates ( potential to change as time progresses ) except age , sex , and education .
the mixed model was chosen for analysis because it best handles unbalanced data , allowing for modeling of both the time - dependent change in variables as well as the time - dependent change in the magnitude of association between variables .
in addition , because h - epese data comprised repeated measures over 6 years , mixed models allowed more flexibility in modeling the effects of time on outcome.31,32 three mixed models were constructed to test the relationship between fall status and cognitive decline over a 6-year period . model 1 included time , age , sex , marital status , years of formal education , and fall status . in model 2 , depressive symptomatology was added to the variables in model 1 .
medical conditions ( stroke , heart attack , hypertension , and diabetes ) were added to variables in model 3 .
models 2 and 3 were performed to test whether high depressive symptoms mediate the relationship between falls and cognitive decline ( model 2 ) and to test whether the relationship between falls and cognitive decline remains statistically significant after adjusting for all covariates ( model 3 ) .
all analyses were performed using the sas system for windows , version 9.2 ( sas institute , inc . ) .
at baseline , the mean age for the 1,119 participants was 80.8 years ( range , 74109 ) . sixty percent of the participants were female , 45.6% were married , and the mean years - of - education was 6.3 years ( range , 017 ) .
sixty five percent of participants reported hypertension , 32.9% diabetes , 14.9% heart attack , 10.7% stroke , 5.6% hip fracture , and 11.9% had high depressive symptoms at baseline .
the prevalence of participants who fell once was 16.6% and was 14.7% for those who fell two or more times .
the mean mmse score was 25.53.1 for no falls , 24.93.0 for one fall , and 24.52.9 for two or more falls .
the difference was statistically significant between those who fell two or more times and those who did not fall ( p<0.05 ) .
participants who fell two or more times were significantly more likely to be older , female , and to report higher levels of depressive symptoms , hypertension , diabetes , heart attack , and stroke compared to those with one fall or no falls .
no statistically significant association was seen with hip fracture . those who fell two or more times had lower performance in handgrip strength when compared with those with no falls ( p<0.05 ) .
table 2 shows the general linear mixed model estimates for mmse score as a function of fall over a 6-year period
. the adjusted rate of decline in the total mmse score was 0.25 points per year ( model 1 ) .
participants who fell two or more times had a greater decline in mmse score of 0.81 points per year ( p<0.0001 ) compared with those who did not fall , after adjusting for age , sex , marital status , and education ( model 1 ) .
participants who fell one time had a decline in mmse score of 0.14 points per year compared to the those who did not fall , but this difference was not statistically significant ( p=0.4261 ) . in model 2 ,
when high depressive symptoms was added to all variables in model 1 , the association between two or more falls decreased but remained statistically significant ( estimate = 0.64 , standard error = 0.19 , p=0.0007 ) : a decline of 0.64 points per year compared with those who did not fall . in model 3 , after adding medical conditions and hand grip muscle strength to model 2 , the association between two or more falls and mmse score remained statistically significant ( estimate = 0.46 , standard error = 0.19 , p=0.0161 ) : a decline of 0.46 points per year compared with those who did not fall .
female sex , high level of education , and high performance in hand grip muscle strength were associated with a lower decline in mmse scores .
figures 1 and 2 shows the unadjusted and adjusted mean distribution for the total mmse score over the 6-year period by falls status .
participants with two or more falls had lower total mmse scores than subjects with one fall or no falls over the follow - up period .
we also found that , among a cohort of cognitively normal elders , those with history of recurrent falls experienced a greater decline in their cognitive function over a 6-year period compared to those with no history of falls .
the fall cognitive - decline association was independent of age , sex , marital status , and education .
presence of high depressive symptoms partially mediated the relationship between falls and cognitive decline . as expected , and consistent with prior studies , a high level of education and high performance in hand grip muscle strength
our findings are similar to prior studies and different from others.912,3335 most studies have looked at the falls cognition association from the perspective of falls - related brain injury versus no injury as predictors of cognitive decline.3335 others have studied risks and consequences of falls in the dementia population , or gait disorders as predictors of cognitive decline or dementia , mostly in non - hispanic populations.36,37 ours is the first study to show that recurrent falls independently predict future cognitive decline in a cohort of community - dwelling hispanic elders with normal baseline cognitive measure .
it is not clear how recurrent falls predict cognitive decline , but one possibility is a shared risk factor for both falls and cognitive decline ( eg , strokes , shared brain network for cognition and gait control , low vitamin d , and brain amyloids).3841 for example , low vitamin d levels are associated with falls , depression , and cognitive impairments.38,39 a 12-month prospective cohort study of cognitively normal , community - dwelling older adults ( n=125 , mean age = 75 years ) showed that higher levels of brain amyloid ( pittsburgh compound b retention ) and of cerebrospinal fluid tau / a42 biomarker ratios were significantly associated with increased risk of falls.42 these amyloid imaging markers have been well described in persons with mild cognitive impairment and dementia.43,44 lack of amyloid imaging data precludes us from testing the association between amyloid burden and recurrent falls in our subjects with incident cognitive decline .
this is an important area for further study , using a combination of amyloid imaging and cerebrospinal fluid biomarker .
were such an association proven , recurrent falls might alert clinicians to patients with high risk of future dementia .
the literature supports our findings of an association of falls with depressive symptoms and subsequent cognitive decline.1422,45 a 12-year longitudinal study of 2,425 cognitively intact adults showed that high depressive symptoms were associated with accelerated decline in memory , compared to low depressive symptoms as measured with the center for epidemiologic studies depression scale.45 this association suggests that older adults who fall should be monitored for depression .
another possible explanation for falls cognitive - decline association is that recurrent fallers are simply sicker than non - fallers , with more conditions ( eg , diabetes , heart attacks ) that contribute to the mmse decline seen in recurrent fallers .
our study indicated that recurrent fallers at baseline were more likely than non - fallers to have lower handgrip muscle strength , hypertension , diabetes , heart attacks , and stroke .
our findings linking falls to cognitive decline still remained significant after adjustment for these conditions .
clearly , efforts to prevent falls - related disabilities require multidimensional approaches to address not just multiple risk factors , but also cognitive , physical , and emotional consequences of falls .
the world alzheimer report states that the earlier the diagnosis of dementia , the better the patients outcome.46 since falls appear to be a predictor of subsequent cognitive decline , health care professionals may be able to detect patients at risk for dementia and implement preventive strategies .
possibilities of such strategies include , but are not limited to , physical activity , mediterranean diet , and smoking cessation.6,21,26,43,46 clinically , our study also suggests that those who fell , even if cognitively intact , should be screened over time not only for cognitive impairment , but also for emergence of depressive symptomatology , with the expectation that such screening will trigger timely interventions to improve psychological health and reduce future falls .
depression in fallers could be caused by a fear of having another fall , a syndrome that may be amenable to interventions .
after a first fall , the fear of falling increases and could cause an adult to withdraw from activities as a defense against future falls .
this activity restriction may manifest as depression.1720 depression is thought to increase the likelihood of cognitive decline.10,11,47 however , caution should be used while prescribing antidepressants to patients who have fallen as studies have shown that these medications cause an increased risk in falling equal to that of untreated depression.16,48 nonpharmacological treatment of these patients may be the most effective option.16 our study has several limitations .
generalizability of our findings to other populations is limited because of the low educational attainment ( mean years = 6.3 ) and advanced age ( mean age = 80.8 years ) of our subjects .
the h - epese data for falls and comorbidities were self - reported , raising the possibility of recall bias , especially if participants have impaired cognition .
. we also could not confirm participant self - reports of medical conditions because of unavailability of participants medical records , radiologic imaging , and blood tests .
however , recent studies have validated the reliability of self - reports in studies on fears of falling in cognitively intact and impaired older adults and on barriers to mobility after stroke.49,50 the mmse is used primarily as a screening assessment for cognitive function and has limited sensitivity to detect changes over time , particularly in specialized areas of cognition .
thus , the rate of cognitive decline in our study may tend towards an underestimation .
our study has several strengths : large community - based sample , prospective design , and exploration of the potential role of falls and depression in cognitive disablement in older mexican americans ( a rapidly growing segment of us elderly population ) .
another important strength of our study is the use of mixed models , an analytic approach that allowed the use of all available data and evaluation of time - dependent effects .
in conclusion , having recurrent falls was independently associated with steeper decline in cognition over 6 years compared to having no history of falls , with a mediating effect of depression on the association between falls and cognitive decline .
the findings suggest that screening for cognitive and emotional problems in recurrent fallers might help stem the onset of falls - related physical and cognitive disability .
future studies should explore the best way to screen for and treat depression and fear of falling in older adults who have fallen . | backgroundlittle is known about long - term emotional and cognitive consequences of falls .
we examined the association between falls and subsequent cognitive decline , and tested the hypothesis that depression would mediate any falls
cognition association among cognitively intact hispanic elders.methodswe used data from the hispanic established population for the epidemiological study of the elderly to examine change in mini mental state examination ( mmse ) scores over the 6-year period according to number of falls .
all participants ( n=1,119 ) had mmse scores 21 and complete data on center for epidemiologic studies of depression scale , social and demographic factors , medical conditions ( diabetes , heart attack , stroke , and hypertension ) , and hand grip muscle strength.resultsat baseline , participants mean age was 80.8 years ( range , 74109 ) , mean education was 6.3 years ( range , 017 ) , and mean mmse was 25.2 ( range , 2130 ) . of the 1,119 participants , 15.8% experienced one fall and 14.4% had two or more falls . in mixed model analyses , having two or more falls was associated with greater decline in mmse score ( estimate = 0.81 , standard error = 0.19 , p<0.0001 ) compared to having no fall , after adjusting for age , sex , marital status , and education .
the magnitude of the association decreased ( estimate = 0.65 , standard error = 0.19 , p=0.0007 ) when adjustment was made for high depressive symptoms , suggesting a possible mediating effect of depression on the falls cognition association .
female sex , high level of education , and high performance in hand grip muscle strength were associated with a slower decline in mmse scores.conclusionhaving two or more falls was independently associated with steeper decline in cognition over 6 years , with a possible mediating effect of depression on the falls cognition association . |
esophageal atresia is an anatomical and physiological disorder of the esophagus requiring urgent surgical intervention .
the most common early complications of esophageal atresia repair include leakage of the anastomosis , recurrent tracheoesophageal fistula , and anastomotic stricture .
we present a case of atypical postoperative complication in the form of pulmonary torsion comprising the middle lobe of the right lung .
a 3,260 g preterm female neonate , delivered at 38 weeks ' gestation , evaluated at 7/8 points in apgar scale , was referred to our department with prenatally diagnosed esophageal atresia .
on contrast , chest x - ray performed directly after birth the presence of congenital defect esophageal atresia with tracheoesophageal fistula was confirmed ( the upper end of esophagus visible at the level of th45 , dilated intestinal loops filled with air ) .
the child was operated on the second day of life by the right laterodorsal thoracotomy .
the tracheoesophageal fistula was dissected and sutured , and esophago - esophageal anastomosis was performed . before closure , the lungs were aerated by forced ventilation , continued until all collapsed regions fully expanded . during postoperative course , on control chest x - rays , there was atelectasis in the upper lobe and the middle lobe of the right lung ( fig .
contrast - enhanced computed tomographic ( ct ) scan was performed and revealed abrupt termination of the bronchial airways with atypical course of horizontal interlobal fissure ( fig .
right pulmonary artery was visible , its lobar branches hardly visible ; right main bronchus showed typical diameter for approximately 7 mm from the bifurcation , then narrowed to approximately 1.5 mm ( compression from outside ? ) and continued as a single bronchus to the lower lobe .
thus , bronchography was performed , which showed obstruction of the upper lobe bronchus and bronchus intermedius and the middle lobe bronchus of the right lung ( fig .
the patient was qualified for tracheobronchoscopy , which confirmed occlusion of the upper and middle lobar bronchi ( trachea wide , smooth , site of sutured fistula wide , patent , tracheal bifurcation normal .
main bronchus : left , normal ; right , narrowing at 10 mm from bifurcation , with a small fissure in the sagittal plane ) .
the neonate was qualified for second thoracotomy on the ninth day of life . on exploration , massive infarction of the upper and middle lobe of the right lung was revealed .
a 180-degree rotation of the right middle lobe in a clockwise direction was found , which caused traction and compression of vessels to the upper lobe .
the lobe was untwisted , with return of inflation in the upper lobe under vision . in the middle lobe after detorsion inflation
was seen only in fragments , however significant decrease of edema and hemostasis was seen , thus no resection was attempted .
postoperatively , the patient recovered normal respiratory function ( initially ventilation synchronized intermittent mandatory ventilation with low parameters , from 20th postoperative day spontaneous respiration , chest drain closed on 3rd postoperative day , removal of the drain on the 5th postoperative day , feeding via gastric tube from 3rd postoperative day , discharged home on 35th postoperative day ) .
the patient was then transferred to a cardiology department for diagnostics of arrhythmia and later , at the age of 5 months , to a specialist pulmonary hospital , where the middle lobe was removed because of atelectasis .
torsion of a lobe of a lung is a severe , life - threatening event , though extremely uncommon .
it may occur spontaneously after thoracic trauma , thoracic or cardiac surgery.1
2
3
4
5
6
7 in children , it is considerably less frequent than in adults . so far , according to our knowledge , only nine cases have been described in pediatric literature , including one case of lung torsion complicating tracheoesophageal fistula repair.1
2 mortality in adult patients ranges from 12 to 16%.8 pulmonary torsion causes clinical deterioration in the face of normal arterial blood gases and requires a high index of suspicion for diagnosis .
the diagnosis is usually suggested on chest x - ray and confirmed by ct scans . on chest x - rays consolidation and opacification of the affected lung fragment , bronchial cutoff or distortion with mediastinum shift and lobar air trapping
can be seen.9
10 the diagnosis was delayed by 14 days in some reports8
11 because of unequivocal results of imaging studies and the fact that clinical findings may be seemingly inconspicuous . pediatric patients may present with tachycardia , fever , tachypnea , abnormal blood tests , reduced breath sounds or chest movements , dullness to percussion , and increased respiratory effort.5
7
8 if pulmonary torsion is suspected basing on clinical history and plain radiographs , chest ct scan should be performed .
ct findings comprise bronchial obstruction or distortion and abnormal pattern of blood vessels.3
4
7
9
10 bronchoscopy is a useful tool in the diagnosis of pulmonary torsion.9
10 a complete interlobar fissure , with no parenchymal bridges between the neighboring lobes , and an absent inferior pulmonary ligament have been described as predisposing factors.2 the accepted treatment of pulmonary torsion is detorsion or removal of the affected lobe.10
11 so far , among the eight cases described in the literature , seven cases have been operated , with five resections2
4
5
7
12 and two detorsions.1
6 detorsion of a lung or a lobe of doubtful viability is controversial , as it may be connected with release of inflammatory mediators and necrotic tissue , leading to a systemic response and multiorgan failure.1
13
14 in the two described cases of detorsion , one patient recovered completely,1 whereas in the second case the lung remained shrunken and nonfunctioning.6 in this case , detorsion allowed for better assessment of the lobe viability , and decrease in edema and hemostasis were observed . in the case of our patient ,
the torsion was probably because of an anatomic anomaly absence of parenchymal bridges between contiguous lobes .
earlier reoperation would have probably been beneficial , but as this complication is extremely rare in the pediatric population , diagnostic process may take longer .
persistent atelectasis of a lung / lobe after thoracotomy should call attention to a possibility of torsion .
the following can be concluded from this case report : pulmonary torsion is a severe , life - threatening event , rarely occurring in children
. early diagnosis ( x - ray / ct / bronchography / bronchoscopy ) may be lifesaving . clinical deterioration in the face of normal arterial blood gases
treatment is surgical , detorsion if possible , or if the lobe is not viable resection . | pulmonary torsion is a severe , life - threatening event , rarely occurring in children .
we present a case of atypical postoperative complication of esophageal atresia repair in the form of pulmonary torsion comprising the middle lobe of the right lung .
clinical deterioration in the face of normal arterial blood gases should rise a high index of suspicion for pulmonary torsion .
early diagnosis is crucial , treatment is surgical , detorsion if possible , or if the lobe is not viable resection . |
the phosphatidylinositol-3-kinase ( pi3k ) signaling pathway is one of the pathways most commonly activated in human cancers .
it is a major downstream signaling component of receptor tyrosine kinases ( rtks ) and is critical for the regulation of cell proliferation , growth , differentiation , migration , and survival .
thus , it represents one of the most promising targets for cancer prevention and therapy .
mounting reports of original studies and reviews have been published , highlighting the paramount importance of this pathway in human cancers . to avoid redundancy with previous publications , in this paper
we will focus on summarizing recent progress in pi3k pathway research in head and neck squamous cell carcinoma ( hnscc ) . specifically , this will include considerations of the molecular alterations seen in some components of the pi3k pathway , as well as functional studies of the role of the pi3k pathway in hnscc initiation , invasion , and metastasis studied both in vitro and in vivo , with a particular focus on the use of genetically engineered mouse models ( gemms ) .
finally , we will explore the potential of the pi3k pathway as a target for chemoprevention and cancer therapy .
hnscc refers to squamous cell carcinomas ( sccs ) arising from the oral cavity , tongue , pharyngeal , and laryngeal regions . as the 6th most common human cancer worldwide
, they generate about 600,000 new cases and 350,000 cancer deaths each year [ 4 , 5 ] .
hnsccs usually occur at a relatively late age and at a higher frequency in males possessing the well - known etiological factors of tobacco and/or alcohol usage [ 4 , 5 ] .
recently , however , the incidence of hnscc is increasing in women of a relatively young age , correlating with human papilloma virus ( hpv ) infection [ 4 , 5 ] .
historically , the best known molecular alterations in hnscc were the inactivation of tumor suppressors , such as p16 and p53 , and activation of oncogenes , such as egfr and stat3 [ 5 , 6 ] . we have studied the role of transforming growth factor beta ( tgf ) pathway in hnscc using both human hnscc samples and gemm approaches [ 710 ] .
our studies indicate that inactivation of the type ii receptor of tgf ( tgfrii ) and the downstream signal mediator of tgf , smad4 , plays a crucial role in hnscc development and progression [ 8 , 10 ] .
perhaps the most comprehensive studies on molecular alterations of hnscc came from two recent publications describing whole exome sequencing on human hnscc samples [ 11 , 12 ] .
two major results were generated from these papers : ( 1 ) the discovery of novel molecular alterations of the notch signaling pathway in human hnscc samples and ( 2 ) the validation of the pi3k pathway as one of the major targets for molecular alterations in human hnscc samples , including alterations of the oncogene pik3ca and the tumor suppressor gene pten .
these two papers , together with many previous reports , clearly demonstrate the importance of the pi3k pathway in hnscc .
furthermore , they suggest its possible involvement in every aspect of hnscc development and progression , including tumor initiation , invasion , recurrence , resistance to therapeutics , and metastasis .
there are three classes of pi3ks , with ia pi3k being the type most widely implicated in human cancers .
class ia pi3k primarily phosphorylate phosphatidylinositol-4,5,bisphosphate [ pi(4,5)p2 ] in the plasma membrane to generate the second messenger phosphatidylinositol-3,4,5,trisphosphate [ pi(3,4,5)p3 ] .
class ia pi3k are most often activated by rtk signaling and indirectly by ras . upon rtk signaling , p85 binds to either phosphotyrosine residues or adaptor molecules .
this binding serves both to recruit the p85-p110 heterodimer to the plasma membrane and to relieve the basal inhibition of p110 by p85 .
the 3-phosphatase pten ( phosphatase and tensin homologue ) dephosphorylates pi(3,4,5)p3 and catalyses the reverse reaction .
pi(3,4,5)p3 binds a subset of pleckstrin homology domain - containing proteins , including 3-phosphoinositide - dependent protein kinase ( pdk1 ) and protein kinase b ( also called akt ) to the plasma membrane .
once there , akt is phosphorylated at thr308 by pdk1 and ser473 by the mammalian target of rapamycin ( mtor ) complex 2 ( mtorc2 ) .
it is believed that akt is the central signal mediator of the canonical pi3k signaling pathway .
however , recent studies also suggest that the link between pi3k and akt can be uncoupled [ 1518 ] and that oncogenic pi3k signaling can be transmitted through an akt - independent pathway , further adding to the complexity of pi3k signal transduction .
akt phosphorylates numerous downstream targets that regulate a wide array of cellular processes important in tumor development and progression .
one of its major effectors is mtor complex 1 ( mtorc1 ) , which is activated in multiple human cancers and is one of the major targets in the pi3k pathway for chemoprevention and therapy ( figure 1 ) . in human hnscc ,
these include gain - of - function mutations and amplifications in pik3ca ( the gene coding p110 , the catalytic subunit of pi3k ) , loss of heterozygosity and inactivating mutations in pten , and overexpression / activation of akt and mtor signaling [ 5 , 6 ] .
several reports utilizing the gemm approach , including our own studies , have confirmed the functional importance of these molecular alterations in hnscc development and progression . in the following sub - sections
, we will summarize several molecular alterations and functional studies , particularly those using gemms , involving molecular components of the pi3k pathway in hnscc tumorigenesis .
pik3ca , the gene coding for the catalytic subunit p110 of pi3k , is one of the most commonly mutated oncogenes in multiple human malignancies ( 3441 mutated samples among a total of 27725 samples , about 12% , according to the catalogue of somatic mutations in cancer ( cosmic ) database ( http://www.sanger.ac.uk/genetics/cgp/cosmic/ ) ) .
most of these mutations are clustered in exon 9 and exon 20 , which corresponds to the helical domain mutant e545k , and the kinase domain mutant h1047r , respectively .
almost all pik3ca mutations are gain - of - function mutants , further supporting its oncogenic role in human malignancies . in human hnscc samples ,
the pik3ca mutation rate is about 10% but is relatively higher ( 20% ) in hnscc arising from a pharyngeal site .
in addition to somatic mutations , genomic amplification of pik3ca has also been reported in several human cancers .
interestingly , a significantly higher percentage of pik3ca gene amplification was noted in squamous cell carcinoma , compared to adenocarcinoma in lung . in human hnscc tissue samples , over 30% of cases involving pik3ca amplification involve the candidate gene residing in the common amplification region of 3q26.3 in human hnscc samples [ 23 , 24 ] .
pik3ca alterations have been associated with cancer recurrence , metastasis [ 26 , 27 ] , and poor prognosis [ 28 , 29 ] in a variety of human cancers . in hnscc ,
pik3ca alterations correlate with an advanced stage [ 30 , 31 ] , vascular invasion , and lymph node metastasis .
interestingly , in breast cancer cell motility and metastatic potential are differentially enhanced depending on whether their mutations are localized at the helical or kinase domain .
an overexpression of the helical domain through mutation e545k of pik3ca produces a more severe metastatic phenotype compared to that of the kinase domain mutation h1047r .
although the activated forms of pik3ca , generated through either mutation or amplification , are transforming in vitro [ 1 , 13 ] , their oncogenic potential in vivo has only recently been assessed through the gemm approach .
while deletion or inactivation of pik3ca significantly impairs oncogenic transformation and produces a significant resistance to ras - oncogene - induced tumorigenesis , overexpression of pik3ca results in hyperplasia in ovarian surface epithelium and can predispose mammary glands to neoplastic transformation .
moreover , a knock - in pik3ca h1047r mutant is sufficient to induce lung and breast cancer development [ 3840 ] .
work being done in our own lab suggests that these trends apply to hnscc as well . using a hnscc inducible transgenic mouse line that we had previously developed , we observe a strong oncogenic role of pik3ca in hnscc at both initiation and progression when it is overexpressed in head and neck epithelium ( l. du et al .
although akt is largely regarded as the dominant mediator of oncogenic pi3k signaling , recent studies suggest that the link between pi3k and akt can be uncoupled [ 1518 ] .
for example , pdk1 , but not akt , is activated in some breast cancers with pik3ca mutations ( figure 1 ) . using a mouse model of breast cancer conditionally expressing the pik3ca h1047r mutant , liu et al .
have shown that pik3ca - driven mammary tumors occur via both pi3k - pathway - dependent and pi3k - pathway - independent mechanisms , suggesting the complexity of the pi3k - driven oncogenic mechanisms . to this point ,
sophisticated pik3ca - gemms for a variety of cancer types may prove to be powerful tools in revealing the role of pik3ca in a context- and stage - specific manner .
besides the common alterations of the pik3ca gene encoding the catalytic p110 subunit of class ia pi3k , somatic mutations in the pik3r1 gene encoding the regulatory subunit p85 have been detected in multiple human cancers , including endometrium ( 26% ) , colon ( 5% ) , central nervous system ( 4% ) , breast ( 2% ) , pancreatic ( 2% ) , and skin ( 1% ) ( adapted from the cosmic database ) .
interestingly , somatic mutations of pik3r1 are fairly common ( 7% , 3/41 ) in human hnscc samples , with two missense mutations and one in - frame insertion .
the functional consequence of these mutants seems oncogenic as the mutants weaken an inhibitory interaction while retaining a stabilizing interaction between p85 and p110 , resulting in an activation of pi3k signaling .
however , p85 has also been shown to positively regulate pten , and reduced expression of p85 correlates with decreased pten expression .
furthermore , deletion of pik3r1 in mouse liver resulted in aggressive hepatocellular carcinomas with pulmonary metastasis , suggesting a tumor suppressor role .
thus , further characterization of pik3r1 mutants using both in vitro and in vivo approaches is warranted to reveal its role in hnscc tumorigenesis .
similar to the oncogenic role of the p110 catalytic subunit , the other isoforms of the catalytic subunit , p110 , p110 , and p110 , have been shown to be oncogenic in experimental settings although there are no reports of molecular alterations of these isoform subunits in human cancer samples .
p110 has been studied the most among these isoform subunits , and most of the results came from the p110-gemm approaches . using a pten - gemm for prostate cancer , jia et al
. showed that ablation of p110 , but not p110 , impeded prostate tumorigenesis . on the other hand , overexpression in a constitutively
furthermore , knock - in of a catalytically inactive form of p110 blocked tumor development in an erbb2-gemm for breast cancer .
future research , using both human samples and gemm approaches and aiming to assess the roles of these isoform subunits in hnscc , will surely produce interesting results .
pten acts as a negative regulator for the pi3k signaling by dephosphorylating pi(3,4,5)p3 and is the second most commonly mutated tumor suppressor in human cancers .
it is estimated that the overall frequency of pten mutations in sporadic human cancers is about 12% ( 2044 mutated samples among a total of 17452 samples according to the cosmic database ) , with endometrium cancer displaying the highest frequency among those considered ( 38% , 690 mutated samples among a total of 1837 samples in the cosmic database ) .
somatic mutation of pten in sccs of the head and neck is about 3% ( 22 mutated samples among a total of 745 samples in the cosmic database ) but is higher in sccs of skin ( 14% , 92 mutated samples among a total of 658 samples in the cosmic database ) .
compared to the relatively less common somatic mutation rate , loss of pten expression was more common ( ~30% ) in human hnsccs .
loss of heterozygosity at chromosome 10q near pten was detected in over 70% of the pten - mutated hnsccs , suggesting an inactivation of a typical tumor suppressor .
promoter hypermethylation of pten has also been detected in multiple pten expression - lacking human cancers [ 51 , 52 ] .
nonetheless , loss of pten expression has been correlated with tumor prognosis and incorporated into the grading system used for human hnscc patients [ 49 , 54 ] .
posttranscriptional regulation of pten by the developmental transcription factor grhl-3 has been shown to correlate with pten loss in sccs in both the skin and the head and neck .
another potential mechanism for pten loss is through posttranscriptional regulation by recently discovered mrnas , namely mir-21 , mir-26a , and mir-106b-25 , all of which have been identified as pten - targeting mrnas [ 5658 ] .
we have shown recently that mir-9 level is positively correlated with pten level in human hnscc cell lines .
for example , the ubiquitin ligase nedd4 - 1 has been shown to negatively correlate with pten level . whether nedd4 - 1 overexpression accounts for a subset of pten loss in human hnscc samples requires further investigation ( figure 1 ) .
other molecules closely related to pten have also been found to be altered in multiple human cancers .
pip3 rac exchanger 2a ( p - rex2a ) has been implicated as a pten - interacting protein and antagonizes pten in human cancers .
similar to the phosphatase activity of pten in the pi3k / akt signaling pathway , inositol polyphosphate 4-phosphatase type ii ( inpp4b ) is able to suppress the pi3k / akt signaling pathway and behaves as a tumor suppressor in at least breast and ovarian cancers .
lastly , the pten pseudogene ptenp1 has been shown to regulate pten level and acts as a tumor suppressor in human cancers ( figure 1 ) .
one of the major consequences of pten alteration is the activation of its main downstream targets akt and mtor , which are oncogenic in hnscc tumorigenesis and are attractive targets for cancer therapies .
for example , the jnk signaling pathway has been found to be a functional target of pten and is significantly associated with pten loss .
protein synthesis by the rna - dependent protein kinase ( pkr ) and the subunit of eukaryotic translation initiation factor 2 ( eif2 ) phosphorylation pathway is also required for tumor suppression by pten .
these results generate potential therapeutic targets to act alongside targeting of the canonical pi3k signaling pathway . given the potent tumor suppressor role of pten in multiple human cancers , gemms possessing tissue - specific deletion of pten have been created to better understand pten in tumorigenesis .
the most striking pten - gemm for human cancer is the prostate pten deletion model , in which mice with a single deletion of pten in prostate cells developed metastatic prostate cancer .
pten deletion also resulted in spontaneous tumor development in other organs , such as breast , lung , bladder , and skin , with a wide range of tumor onset pattern and penetrance .
furthermore , pten deletion increases susceptibility of mice to the induction of lung cancer by the tobacco carcinogen nnk [ 4-(methylnitrosamino)-1-(3-pyridyl)-1 - 1-butanone ] suggesting a role for pten in tobacco - induced tumor initiation .
additionally , pten deletion enhances tumor development and progression in the presence of additional molecular changes , such as ras and p53 [ 68 , 69 ] . using head - and - neck - specific gemms , we deleted pten specifically in the head and neck region and observed both premalignant lesions and tumor development .
this head - and - neck - specific pten - gemm can be utilized as a model for testing chemoprevention and therapeutic approaches targeting pi3k pathway ( j. p. shen et al .
the serine / threonine kinase akt is the central mediator of the canonical pi3k pathway and mediates multiple cellular processes , including cell survival , proliferation , angiogenesis , metabolism , and protein translation through numerous downstream signaling proteins .
for this particular paper , we will focus on the following topics : ( 1 ) molecular alterations of akt in human cancers , ( 2 ) isoform - specific role of akt in human cancers , ( 3 ) tobacco carcinogen - induced akt activation , ( 4 ) in vivo role of akt in human cancers , and ( 5 ) regulation of akt by interacting proteins . given its central node position in the canonical pi3k pathway ,
this subsequent akt activation , together with molecular alterations of akt itself , represents one of the most frequent molecular changes in human cancers and provides rationale for targeting akt as a therapeutic approach .
all three isoforms of akt , that is akt1 , akt2 , and akt3 , have been reported to be altered in various human cancers [ 70 , 71 ] .
somatic mutation occurs most frequently in akt1 and almost exclusively manifests as the e17k mutation [ 72 , 73 ] .
this akt1 e17k somatic mutation was detected in about 5% of breast cancers and 3% of both thyroid and urinary cancers
although the akt1 e17k mutation has not been identified so far in human sccs of head and neck according to a single report , this mutation has been found in human sccs of lung .
mutations of akt2 have been sporadically reported in various human cancers , but none of these mutations occur at the corresponding position of the e17k in akt1 . of particular note , somatic mutation of akt2
mutations of akt3 at both e17k and other sites were reported in melanoma and endometrial carcinoma . unfortunately , as of yet there are no studies examining akt2 and akt3 mutations in human hnscc samples .
it is also worth noting that , although several studies have shown the oncogenic properties of these akt mutations in vitro , a confirmation of their functional significance requires further investigation and demonstration of validity in vivo .
in addition to somatic mutation , overexpression of akt isoforms , particularly akt2 , has been reported in multiple human cancers .
also , overexpression of akt2 at the messenger level has been shown in breast and colon cancers and seems to correlate with cancer migration , invasion , and metastasis [ 78 , 79 ] .
interestingly , akt2 has been shown to be transcriptionally regulated by the master regulator of epithelial - mesenchymal transition ( emt ) , twist , and is associated with tumor progression and metastasis [ 78 , 79 ] .
overexpression of akt1 and akt3 has only been shown in human gastric cancer and melanoma , respectively . in human hnscc samples
the overexpression of akt2 , but not akt1 or akt3 , has been reported in one study .
pan - akt activation through phosphorylation of ser437 and thr308 is fairly common in multiple human cancers [ 19 , 70 ] .
evaluation of these phosphorylation sites yields prognostic value in human lung cancer and predicts chemotherapeutic benefit in breast cancer .
persistent akt activation is also common in human hnscc samples , and occurs as early as the premalignancy stage , including dysplasia and carcinoma in situ , suggesting that akt activation is an early event in human hnscc tumorigenesis [ 6 , 84 , 85 ] .
however , reports have also shown akt activation to correlate with a poor clinical outcome in human hnscc patients [ 86 , 87 ] .
the role of akt activation in human hnscc development and progression is still in need of further investigation .
although akt1 , 2 , and 3 share high sequence homology , clinical studies suggest the existence of isoform - specific roles of akt in multiple human cancers [ 19 , 70 ] .
this is further validated by experimental studies using both in vitro and in vivo approaches . while akt1 and akt2 play a similar role in regulating cell survival and proliferation , they behave distinctly in their regulation of cell migration and emt [ 88 , 89 ] . for example , akt1 knockdown induces cell migration and emt in breast cancer cell lines , while akt2 knockdown suppresses these behaviors .
this is further exemplified in breast cancer mouse models : while overexpression of akt1 accelerates erbb-2 mediated mammary tumorigenesis and suppresses tumor invasion , overexpression of akt2 markedly increases the incidence of pulmonary metastases in breast cancer .
these data suggest that akt1 acts as a metastasis suppressor , while akt2 as a metastasis promoter , further warranting the need to use isoform - specific akt inhibitors in clinical management of cancer patients .
distinct downstream targets of each akt isoform might mediate this separate signaling transduction and be responsible for the distinct behavior of akt isoforms in human breast cancer progression and metastasis . a recent report showing regulation of mrna-200 , which plays a critical role in cell migration and emt , by the ratio of akt1 to akt2 suggests another potential mechanism for the isoform - specific roles of akt .
yet , there are no human hnscc studies that address these questions though they are needed to guide future clinical trials on hnscc patients using akt isoform - specific inhibitors .
though tobacco exposure is one of most important etiological factors in hnscc tumorigenesis , its underlying molecular mechanisms remain poorly understood .
in addition to the formation of dna adducts , tobacco carcinogens , such as nnk , activate several signal transduction pathways , including akt , in both normal and cancer cells in the lung .
we have shown that , in both hnscc tumors and the adjacent mucosa , akt is activated at a higher frequency in hnscc patients who are smokers compared to those who are nonsmokers .
also , adding physiologically relevant concentrations of nnk to normal head and neck epithelial cells and hnscc cell lines will rapidly and constitutively activate akt in a dose - dependent and time - dependent manner .
finally , we demonstrated that nnk exposure to mouse head and neck epithelium results in epithelial hyperproliferation and reduced apoptosis , which is correlated with akt activation .
these studies suggest that akt activation plays a pivotal role in mediating tobacco - induced hnscc carcinogenesis and that it may be an effective target for chemoprevention .
the precise functional consequence of akt activation can not be assessed without in vivo studies .
this is particularly critical for the evaluation of the isoform - specific role of akt in context- and stage - specific manners .
current in vivo models of akt activation overwhelmingly confirm its oncogenic role at differing levels of potency in various cancer types .
however , at least in breast cancer models , the distinct roles of different akt isoforms in cancer progression and metastasis have been observed as described previously .
mice constitutively overexpressing an activated akt driven by a keratin 5 promoter developed both skin and head and neck tumors and an increased sensitivity to skin carcinogenesis [ 96 , 97 ] .
the oral lesions seen in these specimens were mostly epithelial dysplasia , their malignant transition hampered by the induction of premature senescence .
this suggests that akt activation is an early event but not sufficient by itself to hnscc tumorigenesis .
this is further supported by the introduction of p53 loss , which synergizes with akt activation to develop metastatic hnscc .
numerous proteins similar to pten have been identified as regulators of akt activation and stability .
ph domain leucine - rich repeat protein phosphatase ( phlpp ) has been shown to attenuate akt signaling through the regulation of distinct akt isoforms .
deletion or loss of expression of phlpp has been reported in a significant fraction of colon and prostate cancers [ 99 , 100 ] .
fk506-binding protein 51 ( fkbp51 ) has been shown to act as a scaffolding protein for akt and phlpp , as well as promote the dephosphorylation of akt .
posttranslational modifications of akt through ubiquitinating proteins such as ttc3 have also been reported ; however , this protein role in oncogenic akt activation has not been studied .
lastly , the p53 target gene ph domain - only protein ( phlda3 ) has been found to compete with the ph domain of akt for binding to membrane lipids , thereby inhibiting akt translocation to the cellular membrane and , thus , its activation .
consistent with its function , loss of the phlda3 genomic locus is frequently observed in primary human lung cancer samples .
however , such studies have not yet been reported in hnscc cases ( figure 1 ) .
although it is widely accepted that akt activation acts as the primary oncogenic mediator in canonical pi3k signaling , as has been stated previously in this paper , recent studies suggest that the link between pi3k and akt can be uncoupled [ 1518 ] .
for example , akt signaling is diminished in human breast cancer cell lines and clinical samples harboring pik3ca mutation . in lieu of akt , these cells make use of a signaling pathway involving the pi3k effector pdk1 and its downstream substrate sgk3 .
increased gene copy numbers of pdk1 have been found in 21% of breast cancer samples , and total pdk1 mrna and protein have been observed to be overexpressed in the majority of human breast cancer .
overexpression of pdk1 promotes invasion and activation of matrix metalloproteinase , while downregulation of pdk1 inhibits migration and experimental metastases of human breast cancer cells .
introduction of a hypomorphic mutation of pdk1 in a pten cancer mouse model suppresses tumorigenesis , confirming the oncogenic role of pdk1 as one of the downstream effectors of the pi3k / pten signaling , and suggesting pdk1 as a promising anticancer target .
however , the role of pdk1 and its correlation with the canonical pi3k / pten / akt pathway in hnscc development and progression has not been assessed yet . among the numerous molecules that could act as downstream effectors of the pi3k / akt pathway , mtor is of particular interest .
mtor assembles into at least two distinct complexes , that is , mtorc1 and mtorc2 .
mtorc2 contains rictor , sin1 and mlst8/gbl and acts upstream of akt to phosphorylate the ser473 of akt .
in contrast , mtorc1 contains raptor , pras40 , and mlst8/gbl and acts as a major downstream target of akt .
mtorc1 regulates cell growth by controlling key eukaryotic translational regulators , including p70-s6 kinase , and the eukaryotic translational initiation factor , 4e binding protein 1 ( 4e - bp1 ) .
in addition , the growth factor receptor - bound protein 10 ( grb10 ) has been recently identified as an mtorc1 substrate . the mtorc1-mediated phosphorylation stabilizes grb10 , leading to the inhibition of the pi3k and erk - mapk pathways .
interestingly , grb10 is frequently downregulated in various human cancers , with the loss of grb10 and pten being mutually exclusive .
this indicates grb1 as being a tumor suppressor , potentially regulated by mtorc1 ( figure 1 ) .
in addition to directly activating mtorc1 , akt also phosphorylates tuberous sclerosis complex ( tsc ) 1 and tsc2 , releasing their inhibition of the ras - like small g protein , rheb , which in turn activates mtorc1 . during conditions of low nutrient availability ,
mtor signaling is normally inhibited by amp activated protein kinase ( ampk ) , which is activated by its upstream serine / threonine kinase lkb1 ( figure 1 ) .
interestingly , these negative regulators of mtor , that is , tsc1 , tsc2 , and lkb1 , are tumor suppressors , and germline mutations of tsc1/2 or lkb1 cause hamartomas and predisposition to multiple malignancies in humans .
the critical connection of mtor to the pi3k / akt pathway has led to the prediction that the targeting of mtor may be useful in cancer therapy . indeed , using the mtor inhibitor , rapamycin , yields promising results for multiple human cancers . in human hnscc ,
activation of mtor / p70-s6/4e - bp1 pathway is a frequent event in clinical specimens and cell lines .
loh of tsc1/2 and dna methylation of tsc2 have been reported in human hnscc samples , and overexpression of tsc2 inhibits cell growth both in vitro and in vivo .
a somatic mutation of lkb1 , which leads to the loss of growth inhibition , has been found in a human hnscc patient . decreased nuclear lkb1 levels
consistent with the effects of anti - mtor therapy in other cancers , inhibition of mtor by rapamycin displays a potent antitumor effect in hnscc in vitro , in oral carcinogenesis model , and in a hnscc - gemm .
targeting mtor has also been shown to be a possible adjuvant therapy for microscopic residual disease in human hnscc patients .
however , studies exploring the frequencies of these molecular alterations and their mechanisms in hnscc tumorigenesis are still lacking .
activation of pi3k / akt / mtor pathway has been illustrated as an early event in multiple human cancers , suggesting that targeting the pi3k / akt / mtor pathway may have chemopreventive value .
this is further exemplified by tobacco exposure activation of akt / mtor in multiple tobacco - related malignancies , including hnscc [ 95 , 119 ] .
the tobacco - associated nnk and the dna adduct - forming agent 4-nitroquinoline-1-oxide ( 4nqo ) activate akt / mtor as early as premalignancy stage [ 95 , 119 , 120 ] .
inhibition of this pathway by either deguelin , a natural compound belonging to the rotenoid family , or metformin , an antidiabetes medicine , has been shown to possess chemopreventive effects on a tobacco carcinogen - induced lung tumorigenesis model .
in addition , mtor inhibition by rapamycin has been shown to prevent early onset of hnscc tumorigenesis in both the 4nqo - induced hnscc mouse model and a hnscc - gemm .
additionally , resveratrol , a phytoalexin enriched in red grapes , strawberries , and peanuts , has been found to be a potent chemoprevention agent for many cancers .
one of the major mechanisms of its chemopreventive effect is through inhibition of the pi3k / akt / mtor pathway
. it will be interesting to study its chemopreventive effect on both the 4-nqo - induced hnscc mouse model and the hnscc - gemms .
personalized cancer therapies with selective molecular targets have emerged as a novel class of anticancer agents , with demonstrated clinical efficacy and less toxicity than conventional therapies . in this situation ,
the pi3k / akt / mtor pathway has been extensively studied in almost all human malignancies including hnscc and in both experimental and clinical settings .
multiple drugs have been designed to target this pathway , making it the most druggable pathway for targeted therapies of human cancers .
given the scope of this paper , we will only comment on a few aspects . for a more detailed explanation of progress in pi3k targeted therapy on hnscc
, please refer to any of the several excellent reviews available on the topic [ 3 , 5 , 126 , 127 ] .
( 1 ) identification of biomarkers for the personalized cancer therapy targeting the pi3k pathwaysince the concept of personalized cancer therapy is based on the identification of a subset of patients whose tumors carry specific molecular alterations , biomarker identification is critical for predicting the effectiveness of targeted therapy .
for example , both pik3ca mutations and nuclear phosphorylation of akt are shown as biomarkers for the effectiveness of pi3k inhibitors for human cancer patients [ 128 , 129 ] .
in addition , human cancer patients harboring pik3ca mutations are sensitive to targeted therapy using the mtor inhibitor everolimus , while human cancer patients carrying kras mutations are resistant to the treatment .
this is further confirmed in a pik3ca - gemm and kras - gemm for lung cancer .
while nvp - bez235 , a dual pan - pi3k and mtor inhibitor , led to marked tumor regression in the pik3ca - gemm , it did not affect tumor growth significantly when treating the kras - gemm unless it was used in combination with a mek inhibitor . finally , a recent report of the screening of over three hundred nonredundant pi3k - pathway - relevant phosphopeptides identified pras40 , a molecule involved in protein phosphorylation , as a biomarker correlated with pi3k pathway activation and akt inhibitor sensitivity .
since the concept of personalized cancer therapy is based on the identification of a subset of patients whose tumors carry specific molecular alterations , biomarker identification is critical for predicting the effectiveness of targeted therapy .
for example , both pik3ca mutations and nuclear phosphorylation of akt are shown as biomarkers for the effectiveness of pi3k inhibitors for human cancer patients [ 128 , 129 ] .
in addition , human cancer patients harboring pik3ca mutations are sensitive to targeted therapy using the mtor inhibitor everolimus , while human cancer patients carrying kras mutations are resistant to the treatment .
this is further confirmed in a pik3ca - gemm and kras - gemm for lung cancer .
while nvp - bez235 , a dual pan - pi3k and mtor inhibitor , led to marked tumor regression in the pik3ca - gemm , it did not affect tumor growth significantly when treating the kras - gemm unless it was used in combination with a mek inhibitor . finally , a recent report of the screening of over three hundred nonredundant pi3k - pathway - relevant phosphopeptides identified pras40 , a molecule involved in protein phosphorylation , as a biomarker correlated with pi3k pathway activation and akt inhibitor sensitivity .
( 2 ) activation of pi3k / akt / mtor pathway as a resistance mechanism to targeted therapy or radiotherapyegfr targeted therapy is the first fda - approved protocol for treating human hnscc patients .
activation of the canonical pi3k / akt / mtor pathway seems to be associated with resistance to egfr inhibitor in multiple human cancers .
however , a recent study showed that an egfr - activating mutation resistant to targeted therapy activates the mtorc2-nf-b signaling pathway in an akt - independent manner in glioblastoma patients .
further studies are necessary to investigate the role of both canonical and noncanonical pi3k pathways in resistance to egfr therapy in human hnscc patients .
however , activation of the pi3k / akt / mtor pathway is implicated in all major mechanisms of radioresistance , including intrinsic radioresistance , tumor cell proliferation , and hypoxia .
thus , blocking the pi3k / akt / mtor pathway has great potential to enhance the effectiveness of radiotherapy for hnscc patients .
egfr targeted therapy is the first fda - approved protocol for treating human hnscc patients .
activation of the canonical pi3k / akt / mtor pathway seems to be associated with resistance to egfr inhibitor in multiple human cancers .
however , a recent study showed that an egfr - activating mutation resistant to targeted therapy activates the mtorc2-nf-b signaling pathway in an akt - independent manner in glioblastoma patients .
further studies are necessary to investigate the role of both canonical and noncanonical pi3k pathways in resistance to egfr therapy in human hnscc patients .
however , activation of the pi3k / akt / mtor pathway is implicated in all major mechanisms of radioresistance , including intrinsic radioresistance , tumor cell proliferation , and hypoxia .
thus , blocking the pi3k / akt / mtor pathway has great potential to enhance the effectiveness of radiotherapy for hnscc patients .
( 3 ) synergistic effect of combination with other receptor tyrosine kinase targeted therapiesrecent evidence of multiple feedback loops and interactions with other signaling pathways highlights the complexity of pi3k signaling .
using an inducible pik3ca - gemm for breast cancer , liu et al . identified c - myc elevation as a potential mechanism by which tumors develop resistance to pi3k - targeted therapies . moreover ,
inhibition of akt induces activation of upstream rtk signaling pathways , such as her3 , and mtor inhibition causes activation of akt signaling or mapk pathway .
these studies suggest that combination therapies of pi3k - targeted therapy together with targeting c - myc , her3 , or mapk pathway , may be more effective for the treatment of certain human cancer patients .
recent evidence of multiple feedback loops and interactions with other signaling pathways highlights the complexity of pi3k signaling . using an inducible pik3ca - gemm for breast cancer , liu et al . identified c - myc elevation as a potential mechanism by which tumors develop resistance to pi3k - targeted therapies .
moreover , inhibition of akt induces activation of upstream rtk signaling pathways , such as her3 , and mtor inhibition causes activation of akt signaling or mapk pathway .
these studies suggest that combination therapies of pi3k - targeted therapy together with targeting c - myc , her3 , or mapk pathway , may be more effective for the treatment of certain human cancer patients .
mounting evidence clearly shows both the paramount importance of the pi3k pathway in the tumorigenesis of many human malignancies including hnscc , and the promising results of targeting this pathway for treatment of human cancer patients . however , there are still many questions that need to be answered . compared to the extensive studies on pik3ca , there are few studies on the other subunits of class ia pi3ks and their interactions with pik3ca and pten
. studies of classes ii and iii of pi3ks in human cancers are generally lacking .
although several proteins interacting with pten or akt have been shown to play a role in tumorigenesis , more studies must be undertaken to discover novel molecules modulating the pi3k pathway , and assess their roles in tumorigenesis .
in addition to the relatively linear canonical pi3k / akt pathway , more and more noncanonical pathways are expected to be identified .
furthermore , the newly discovered mrnas described in this paper add yet another layer of complexity to our understanding of the molecular regulation of the pi3k signaling pathway .
integrative mapping of molecular alterations in human cancers , particularly in hnscc samples , is highly demanding .
utilization of multiple molecular approaches , especially gemms of the pi3k signaling pathway , will help us to better understand the complexity of this pathway in human cancers , as well as in context and stage - specific manners .
ultimately , these studies will yield identifiable biomarkers for improved clinical diagnosis and prognosis , contributing to strategies of therapy and prevention that will allow for the better management of human cancers and better outcomes for human patients . | activation of the phosphatidylinositol-3-kinase ( pi3k ) pathway is one of the most frequently observed molecular alterations in many human malignancies , including head and neck squamous cell carcinoma ( hnscc ) .
a growing body of evidence demonstrates the prime importance of the pi3k pathway at each stage of tumorigenesis , that is , tumor initiation , progression , recurrence , and metastasis .
expectedly , targeting the pi3k pathway yields some promising results in both preclinical studies and clinical trials for certain cancer patients . however , there are still many questions that need to be answered , given the complexity of this pathway and the existence of its multiple feedback loops and interactions with other signaling pathways .
in this paper , we will summarize recent advances in the understanding of the pi3k pathway role in human malignancies , with an emphasis on hnscc , and discuss the clinical applications and future direction of this field . |
prion diseases are a family of progressive fatal neurodegenerative diseases of infectious , sporadic or inherited origin , which affect humans and other animals .
central to the pathogenesis is the conversion of a host - encoded prion protein , prp , into a misfolded , protease resistant isoform , prp , which accumulates in the brain and constitutes the infectious agent responsible for the disease .
prion diseases are typically diagnosed by manifestation of dementia and locomotor symptoms , which correlate with extensive neuronal loss and prp deposition . at the histological level , neuronal loss is preceded by synaptic pathology and early synaptic failure is a well - documented component of prion diseases .
however , little is known about the underlying mechanisms of synaptic degeneration , and the sequence of events involved in the neurodegeneration is not yet completely understood .
it has been proposed that impairment in mitochondrial function could be responsible for the synaptic pathology observed in prion diseases . by investigating a wide range of mitochondrial parameters , it was shown that mitochondrial function was impaired in prion diseases and that mitochondrial damage could potentially be the initial cause of synaptic pathology .
additionally , several neurodegenerative diseases marked by the accumulation of misfolded proteins , such as alzheimer and parkinson diseases , are associated with synaptic dysfunction and mitochondrial abnormalities .
evidence that mitochondria could have a role in prion pathology comes from the finding that prp can interact with neuronal mitochondria and impair their function . in transgenic mice overexpressing prp ,
redistribution of prp to the mitochondria induces the loss of mitochondrial membrane potential ( mmp ) and cytochrome c release , resulting in caspase-3-dependent apoptosis , predominantly in hippocampal neurons .
moreover , the expression of prp as a cytosolic protein induces the formation of cytosolic aggregates , mitochondrial clustering and promotes cell death by a process that involves depolarization of the mitochondrial membrane , release of cytochrome c and caspase activation .
finally , the synthetic peptide prp-(106126 ) , which has been shown to be neurotoxic , can induce the rapid depolarization of mitochondrial membranes , cytochrome c release and apoptosis in a panel of different neuronal cells .
while the mechanism of prion disease development remains unclear , mitochondrial damage and dysfunction has emerged as the possible initial cause of synaptic degeneration , which eventually results in apoptotic neuronal cell death .
the possibility that the mitochondria constitute the primary site of the proapoptotic pathway opens up new avenues for early diagnosis and particularly for early treatment .
prp is a glycoprotein that localizes to the plasma membrane via a c - terminally linked glycosylphosphatidylinositol ( gpi ) anchor .
this posttranslational modification concerns over 100 different proteins in humans , including adhesion molecules , surface receptors and enzymes .
gpi anchor addition occurs in the lumen of the endoplasmic reticulum ( er ) , where the pre - assembled gpi anchor is transferred to the acceptor amino acid ( called the omega site ) by the action of a multimeric gpi transamidase .
the gpi transamidase recognizes a c - terminal gpi - anchor signal peptide ( gpi - sp ) , which is cleaved concomitantly to the attachment of the gpi - anchor .
the gpi - sp consists of a hydrophilic region ( 510 amino acids ) followed by a hydrophobic sequence ( 1520 amino acids ) , but overall lacks a well - defined consensus sequence . as such
, they are not usually conserved across species , with the significant exception of prp , whose gpi - sp shows a remarkable degree of conservation .
additionally , prp gpi - sp harbors 2 autosomal dominant pathogenic mutations responsible for genetic forms of prion diseases ( m232r and p238s ) ; these mutations are particularly perplexing since the gpi - sp is replaced by a gpi anchor and is absent in the mature prion protein . because of its unusual level of conservation , and because of the presence of unexplained pathogenic mutations in its sequence , prp gpi - sp represents an ideal model to address the fate of the gpi - anchor signal peptides . to investigate the metabolism of prp gpi - sp we recently created a hybrid
construct where the c - terminus of gfp had been fused to the gpi - sp of prp , which had then been linked to the double tag myc - his via a flexible gly - ser linker . as a control , we fused gfp to the gpi - sp of folate receptor ( fr )
; the gpi - sps of fr and prp have the same length ( 24 amino acids ) and an identical size of the hydrophilic ( 9 amino acids ) and the hydrophobic ( 14 amino acids ) regions .
we were able to show that , while fr gpi - sp was retained within the lumen of the er , prp gpi - sp was selectively transported to the cytoplasm , where it was targeted for degradation by the proteasome .
interestingly , when the pathogenic point mutation p238s was introduced into our construct , prp gpi - sp was spared from degradation . linking the p238s point mutation to the accumulation of prp gpi - sp potentially
establishes a link between the accumulation of prp gpi - sp and prp induced neurodegeneration , opening up the possibility that prp gpi - sp could play a role in prion diseases .
we show that , differently form the control gpi - sp of folate receptor , the gpi - sp of prp is targeted to the mitochondria , and causes their fragmentation and loss of mmp .
to further explore the function of prp gpi - sp , we decided to investigate the fate of prp gpi - sp following its retrotranslocation into the cytoplasm . because the retrotranslocation of proteins into the cytosol is ubiquitination - dependent
, we reasoned that the expression of a cytosolic form of prp gpi - sp ( cprpgpi - sp ) would allow prp gpi - sp to escape ubiquitination .
indeed , it has been shown that er proteins that are targeted for proteasomal degradation accumulate in the cytosol when degradation is prevented by proteasome inhibitors ( as is the case for prp gpi - sp ) , but remain restricted to the lumen of the er under ubiquitination - deficient conditions , confirming that the release of the protein into the cytosol is linked to ubiquitination .
the cytosolic versions of prp 's and fr 's gpi - sps ( cprpgpi - sp and cfrgpi - sp respectively ) were cloned starting from gfp - fr - mh and gfp - prp - mh ( described in ref .
constructs the gpi - sp was linked to a myc - his ( mh ) tag via a gly - ser flexible linker , as described in ref .
the scheme in figure 1a represents the starting construct ( gfp - gpi - tag ) and circled in red is the part expressed as cytosolic protein .
the constructs were expressed in hela cells and analyzed by western blot ( wb ) and immunofluorescence ( if ) .
wb showed that both constructs produced a single band at the expected molecular weight ( fig .
1b ) ; by if analysis , the 2 constructs showed significantly different localization patterns .
cfrgpi - sp appeared to localize to a reticular network that extended across the cytoplasm and around the nucleus ( fig .
1c ) , most likely representing the er . this localization is not surprising , as the hydrophobic region of gpi - sps can potentially function as an er - signal sequence , explaining why cfrgpi - sp localizes to the er . specifically , prp gpi - sp has been shown to work as an er - signal sequence when linked to the n - term of gfp .
however , when expressed in hela cells , cprp - gpi - sp localized to discrete tubular elements ( fig .
1d ) , reminiscent of the mitochondrial network . to confirm the localization of our constructs , cells expressing cfrgpi - sp were co - stained with the er marker calnexin ( fig .
g ) and cells expressing cprpgpi - sp were labeled with the mitochondrial marker tom20 ( fig . 1h
j ) . as shown in figure 1e j , complete colocalization confirmed that cprpgpi - sp localizes to the mitochondria , whereas cfrgpi - sp localizes to the er .
figure 1.expression and localization of cprpgpi - sp and cfrgpi - sp .
wb ( a ) and if ( b , c ) of hela cells expressing cfrgpi - sp and cprpgpi - sp .
( d f ) colocalization between cfrgpi - sp ( green ) and the er marker calnexin(red ) .
( g i ) colocalization between cprpgpi - sp ( green ) and the mitochondrial marker tom20 ( red ) .
nuclei are in blue .
expression and localization of cprpgpi - sp and cfrgpi - sp .
wb ( a ) and if ( b , c ) of hela cells expressing cfrgpi - sp and cprpgpi - sp .
( d f ) colocalization between cfrgpi - sp ( green ) and the er marker calnexin(red ) .
( g i ) colocalization between cprpgpi - sp ( green ) and the mitochondrial marker tom20 ( red ) .
while cfrgpi - sp er localization had no effect on the structure of the compartment or on the viability of the transfected cells ( data not shown ) , we noticed that in cells expressing cprpgpi - sp the mitochondria were affected in a time - dependent fashion : within the first hours after transfection , cprpgpi - sp localized to intact active mitochondria , as shown by costaining with mitotracker red cmxros .
mitotracker red cmxros was used to indicate the alteration of mitochondrial membrane potential ( mmp ) : cells were transfected with cprpgpi - sp and incubated with mitotracker red cmxros ( 100 nm , 30 min ) 12h , 24h and 36h after transfection ( fig .
after fixation , the degree of mmp was visualized qualitatively as red fluorescence while the morphology of nuclei could be observed as blue fluorescence ( dapi staining ) . because the uptake of mitotracker red depends on mmp , loss in mmp causes a dramatic decrease of red fluorescence . as shown in figure 2
, the shape of mitochondria dramatically changed , appearing fragmented at 24h and extensively vacuolarized at 36h after transfection ; at the same time the nuclei appeared condensed , a characteristic morphology of cells undergoing apoptosis ( fig .
moreover , the degree of red fluorescence in mitochondria containing cprpgpi - sp decreased from 12h to 24h and almost completely disappeared 36h after transfection .
the degree of colocalization between mitochondria and cprpgpi - sp was quantified by calculating manders overlap coefficient ( fig .
manders m1 and m2 coefficients measure the portion of the pixels in each channel ( here red and green ) that coincides with a signal in the other channel .
the m coefficient varies from 0 to 1 , where 1 indicates complete overlap . after each time - point
( 12h , 24h , 36h ) , the value of m1 ( amount of mitotracker colocalizing with cprpgpi - sp ) was 0.9 0.05 , indicating that virtually all mitotracker signal overlaps with cprpgpi - sp 's signal .
however , the value of m2 ( amount of cprpgpi - sp colocalizing with mitotracker ) decreased from 0.9 ( at 12h after transfection ) to 0.4 and 0.1 ( 24h and 36h after transfection respectively ) , indicating that the majority of cprpgpi - sp had lost mitotracker staining , which is indicative of loss in mmp .
hela cells transfected with cprpgpi - sp for 12h ( a c ) , 24h ( d f ) and 36h ( g i ) were stained to visualize cprpgpi - sp ( green ) and the status of mmp via mitotracker ( red ) .
colocalzation efficiency was measured using imagej software and shown by mander 's coefficient ( j ) .
m1 = amount of mitotracker colocalizing with cprpgpi - sp ; m2 = amount of cprpgpi - sp colocalizing with mitotracker .
hela cells transfected with cprpgpi - sp for 12h ( a c ) , 24h ( d f ) and 36h ( g i ) were stained to visualize cprpgpi - sp ( green ) and the status of mmp via mitotracker ( red ) .
colocalzation efficiency was measured using imagej software and shown by mander 's coefficient ( j ) .
m1 = amount of mitotracker colocalizing with cprpgpi - sp ; m2 = amount of cprpgpi - sp colocalizing with mitotracker .
the average and standard deviation were obtained by the analysis of 5 images . in conclusion
, we were able to show that prp gpi - sp , when expressed as a cytosolic peptide , is able to localize to the mitochondria and to induce mitochondria fragmentation and vacuolarization , which are accompanied by loss of mmp and ultimately apoptosis .
this phenotype of mitochondrial damage and mitochondrial - dependent apoptosis strengthens the evidence that mitochondria play a role in prion pathology .
our results , for the first time , identify the gpi - sp of prp as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases .
it is conceivable that during prion infection prp gpi - sp may accumulate in the cytoplasm , bind to mitochondria and induce depolarization of the mitochondrial membrane , release of cytochrome c and caspase activation , resulting in neuronal cell death .
p. cossart , institut pasteur , paris , france ) were maintained in dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen ) supplemented with 10% fetal calf serum ( fcs ) in a 5% co2 incubator at 37c .
cells were transfected at 70% confluence using fugene6 ( roche diagnostic ) according to manufacturer 's protocol . for immunofluorescence and western blot
the following antibodies were used : primary antibodies : rabbit anti - his ( a gift from dr .
dautry , institut pasteur ) ; tom20 ( bd transduction laboratories , # 612278 ) ; calreticulin ( stressgen # spa-600 ) ; myc9e10 ( sigma # m4439 ) .
secondary antibodies for if : alexa fluor 488 goat anti mouse ( 1:500 ) and alexa fluor 594 goat anti rabbit ( 1:500 ) from molecular probes .
mitotracker red cmx ros ( invitrogen , carlsbad , ca ) was used at a final concentration of 50 nm for 15 min .
dna constructs : gpi - sps were inserted into pcdna3.1mychis expression vector ( invitrogen ) using nhei / bamhi restriction sites .
ssgfp - fr - mh and ssgfp - prp - mh described in were used as templates .
primers : 5-gga gac cca agc tgg cta gcc acc atg aag gaa ttc-3 and bghrv ; the use of a common fw primer resulted in the addition of 8aa upstream of the omega site .
immunofluorescence : hela cells plated on 12 mm glass coverslips were fixed with 4% formaldehyde , permeabilized with 0.1% tritonx100 in pbs for 5 min , then incubated in blocking buffer ( pbs containing 10% fcs ) for 30 min at room temperature .
coverslips were then mounted onto glass slides with aqua / poly mount ( polysciences ) and visualized using a zeiss observer z1 156 inverted microscope with 63 objective controlled by axiovision software ( zeiss , thornwood , ny ) .
the degree of colocalization between mitochondria ( red ) and ga - bodipy ( green ) was quantified using the colocalization finder and jacop plug - in of imagej software ( http://rsb.info.nih.gov/ij/ ) .
western blot : cells were lysed with page loading buffer ( 60 mm tris , ph 6.8 , 5% 2-mercaptoethanol , 2% sds , 0.01% bromophenol blue , and 10% glycerol ) .
page using a 420% gradient tris - tricine gel ( mini - protean tris - tricine precast gels , biorad ) .
proteins were transferred on whatman protran nitrocellulose membranes ( 0.1um pore size ) ( sigma ) .
the membrane was kept in blocking buffer ( 50 mn , tris , ph 7.5 , 150 mm nacl , 0.05% tween 20 , and 3% bsa ) and then incubated with the primary antibody diluted in blocking buffer .
hrp - conjugated secondary antibodies and ecl reagents from amersham ( ge healthcare ) were used for detection on kodak biomax films .
the work in c.z . lab was supported by fellowships from the federation of european biochemical societies ( febs ) , the fondation pour la recherche mdicale ( frm ) , the european union [ grant number fp7-kbbe-20072a-22287 ] , the anr ( agence nationale de la recherche ) ( grant numbers anr-09-blan-0122 ) and the pasteur weizmann foundation ( 20102012 ) | we previously reported that prp gpi - anchor signal peptide ( gpi - sp ) is specifically degraded by the proteasome .
additionally , we showed that the point mutation p238s , responsible for a genetic form of prion diseases , while not affecting the gpi - anchoring process , results in the accumulation of prp gpi - sp , suggesting the possibility that prp gpi - anchor signal peptide could play a role in neurodegenerative prion diseases .
we now show that prp gpi - sp , when expressed as a cytosolic peptide , is able to localize to the mitochondria and to induce mitochondrial fragmentation and vacuolarization , followed by loss in mitochondrial membrane potential , ultimately resulting in apoptosis .
our results identify the gpi - sp of prp as a novel candidate responsible for the impairment in mitochondrial function involved in the synaptic pathology observed in prion diseases , establishing a link between prp gpi - sp accumulation and neuronal death . |
urinary bladder rupture in the adult mare is rare , but has been reported to occur associated with parturition .
repair via celiotomy has been described , but it can be difficult to exteriorise the bladder sufficiently , particularly in the case of caudal or dorsal tears .
previous standing techniques have involved approaching the bladder via urethrotomy or using a urethral sphincterotomy to place a hand into the bladder . here , we describe a non - invasive urethral approach that was successfully used to repair a 10 cm tear in a postparturient mare .
the mare had foaled uneventfully 36 hours previously and since then had been depressed , had experienced mild colic and had developed a swollen abdomen .
on arrival , the mare was mildly uncomfortable with a heart rate of 72 beats / min , respiratory rate 12 breaths / min and rectal temperature 38.1c .
gastrointestinal borborygmi were reduced in all quadrants . on examination per rectum , thickened small intestine could be palpated .
trans - abdominal ultrasonography demonstrated free fluid in the peritoneal cavity and some thickened loops of small intestine .
analysis of the peritoneal fluid showed : a nucleated cell count count of 23.8 10/l ; 76.9% neutrophils ; 16.8% lymphocytes ; 8.3% monocytes ; a total protein concentration of 16
the ratio of peritoneal to plasma creatinine concentrations of just over 4:1 was indicative of uroperitoneum .
haematology revealed a leukopaenia ( peripheral white cell count 4.8 10/l ) with an increased neutrophil percentage ( 82% ) .
in addition to increased creatinine concentration , plasma biochemistry revealed increased urea ( 14 mmol / l ) and lactate ( 2.4 mmol / l ) concentrations .
plasma sodium ( 133.8 mmol / l ) , potassium ( 3.64 mmol / l ) and chloride ( 97.6 mmol / l ) concentrations were normal .
endoscopy of the bladder showed a large rent in the dorsal - cranial part of the bladder ( figure 1 ) .
a 28f chest drain was placed midline in the most ventral part of the abdomen and 15 l of proteinaceous fluid was drained off .
the abdomen was then lavaged with 12 l of warm sterile hartman 's solution through the chest drain ( figure 2 ) .
the mare was treated with flunixin meglumine ( 1.1 mg / kg iv bid ) , cefquinome ( 2 mg / kg iv sid ) and intravenous fluids ( hartmann 's solution at 1.7 l / hr ) . a 30 ml foley catheter
was placed into the bladder and secured to the perineum with a urine collection bag to monitor urine production ( figure 3 ) .
there is an area of intraluminal purple discolouration leading to a rent in the bladder wall .
a continuous infusion of detomidine ( decreasing dose from 0.6 to 0.1 ug / kg / min ) was given for sedation .
a mixture of 9 ml of 2% mepivicaine and 4 ml of 2% xylazine were placed into the epidural space after sterile preparation of the skin .
a rectal examination was performed to evacuate all faeces and the tail was tied dorsally .
a balfour retractor was placed in the dorsal vagina to provide exposure . on examination ,
three fingers could be easily inserted into the urethral orifice . using sterile lubricant and moderate force it was possible to insert a hand into the bladder .
two fingers were inserted through the tear and used to provide traction to evert the bladder completely into the vagina where it could grasped with the surgeons other hand to prevent further trauma .
this was repaired using a single layer of size zero pds ( polydioxanone ) suture in a simple continuous pattern ( figure 4 ) .
as soon as the bladder was repaired , it was replaced via the urethra . during surgery
three vials ( 180 mg ) of morphine sulphate were administered intramuscularly and topical 2% mepivicaine was placed into the urethra .
after surgery , a foley catheter was maintained in the bladder for 24 hours , urine output was measured and urine specific gravity was monitored .
flunixin meglumine was administered as an analgesic and the mare received one further abdominal lavage .
trans - abdominal ultrasound 24 hours after surgery showed no excess fluid in the peritoneal space , and that the thickening of the small intestine had resolved . after the surgery , the mare was administered domperidone ( 1.1 mg / kg po q24h ) to maintain lactation .
after the procedure , the foal was nursing well , and appeared to be receiving enough milk .
the mare was discharged after 48 hours with instructions to administer oral potentiated sulphonamides for five days and was urinating normally at this time .
one theory is that bladder rupture or trauma occurs as a result of impingement of the bladder between the pelvic rim and the foal during labour .
the bladder may rupture at parturition , or later , due to necrosis of the bladder wall .
after parturition , the symptoms that have been described include depression , anorexia , anuria , fever and abdominal distension . diagnosis can be made by detection of the bladder defect with endoscopy , trans - abdominal ultrasonography demonstrating free fluid in the abdomen , evaluation of serum biochemical variables and comparison of serum and abdominal fluid creatinine concentrations .
a number of different surgical approaches have been described for repair of the bladder in the adult horse .
a ventral median abdominal approach requires full traction on the bladder to observe the defect . in some cases ,
. this approach would be especially unsuitable for dorsal bladder tears such as the one described here .
the urethral sphincterotomy approach has been associated by some authors with post operative urine dribbling and urine pooling , although other authors have performed this successfully without long term complications .
in one , the vaginal floor was incised 5 - 10 cm caudal to the cervix to retract the bladder .
the second technique was to incise the vaginal floor and dorsal urethra 5 cm cranial to the urethral orifice .
obviously , both these approaches require careful repair of the incisions made to close the abdomen . in the mare examined in this case report ,
there was marked bruising and swelling in the vagina , particularly at the urethral orifice .
this bruising would have certainly have complicated both performing the procedures described by rodgerson and potentially healing in the postsurgical period . we were able to exteriorise the bladder by inserting a hand directly through the urethral orifice .
the small size of the author 's hand ( 9 cm 17 cm ) might have been favourable in performing this . while the technique described sounds simple , having two experienced surgeons greatly facilitated the success of this procedure .
gentle , yet firm tissue handling was essential to prevent tearing of the damaged tissue .
eversion of the bladder required very careful tissue handling in order to exert enough force to bring the bladder out without causing further damage or tearing .
having one surgeon concentrating on bladder exposure allowed repair without stay sutures or surgical retraction which may have caused further trauma . suturing
as this was a dorsal tear , care had to be taken not to suture dorsal to ventral bladder .
suture ends were kept as short as possible to avoid leaving a future nidus for urinary calculi formation .
the technique used here meant there was no need to open the abdominal cavity , it also minimised potential complications and preserved the integrity of the vaginal structures .
the authors would like to thank peter bakonyi and jemima ripman for their assistance with this procedure . | an 11-year - old mare presented 36 hours after foaling with a ruptured bladder .
uroperitoneum was diagnosed on ultrasound and from the creatinine concentration of the peritoneal fluid .
bladder endoscopy demonstrated tissue necrosis and a rent in the dorsocranial aspect of the bladder .
following stabilisation , including abdominal drainage and lavage , the mare was taken to standing surgery . under continuous sedation and epidural anaesthesia , and after surgical preparation , a balfour retractor was placed in the vagina . using sterile lubricant and moderate force
, it was possible to insert a hand into the bladder .
the tear was easily palpable on the dorsal portion of the bladder .
two fingers were inserted through the tear and used to provide traction to evert the bladder completely into the vagina where it could grasped with the surgeons other hand to prevent further trauma .
a second surgeon could then visualise the entire tear and repaired this using a single layer of size zero pds suture in a single continuous pattern .
as soon as the bladder was repaired , it was replaced via the urethra .
the mare did well after surgery and was discharged after 48 hours , apparently normal.this report is the first describing repair of the bladder without an abdominal incision or incision into the urethral sphincter .
this greatly reduces the chance of possible complications such as urine pooling after surgery with the previously described standing technique or bladder trauma due to traction with abdominal surgery . |
these psychosocial interventions are valuable adjuncts to physical treatment for individuals who have been diagnosed with cancer .
it has been determined that 33% of individuals diagnosed with cancer experience severe psychological distress and up to 70% exhibit some degree of anxiety and depression [ 13 ] .
relationships , work life , and sense of self are all impacted by a cancer diagnosis .
excellent care must include interventions that focus on the informational and psychosocial needs of patients .
psychosocial interventions including therapeutic communication have been used with success to minimize stress , improve quality of life , treat depression , and support cancer patients throughout the course of their diagnosis and recovery [ 1 , 7 ] . a national symposium organized by the agency for health care research and quality ,
the national cancer institute , and experts in the field concluded that communication and interventions targeting psychosocial issues were among the 8 key domains that are of vital importance in cancer care .
these domains highlight the challenges that cancer patients face in coping with their emotions and navigating life disruptions associated with their treatment .
a number of meta - analysis and reviews of the literature have focused on the effectiveness of psychosocial interventions with cancer patients .
a meta - analysis conducted by meyer and mark concluded that relaxation and behavioral modification improved functional adaptation and symptom control but did not affect medical outcomes .
newell et al . reported that group - based and individual therapy , educational interventions , and guided imagery were effective with cancer patients .
most of these interventions were provided by a psychiatrist or psychologist , not nurses , and involved at least six contact hours .
conducted a systematic review of 36 studies to conclude psychoeducational interventions as well as behavior therapy reduced depression in cancer patients .
rehse and pukrop conducted a meta - analysis of 37 controlled studies and found that psychosocial interventions improve quality of life among cancer patients .
most studies were conducted in university settings and included primarily well - educated white participants .
females were twice as likely to be included given the frequency of breast cancer diagnoses .
an overall moderate effect sized of 0.31 was reported ( even when rosenthal 's fail safe method was used to estimate unpublished studies ) .
the duration of the intervention ( greater than 15 weeks ) was the only significant predictor that remained after controlling for other variables .
the authors suggested that educational programs were more effective because other psychosocial interventions consisted of heterogeneous techniques while education was more consistently defined and implemented across studies .
. reviewed the literature to examine the effectiveness of psychosocial interventions ( specifically cognitive behavioral therapy ) on quality of life for individuals diagnosed with advanced cancer .
nurses delivered the majority of interventions ( n = 8 studies ) . in 12 of the 13 trials positive quality of life changes including improved levels of depression were seen . also in 2004 ,
conducted a meta - analysis on randomized controlled trials published between 1966 and 2002 and reported that psychosocial intervention did not prolong survival among cancer patients .
only 8 trials were reviewed , patients with differing types of metastatic illness were included , and trials with short follow - up times were reviewed , all of which limited the conclusiveness of the results . williams and dale published a systematic literature review that questioned whether psychotherapeutic interventions are effective in reducing depression among cancer patients .
the major flaw identified by the author was that numerous studies were single - centre trials that did not monitor for use of other interventions that could have confounded the results .
also in 2006 osborn et al . published a meta - analysis of the effectiveness of psychosocial interventions for depression , anxiety , and quality of life among cancer survivors .
cognitive behavioral therapy was described as being effective in reducing depression , minimizing anxiety , and improving quality of life .
jacobsen and jim summarized the results of systematic reviews and meta - analysis of the effects of psychosocial intervention on anxiety and depression among cancer patients .
edwards and hulber - williams published a review of psychological interventions for women with metastatic breast cancer on psychological and survival outcomes .
five studies ( cognitive behavioral and supportive - expressive ) used a group format and showed limited evidence of benefit .
edwards and hulber - williams argued for standardization of outcome instruments , inclusion of cost effectiveness analysis and suggested interventions be designed based on preferences of individuals with cancer . this integrative review of the literature was conducted to examine whether recent research has shown psychosocial interventions to be effective with cancer patients .
the review summarizes randomized controlled studies and integrative reviews that have been published between 2006 and 2010 . during
that period strides were made in medical treatment and knowledge of the influence of cortisol levels .
patient attitudes toward psychosocial intervention may have shifted , reimbursement policies have been modified , and types of practitioners involved in provision of care have changed .
insufficient guidance currently exists for health care providers and researchers regarding the most effective type of psychosocial care .
it is necessary to understand whether , how , and why psychosocial interventions are effective with individuals who have been diagnosed with cancer .
it is also critical to examine published studies if we are to design additional research that builds in a progressive manner on existing studies . if gaps in the literature are narrowed by successive refinements of psychosocial interventions and research methods we will have a more coherent research base from which to design future clinical interventions and research .
an integrative review of the literature , not a meta - analysis , designed to determine effect size was conducted .
electronic searches of pub med , cinahl , and the cochrane library between 1/1/2006 ( since the last published integrative literature review and meta - analysis ) and 2/23/2011 were completed using the key words listed in table 1 .
this was done to identify as many relevant articles as possible and to avoid use of the search terms randomized controlled trial that might have resulted in missing relevant articles .
patients with cancer of any type or stage were included , and the review was not limited to a specific outcome measure such as depression or quality of life .
this was done because given the small number of studies located ( n = 19 ) it was not possible to target specific outcome measures such as depression or quality of life although several studies documented decreased depression and decreased anxiety [ 7 , 2123 ] , improved quality of life [ 23 , 24 ] , and enhanced functional status / well - being [ 25 , 26 ] .
references of all 19 papers were examined using the snowball method to identify additional studies .
initially identified , 42 were located with more than one search term and eliminated for being duplicative .
eight hundred forty - four studies were eliminated for not being randomized controlled trials . only 27 ( 19 included + 8 excluded ) studies identified using search terms were randomized controlled trials .
inclusion criteria were that the study was a randomized controlled clinical trial published in english , involving adult cancer patients living at home who were over the age of 18 ( n = 19 ) .
randomized controlled trials that used volunteers to provide the intervention , focused on cancer patients not living at home , included patient spouses in the intervention , or included pharmacological , physical therapy , and/or complementary and alternative interventions were excluded .
one study was excluded as it was a composite of hypnosis ( an alternative treatment ) and supportive - expressive group therapy .
one study was eliminated because it included an intervention that included conditioning exercises provided by a physical therapist .
two studies were excluded because they combined psychosocial interventions along with use of medications in studies involving cancer patients [ 1 , 30 ] .
one study was excluded because it focused on elderly individuals with cancer living in care homes rather than at home .
one study was eliminated because it reported the qualitative results obtained from a randomized controlled trial . a data extraction form which is labeled table 2
was developed based on consolidated standards of reporting trials ( -consort ) guidelines and used to summarize the included studies and to rank their quality .
nineteen randomized controlled trials focusing on therapeutic communication and supportive interventions were grouped into four main categories : cognitive behavioral interventions , supportive interventions , group interventions , and telephone - assisted interventions .
of the 19 studies reviewed 11 studies demonstrated positive findings [ 7 , 23 , 25 , 26 , 31 , 32 , 34 , 35 , 38 , 40 , 41 ] while an additional 4 demonstrated positive findings only after a post hoc analysis was completed [ 5 , 19 , 33 , 36 ] .
cognitive behavioral interventions were based on the belief that how individuals view situations influences their emotional response and problem - solving ability .
antoni et al . [ 7 , 32 ] reported different outcome measures from a single study of female breast cancer patients . in one publication
the intervention group had lower anxiety and cortisol and higher cytokine production than the control group . in the second article , a greater reduction in cancer - specific thought intrusion , anxiety , and emotional distress
reported quality of life improved for patients with colorectal , head / neck , lung , and breast cancer who participated in 8 sessions of a cognitive behavioral intervention .
pitceathly et al . found high - risk patients with breast , lymphoma , or gynecological cancer who received cognitive behavioral therapies were less likely to become depressed or anxious .
randomized controlled trials focused on supportive psychosocial interventions that included listening , validation , stress management , problem solving , and education related to the diagnosis .
interventions in this category varied substantially including ( 1 ) a one - time meeting with a psychologist for clients with gynecological cancer , ( 2 ) two communication skills training sessions with nurses for clients with gastric , colorectal , or breast cancer , ( 3 ) a coping and communication - enhancing intervention , supportive counseling or usual care intervention offered in six hour - long sessions with a therapist for patients with gynecological cancers , and ( 4 ) an educational and stress management intervention lasting 2 hours for patients with melanoma .
a coping and communication support intervention provided by mental health nurses who offered an in - home meeting and a follow - up telephone contact for patients with lung , pancreatic , or liver cancer is discussed in the telephone intervention section even though it is both supportive and telephone based .
although each of the supportive interventions resulted in improved outcomes , each study used a different type of supportive care , was implemented by varied types of practitioners , relied on a spectrum of different outcome measures , and included clients with several types and stages of cancer .
the use of varied interventions as well as the limitations listed in table 2 made it difficult to make any definitive conclusions about these randomized , controlled trials .
several researchers conducted randomized controlled trials to explore the effectiveness of group approaches that included psychosocial interventions for individuals diagnosed with cancer .
group interventions are summarized in table 5 . as with the supportive approaches the actual group interventions that were used differed significantly .
interventions for individuals with breast cancer included ( 1 ) 26 group sessions offered over a one - year period led by clinical psychologists [ 23 , 31 , 38 ] , ( 2 ) 1 year of group therapy offered by psychologists , psychiatrists , or social workers , and ( 3 ) weekly supportive expressive group therapy and education lead by psychiatrists , psychologists , and social workers .
the length and number of sessions offered , the professionals involved , and the outcome measures used in these studies varied .
this meta - analysis covered the years of 1981 to 2001 and included 20 randomized controlled trials .
results indicated that support group participation results in decreased depression and anxiety , increased illness adaptation , improved quality of life , and enhanced marital relationships .
forty - five percent of principal investigators were psychologists , 35% were nurses , and 20% were physicians .
seventy % of studies involved women , 65% focused on women with breast cancer , 96 % offered group sessions on a weekly basis , and a cognitive - behavioral model was used in 92% of studies .
twelve studies focused on depression , 11 on anxiety , 6 on quality of life , 8 on adaptation to illness , 3 on survival , and 2 on marital relationships .
telephone - assisted interventions typically included in - person contacts to establish rapport , followed by periodic telephone contact and support [ 5 , 6 , 34 , 37 , 42 ] .
in - person contacts among the telephone studies ranged from 1 session [ 5 , 6 ] to 6 to 8 sessions . in these studies challenges such as symptoms limiting travel , geography , and
telephone contact was used because it can be a flexible way to provide timely intervention during a stressful period . the studies by rose et al .
[ 6 , 42 ] were based on the same sample , collected at 6 weeks showing middle - aged patients had more problems communicating with family members and at 2 months showing middle - aged patients averaged more contacts per month compared to individuals aged 61 to 80 .
aranda et al . used a brief , nurse - delivered visit followed by one phone call .
when the results were analyzed only with women with high needs the intervention was effective in reducing psychological and emotional needs .
walker et al . randomized 200 individuals with lung cancer to a usual care or depression care intervention .
compared telephone intervention with care provided by a physician to find that short - term outcomes measured at 2 weeks improved although the long - term outcomes at 6 and 12 weeks were not improved .
compared telephone caseworkers ( oncology nurses ) to a general practitioner model to find no overall intervention effect .
additional research on the frequency , timing of in relation to diagnosis , and length of telephone support is needed to determine what level of intervention is needed .
in addition , whether regional and generational differences exist in response to telephone support needs to be examined .
overall , 11 of the 19 studies included in this integrative review showed positive results [ 7 , 23 , 25 , 26 , 31 , 32 , 34 , 35 , 38 , 40 , 41 ] , and an additional 4 showed positive results [ 5 , 19 , 33 , 36 ] based on a post hoc analysis .
consistent outcomes that were affected by more than one study in this integrative review included decreased depression [ 19 , 26 , 35 , 41 ] , decreased anxiety [ 7 , 26 , 32 , 41 ] , improved quality of life [ 25 , 41 ] , and enhanced functional status / well - being [ 31 , 38 ] .
these positive results demonstrating the effectiveness of psychosocial interventions are consistent with positive results from previous meta - analyses [ 9 , 12 , 16 , 17 , 45 ] and integrative reviews [ 11 , 13 ] .
in contrast , one previous integrative review reported that psychosocial interventions do not improve depression while two integrative reviews reported psychosocial interventions do not prolong life [ 14 , 18 ] .
this outcome of not increasing survival time is consistent with findings from this integrative review that showed survival time in two studies did not increase in the group receiving psychosocial interventions [ 19 , 36 ] while one study reported a decreased risk of cancer reoccurrence in the group receiving psychosocial intervention .
several authors of meta - analyses and integrative reviews have stressed the need for greater consistency within the research trajectory in this area in order to report conclusively that psychosocial interventions are effective [ 15 , 18 ] .
variations among outcome measures , treatment conditions ( cancer stage , treatment type , treatment location , and group / individual approach ) , age , racial / ethnic background , and methodological quality made it difficult to draw definitive conclusions from this integrative review .
what is clear is that both clinicians and researchers would benefit from future studies which build on previous findings , use comparable outcome measures , and adhere to standards of quality research such as those identified in the consort guidelines .
it is important to identify what types and duration of intervention are effective for what population .
as jacobsen and jim commented psychosocial care that is ineffective may be worse than no care at all
all of the 19 studies reviewed included detailed information about the analysis phase of their research . but as is evident in table 2 , not all of the studies reviewed included all of the quality measures that could be expected in a randomized , controlled clinical trial .
fewer studies ( 8 out of 19 ) included a power analysis , fewer studies addressed treatment fidelity ( 8 out of 19 ) , fewer studies ( 9 out of 19 ) specified the method of randomization , fewer studies ( 5 out of 19 ) discussed blinding , fewer studies ( 9 out of 19 ) described reasons for dropping out of the study , and fewer studies ( 8 out of 19 ) used an intent - to - treat analysis .
researchers need to include the quality measures listed in the consort guidelines , incorporate cancer - specific outcomes measures , and explore whether certain interventions are more effective in select racial / ethnic or age groups .
additional research in needed to clarify if differences in time of diagnosis , cancer stage , the influence of age , or geographic area impact the effectiveness of psychosocial interventions .
it is useful to examine individual study quality by comparing a perfect score of 13 based on allocation of 1 point based on whether ( 1 ) a hypothesis was provided , ( 2 ) eligibility criteria are clear , ( 3 ) the intervention is described in sufficient detail for replication , ( 4 ) treatment fidelity is discussed , ( 5 ) a power analysis was provided , ( 6 ) the method of randomization was clear , ( 7 ) blinding was discussed , ( 8) reasons for dropouts were given , ( 9 ) an intent - to - treat analysis was used , ( 10 ) recruitment dates were specified , ( 11 ) study limitations were described , ( 12 ) funding sources were listed , and ( 13 ) a trial registry was mentioned .
these 13 criteria included in the consort guidelines and summarized in table 2 provide guidance when ranking study quality . using these 13-point criteria pitceathy et al . and walker et al . received high scores of 11 , followed by a score of 9 for kravitz et al . , spiegel et al . , and kissane et al . and by a score of 8 achieved by andersen et al . and girgis et al . .
, antoni et al . , manne et al . , and fukui et al . while a score of 6 is seen for antoni et al . and miller et al . .
, andersen et al . , boesen et al . , and powell et al .
the lowest quality scores of 3 for rose et al . and 4 for rose et al
it is , however , important to remember that not all of the above - mentioned scores obtained from the consort guidelines are of strictly equivalent value for determining study quality .
only eight studies [ 19 , 26 , 32 , 34 , 3638 , 40 ] did an adequate job of assuring treatment fidelity including ( 1 ) basing interventions on theory , ( 2 ) offering standardized training to those implementing the intervention , ( 3 ) monitoring to ensure interventions were consistently provided , ( 4 ) reinforcing training to avoid loss of skills or variation in approach , and ( 5 ) minimizing contact between treatment and control groups .
too few studies have included mental health nurses or been designed by mental health nurses [ 6 , 42 ] . although it is a challenge given current economic realities , there is a need for mental health nurses to locate funding for randomized clinical trials and to focus on the effectiveness of mainstay interventions within mental health nursing practice , namely , therapeutic communication and supportive care in minimizing depressive symptoms and improving quality of life measures among individuals who have been diagnosed with cancer .
eleven studies documented positive outcomes from psychological interventions [ 7 , 23 , 25 , 26 , 31 , 32 , 34 , 35 , 38 , 40 , 41 ] and an additional 4 showed positive results [ 5 , 19 , 33 , 36 ] based on ad hoc analysis .
additional research is needed because the literature continues to be full of methodological gaps and discrepancies . as edwards et al .
commented it is difficult to integrate the results of randomized , controlled trials because of the variability of interventions , age groups , ethnic / racial backgrounds , cancer stages , outcome measures , and methodological quality .
it is critical to increase the number of qualitative studies to determine what sort of intervention cancer patients of varied ages , ethnic / racial backgrounds , and cancer stages feel would meet their psychosocial needs . when those perspectives have been assessed researchers can design more effective randomized clinical trials that help close existing gaps in the literature | background . previous integrative literature reviews and meta - analyses have yielded conflicting results regarding the effectiveness of psychosocial interventions for cancer patients .
methods . an integrative review of the literature focused on 19 randomized , controlled trials ( 20062011 ) was completed to examine the effectiveness of psychosocial interventions for cancer patients .
eligibility criteria : inclusion criteria were the study was an english language randomized controlled clinical trial .
results .
seven studies involved nurses .
eleven studies resulted in positive outcomes .
overall , study quality was limited . in eight studies
the intervention was not adequately described , 7 studies did not contain a hypothesis , 4 did not include clear eligibility criteria , 10 studies did not randomize appropriately , 9 did not list recruitment dates , 11 did not include a power analysis , 14 did not include blinded patients or data collectors , 11 did not use an intent - to - treat analysis , 10 did not clarify reasons for drop outs , and 11 did not discuss treatment fidelity . conclusions .
future studies should build on previous findings , use comparable outcome measures , and adhere to standards of quality research .
qualitative studies are needed to determine what cancer patients of varied ages , cancer stages , and racial / ethnic backgrounds believe would be an effective intervention to manage their psychosocial needs . |
clinical information was collected retrospectively for january 1 through july 31 , 2009 , and prospectively from august 1 , 2009 through april 30 , 2011 . retrospectively , we identified patients who had been tested for influenza by use of a rapid test kit and extracted personal and clinical information from medical records .
prospectively , we collected the same information from patients tested for influenza by rapid test kit and used nasopharyngeal swab extracts from rapid test kits for virus typing .
typing was performed by reverse transcription nested - pcr ( rt - npcr ) : multiplex for seasonal influenza virus ( subtypes a / h1 , a / h3 , b- , and a / h5 ) and simplex for influenza a(h1n1)pdm09 virus ( technical appendix table 1 ) . by amplifying product exclusively for influenza a(h1n1)pdm09 and for traditional influenza ( h1n1 ) viruses and by producing a different length product for each subtype ,
# iincludes 1 case for which tamiflu and relenza were prescribed . for the retrospective period ,
we identified 1,066 suspected cases of influenza ; 20 patients were nonresidents of izu - oshima island ; the address for 1 patient was unknown . for the prospective period , we identified 2,348 patients with suspected influenza ; 3 were excluded because they did not consent to study participation .
patients from the prospective period were tested with a rapid test . in total , 97.8% ( 2,293/2,345 ) of the samples
the total number of patients with suspected influenza in the prospective study was 2,219 ( a patient with multiple visits within 7 days was counted as 1 patient ) , of which 78 were not residents of izu - oshima island ( technical appendix table 2 ) .
* age during january 2009 ( for 200809 ) , 2010 ( for 200910 ) , and 2011 ( for 201011 ) .
population recorded in the resident registry in january 2009 ( for 200809 ) , 2010 ( for 200910 ) , and 2011 ( for 201011 ) .
the sum of influenza a(h1n1)pdm09 cases among residents in izu - oshima during the study divided by the population during january 2011 .
the sensitivity of the rapid test kit compared with rt - npcr was 90% for type a but lower ( 80% ) for type b ( technical appendix table 3 ) . among patients with positive rapid test results ,
the same influenza subtypes circulated in izu - oshima as in other areas ( 2,3 ) .
we assessed the period from when a novel virus was introduced through the postpandemic season .
the introduction of a(h1n1)pdm09 virus in izu - oshima occurred 11 weeks after confirmation of the first case in japan ( 4 ) and 10 weeks after confirmation in tokyo ( 5 ) .
after the first case of a(h1n1)pdm09 infection was identified in japan on may 16 ( week 20 ) ( 4 ) , no other influenza a cases were diagnosed by a rapid test in izu - oshima until august 1 ( week 31 ) , when a patient with influenza a ( unspecified ) was determined to have had contact with a person with confirmed a(h1n1)pdm09 infection on mainland japan .
the first outbreaks of influenza a in izu - oshima ceased within 5 weeks ( figure 1 ) .
after 3 weeks with no cases , starting at the end of september ( week 39 ) , clusters of influenza cases were observed in schools and families .
although immediate school or class closures were implemented , the pandemic began in the middle of november and peaked during week 50 , which was 6 weeks later than the tokyo peak ( 5 ) . on the island ,
the incidence of a(h1n1)pdm09 infection during the pandemic season was higher than the incidence caused by other subtypes during the 20092011 seasons ( table 2 ) .
however , the overall incidence on the island ( 5.0% ) during the pandemic season was one third of the estimated incidence for all of japan ( 16.2% ) ( 3 ) .
cases of influenza and influenza - like illnesses on izu - oshima island , japan , from week 1 of 2009 through week 17 of 2011 . the number of influenza cases and influenza - like illnesses are plotted weekly from the disease onset .
influenza cases were defined as illnesses diagnosed by a rapid test combined with a reverse transcription nested pcr ( rt - npcr ) or by a rapid diagnostic test ( rdt ) alone , during the retrospective period ( unspecified ) .
influenza - like illnesses were defined as cases for which influenza was ruled out by negative rt - npcr or cases for which influenza was ruled out by rdt results and further tests were not performed .
disease onset was defined by the date when the patient first reported fever or upper respiratory symptoms .
the disease onset for the case that had no date in the clinical records was defined as the day before the first clinical visit according to the median day of visit from the available study data .
b ( rdt / pcr ) , cases diagnosed by a rdt or rt - npcr . a(h1n1)pdm09 and h3 , co - infection cases with 2 virus subtypes confirmed by rt - npcr .
influenza seasons were defined as follows : week 130 of 2009 was the 200809 prepandemic season , week 31 of 2009week 33 of 2010 was the 200910 pandemic season , and week 34 of 2010week 17 of 2011 was the 201011 postpandemic season . the introduction and dissemination of a(h1n1)pdm09 virus varied by age ( figure 2 ) . on the island , as on the mainland , introduction of the emerging virus preceded the outbreak among high school age children ( 4,6 ) . however , at the end of the season , the incidence among persons 514 years of age exceeded that among persons 1519 years of age ( table 2 ) .
the cumulative incidence of a(h1n1)pdm09 infections for 200910 was calculated for the sum of a(h1n1)pdm09 virus cases among residents on izu - oshima island , japan , divided by the population at the end of december 2009 and plotted by week in the 200910 season .
the numbers adjacent to the lines indicate the age groups , in years . during the postpandemic season
, in addition to a(h1n1)pdm09 virus , epidemics of influenza a / h3 and b viruses occurred . no patient with confirmed a(h1n1)pdm09 infection during the postpandemic season had a history of influenza in the previous season ; but 28% ( 50/180 ) of patients with influenza b and 3% ( 2/58 ) with influenza a / h3 virus did .
the cumulative incidence of a(h1n1)pdm09 infection 2 seasons after the pandemic was estimated at 7.3% and was highest ( 43.1% ) among persons 1014 years of age .
this population - based surveillance study determined the incidence of the influenza virus subtypes circulating in pandemic and postpandemic seasons .
the estimated incidence of symptomatic cases was accurate because of the easy access to health care on izu - oshima island .
care was sought for almost all ( 97% ) children with upper respiratory symptoms and fever , although the proportion of adults who sought clinical care was not high ( 7 ) .
most ( 83.6% ; 1,774/2,122 ) patients for whom disease onset was identified had visited a medical institute within 2 days of disease onset despite their symptoms being only mild to moderate .
furthermore , all suspected cases of influenza , except 2 , were confirmed by a rapid test ; most were tested further and isolates were subtyped by rt - npcr . the cumulative incidence 2 seasons after the pandemic indicates that early introduction of a(h1n1)pdm09 virus to those 1519 years of age was not caused by differential sensitivity to the virus .
rather , it was probably caused by more frequent exposure to the emerging virus , possibly because of higher mobility of persons in this age group .
considering the conservative antigenic property of a(h1n1)pdm09 virus in the postpandemic season ( 8) , the absence of a(h1n1)pdm09 infection in this season among those who had experienced it in the pandemic season suggests that immune memory persisted in the postpandemic season .
the cumulative incidence suggests that nearly half of the school - age children had immunity to a(h1n1)pdm09 virus by infection after 2 seasons .
the remaining virus - naive elderly population should be considered for future preventive intervention , although they might have some immunity against a(h1n1)pdm09 virus ( 912 ) .
the delayed introduction of a(h1n1)pdm09 virus might primarily be explained by the isolated environment of the island ; introduction would be mediated solely by visitors carrying the virus .
the delayed start and peak of the epidemic and the low incidence could be attributed early case identification plus early and extensive therapy ( including prompt initiation of antiviral medication according to results of proactively performed rapid tests ) ; easy access to health care ; and public health interventions ( such as school closures ) .
in addition , unique social features might also have contributed to the delayed pandemic and low disease incidence .
the proportion of children < 15 years of age ( 12% ) is the same on izu - oshima island as in tokyo ; whereas , the proportion of those 65 years of age is 31% on the island and only 20% in tokyo . assuming that persons 65 years of age had preexisting immunity against a(h1n1)pdm09 virus , as suggested by other studies ( 912 ) , the community possibly had a larger number of nonsusceptible persons .
limited public transport and low population density ( 96/km ) might have reduced disease spread .
school closures might have more effectively reduced the chance of transmission in such settings than in other areas .
this study provides a sound basis for modeling studies that consider social structures to help explain the effects of public health interventions for influenza spread in a community .
reverse transcription nested pcr ( rt - npcr ) primers used for seasonal ( multiplex ) and pandemic influenza ( simplex ) detection and typing ( table 1 ) .
diagnosis of influenza cases and influenza - like illnesses from 200809 to 20102011 influenza seasons ( table 2 ) .
sensitivity and specificity of quicknavi - flu kit ( nordic biolabs ab , taby , sweden ) compared with rt - npcr ( table 3 ) . | a population - based influenza surveillance study ( using pcr virus subtyping ) on izu - oshima island , japan , found that the cumulative incidence of influenza a(h1n1)pdm09 virus infections 2 seasons after the pandemic was highest for those 1014 years of age ( 43.1% ) . no postpandemic a(h1n1)pdm09 case - patients had been infected with a(h1n1)pdm09 virus during the pandemic season . |
atrial fibrillation ( af ) is the most common sustained cardiac rhythm disturbance , increasing in prevalence with age .
pulmonary vein ( pv ) isolation with catheter ablation is one of the primary treatments for symptomatic af refractory or intolerant to antiarrhythmic medications . in some cases
, it may be even appropriate to perform af ablation as first - line therapy , particularly in selected symptomatic patients with heart failure and/or reduced left ventricular ejection fraction .
the main goal in catheter ablation is complete electrical isolation of the pvs , and therefore the success of the procedure and the reduction of procedure - related complications require careful identification of the pv ostia and application of ablation to the pv antra .
currently radiofrequency ( rf ) energy is the most commonly used energy source for ablation .
however , this form of energy is associated with various complications including thromboembolic events , pv stenosis , and atrio - esophageal fistula .
recent clinical and preclinical studies demonstrated that the cryothermal energy application using a balloon catheter for pv isolation is an effective treatment for af [ 5 , 6 ] .
the purpose of this paper is to review the current literature for better understanding of the utility of cryoablation for the treatment of patients with af .
the literature was searched in pubmed from 1950 to february 2011 with the keywords atrial fibrillation , cryosurgery , and catheter ablation published in english .
all full - text articles and their references were reviewed and included in this paper if they were relevant to our presentation .
cryothermal energy causes progressive necrosis in the absence of significant alteration of tissue structure at thaw .
the effect of ablation on the cellular ultrastructure has been evaluated in studies using a cryoprobe cooled to 60c with expanding nitrous oxide .
ice crystals were observed to form inside the cells during ablation , with a resulting cell damage that is osmotic rather than mechanical .
acute changes in subcellular organelle structure and mitochondrial destruction occur in 1 minute at 70c .
subsequent changes up to 10 hours after thaw are most notable for progressive mitochondrial damage .
the cryothermal energy lesion formation can be divided in three sequential stages which are the freeze / thaw phase , the hemorrhagic - inflammatory phase , and the replacement fibrosis phase .
the resulting lesions are minimally thrombogenic , have sharp well - demarcated margins , and exhibit minimal tissue disruption and preserved basic underlying tissue architecture [ 8 , 9 ] .
thus the histopathologic changes of cryoablation are likely to be associated with low arrhythmogenic potential .
cryothermal energy has been used in treatment of cardiac arrhythmias for the past 3 decades , especially in supraventricular arrhythmias .
cryoablation was performed as early as 1977 to the atrioventricular ( av ) conduction system .
harrison et al . studied cryoablation using a hand - held nitrous oxide - cooled cryogenic probe in 20 dogs and subsequently in three patients with drug - resistant , life - threatening supraventricular tachycardias . in the same year accessory pathway elimination by cryoablation
since then , numerous experimental and clinical studies have evaluated the efficacy and safety of cryoablation [ 4 , 5 , 9 , 1214 ] . a multicenter clinical trial in patients with av nodal reentry tachycardia ( 103 patients ) , av reentrant tachycardia ( 51 patients ) , and af ( 12 patients ) demonstrated that cryoablation is a safe and effective strategy for the treatment of supraventricular arrhythmias .
catheter - based cryoablation was found to be a safe alternative to rf ablation for slow pathway modification , accessory pathway termination , and av junction ablation .
acute procedural success was achieved in 83% of the overall group , and long - term success after 6 months was 91% . cryomapping ( cooling the catheter tip to 30c ) successfully identified ablation targets in the majority of the patients , and the electrophysiologic effects of cryomapping were reversible within minutes in 94% of the attempts . also , a prospective , randomized trial in patients with recurrent narrow qrs - complex tachycardia suggestive of av nodal reentry tachycardia compared cryoablation with rf ablation and showed a procedural success rate of 91% in the rf group and 93% in the cryoablation group .
the median number of cryothermal applications was significantly lower than the number of rf applications .
no cryoenergy - related complications were observed , and no permanent av conduction block occurred .
currently cryoablation is used to treat various forms of ventricular and supraventricular arrhythmias including av nodal reentry tachycardia , av reentrant tachycardia , atrial flutter , and focal atrial tachycardia [ 1218 ] .
compared to rf ablation , cryoablation has been associated with lack of pain during energy delivery , homogenous lesion formation , less destruction to surrounding vasculature , preserved tissue integrity , and lower risk of thrombus formation [ 59 , 1214 ] .
lesion reversibility is another advantage of cryoablation over rf energy and is useful for ablation of accessory pathways in proximity to the conduction system [ 1215 ] .
moreover , cryoablation can be applied within venous structures , like the coronary sinus and the middle cardiac vein with less risk of thrombosis compared to rf ablation [ 14 , 19 ] .
it has been shown that cryoablation in the coronary sinus within 2 mm of the left circumflex artery produces myocardial lesions similar to rf ablation with lower risk of coronary artery stenosis . the absence of endothelial disruption with cryoablation results in less thrombogenicity and may offer an opportunity for improved safety during ablations in the left side of the heart [ 59 ] .
this may be important when large areas of the endocardium are ablated such as in af ablation procedures .
all these characteristics of cryoenergy , in addition to the ability to use it with a balloon , make it an attractive source of energy for pv isolation .
point - to - point ablation using a focal catheter for pv isolation is associated with technical difficulties and requires experienced operators with high level of skills .
pulmonary vein isolation using a focal cryocatheter has been studied and was found to be associated with long procedure times .
the cryoballoon catheter was designed with the aim of facilitating pv isolation [ 46 , 21 ] .
currently only one balloon design is available for clinical use ( medtronic inc , minnesota , minn ) .
this system includes an over - the - wire balloon catheter which is cooled using nitrous oxide ( n2o ) [ 46 , 2227 ] .
the shaft has a central lumen that can accommodate a wire for support and also used for contrast and saline injection .
the balloon is inflated outside the vein and advanced over the wire to occlude the vein ( figure 1 ) . obtaining
an incomplete occlusion will lead to flow around the balloon and prevents the temperature from reaching low enough levels necessary for adequate ablation .
a complete occlusion is typically confirmed by injecting contrast in the lumen at the balloon tip .
once occlusion is confirmed , the contrast is flushed using saline and freezing is started . if successful , a dip in the temperature curve is observed indicating a better occlusion .
lesions are usually 240300 sec long and pv isolation is usually achieved with one lesion in most patients [ 2224 ] .
currently , two sizes of the cryoballoon catheter are available , 28 mm and 23 mm diameter .
it is advisable to use a 28 mm balloon regardless of the size of the vein in order to reduce the risk of complications such as pv stenosis and phrenic nerve injury .
a quadripolar catheter is usually positioned in the superior vena cava for continuous phrenic nerve stimulation during cryoablation of right pvs .
intracardiac echocardiogram and color doppler can be used to confirm occlusion and the site of gaps if present ( figure 2 ) .
recently , the use of transesophageal echocardiography with color doppler was described allowing real - time visualization of the cryoballoon during ablation .
after ablation , pv isolation is assessed using a circular mapping catheter inserted in the left atrium via a separate transseptal puncture [ 22 , 25 , 29 ] . a small caliber circular mapping that can be advanced in the central lumen instead of the wire
two maneuvers can be helpful if complete occlusion can not be achieved by advancing the balloon .
this can provide a better alignment of the catheter shaft with the axis of the pv .
this can be useful when a gap is present at the inferior border of the vein and can not be closed despite advancing the balloon . with this technique ,
freezing is started despite the presence of the gap . at about 6090 sec into the freeze , the balloon and the sheath are both pulled down gently in order to close the gap at the inferior portion of the vein .
this should be done very carefully because of the risk of severe vascular damage if excessive force is applied .
if occlusion and isolation can not be obtained despite these maneuvers , a standard point - by - point ablation catheter can be used for completion .
the sites of pv reconnection have been analyzed in some studies [ 24 , 2931 ] .
the inferior border of the veins as well as the ridge between the left atrial appendage and the left pv was found to be the sites where most reconnections occurred .
this is likely to be the result of sharp catheter angulation with loss of central cryoballoon alignment with the axis of the pvs . to date over 10,000 pv procedures
there have been many publications describing the clinical experience with the cryoballoon in both paroxysmal and persistent af [ 2241 ] .
an early report by van belle and colleagues described 57 consecutive patients with paroxysmal af who underwent cryoballoon ablation .
eighty - four percent of the targeted pvs were successfully isolated using the cryoballoon and the rest required a standard cryocatheter to achieve isolation .
klein et al . reported the result of cryoballoon ablation in 21 patients with symptomatic paroxysmal af in 2008 .
a total of 95% of pvs were isolated , and 86% of the patients remained free of af after 6 months based on periodic holter monitoring .
subsequently , neumann et al . evaluated the efficacy of cryoballoon ablation in 293 patients with paroxysmal and 53 patients with persistent af .
ninety - seven percent of the pvs were isolated with either cryoballoon alone or in combination with a standard cryocatheter .
maintenance of sinus rhythm was reported in 74% and 42% of the patients with paroxysmal and persistent af , respectively . in a recent study by ahmed et al . , the permanence of pv isolation after cryoballoon ablation was tested in 12 patients with paroxysmal af .
eighty - eight percent of pvs remained isolated at 812 weeks after the initial procedure .
the most comprehensive result regarding the efficacy and safety of the cryoballoon catheter ablation for the treatment of af was obtained from the stop - af trial [ 32 , 33 ] .
this trial randomized patients with paroxysmal drug - refractory af in a 2 : 1 fashion to either pv isolation with cryoballoon ablation or antiarrhythmic drug therapy .
balloon - only isolation of pvs was achieved in 90.8% , and the overall procedural success ( 3 pvs isolated ) was achieved in 98.2% of the patients .
nineteen percent of the patients needed a repeat cryoablation procedure within the 90-day blanking period .
the use of cryoballoon for pv isolation in patients with persistent af has been associated with high rates of arrhythmia recurrence [ 23 , 34 , 35 ] .
this is not surprising knowing that the left atrial substrate and not only the pvs play a role in af maintenance in this subgroup of patients .
a study from our group investigated the combined use of cryoballoon pv isolation and conventional rf ablation of areas of complex fractionated electrograms in 22 patients with persistent af .
after a single procedure , 86% of patients were af - free at 6-month followup . in this
study the followup was performed in most patients using implantable loop monitors allowing automatic detection of asymptomatic af .
in addition to af - free survival , af burden reduction was also analyzed in some studies .
studied 144 patients with symptomatic af refractory to antiarrhythmic drugs who underwent cryoballoon pv isolation .
followup was performed using daily transtelephonic ecg monitoring , 24 h holter - ecg , and an arrhythmia - focused questionnaire to document af .
phrenic nerve palsies ( pnp ) , pv stenosis , and other complications have been reported with cryoballoon pv isolation [ 2241 ] . in the stop
af study , pnp was reported in 29 out of 259 procedures ( 11.2% ) [ 32 , 33 ] . of these
pnp can occur with isolation of the right superior and less commonly the right inferior pv .
another important procedural complication with the cryoballoon is pv stenosis . although earlier studies showed no significant incidence of pv stenosis following cryoballoon ablation , stop - af trial demonstrated 3.1% risk of pv stenosis in its study population [ 32 , 33 ] .
this complication is likely associated with ablation of the tubular portion of the vein . as a result , it is believed that the use of the 28 mm balloon can reduce this complication because this larger balloon is less likely to be positioned inside the vein during ablation . for the same reason
, it is believed that the use of the larger balloon will carry a lower risk of pnp .
luminal esophageal temperature was significantly decreased ( > 1 degrees c ) in 62 of 67 ( 93% ) patients , and reversible esophageal injuries were seen in 17% of patients .
however , no atrial - esophageal fistula was documented and the follow - up endoscopy confirmed healing of all ulcers .
other procedure - related complications with the use of the cryoballoon are similar to rf ablations including stroke , pericardial effusion or tamponade , femoral vein access complications , and cerebral embolism [ 2241 ] .
both conventional focal rf ablation and cryoablation are used for pv isolation and both are shown to be effective [ 13 , 2244 ] .
recent studies compared clinical outcome of cryoballoon ablation for pv isolation with rf ablation in regards to the safety and the efficacy of the procedure [ 30 , 35 , 42 ] .
linhart et al . demonstrated in a case - control study of 40 patients with paroxysmal atrial fibrillation that cryoballoon ablation has similar success rate to rf ablation in addition to similar procedure and fluoroscopy times .
studied the efficacy of a strategy using a large cryoballoon to perform antral pv isolation in 124 patients with paroxysmal and persistent af and compared it with rf ablation . at one - year followup ,
77% of paroxysmal and 48% of persistent af patients remained free from af after a single procedure . in
a recent study from khne at al . confirmed the shorter procedure duration with cryoablation compared to rf ( 166 32 versus 197 52 minutes ) . in this study ,
a total of 55 patients with paroxysmal af were studied : 25 patients underwent pvi using a 28-mm cryoballoon and 25 patients using an open - irrigation rf catheter .
the number of procedures was not different in the 2 groups ( 1.2 0.4 in cryoablation versus 1.3 0.6 in rf ablation ) with a success rate at one - year followup of 88% in the cryoballoon group and 92% in the rf group .
another study , the freeze af trial , is being planned to compare cryoballoon catheter ablation with open - irrigation rf ablation in a randomized clinical study including 244 patients with paroxysmal af .
cryoballoon catheter ablation is a safe and effective technique for pv isolation to treat paroxysmal af
potential procedural complications related to cryoballoon ablation include pnp and pv stenosis as well as rare neurological complications . | pulmonary vein isolation with catheter ablation is an effective treatment in patients with symptomatic atrial fibrillation refractory or intolerant to antiarrhythmic medications .
the cryoballoon catheter was recently approved for this procedure . in this paper , the basics of cryothermal energy ablation
are reviewed including its ability of creating homogenous lesion formation , minimal destruction to surrounding vasculature , preserved tissue integrity , and lower risk of thrombus formation .
also summarized here are the publications describing the clinical experience with the cryoballoon catheter ablation in both paroxysmal and persistent atrial fibrillation , its safety and efficacy , and discussions on the technical aspect of the cryoballoon ablation procedure . |
in senegal , livestock play both social and economic roles . it remains as the only source of livelihood for 30% of rural households .
it is indeed a great potential for wealth creation in the sense that it represents 4.2% of the national gross domestic product and 28.8% of the primary sector ( gdp ) .
nevertheless the enormous potential productivity of small ruminants is still weak , due to several constraints , including malnutrition , management , and diseases .
in fact , diseases caused by parasites are also factors that contribute to reduction of the productivity of these animals around the world .
gastrointestinal helminthes , with their toxic , mechanical , and absorptive actions , constitute a major constraint to health and productivity of domestic animals , especially in developing countries [ 47 ] .
as far as we know , in africa , five species of anoplocephalid cestodes have been reported as major intestinal infections in small ruminants [ 8 , 9 ]
. these are avitellina centripunctata rivolta , 1874 , moniezia expansa rudolphi , 1810 , moniezia benedeni moniez , 1879 , stilesia globipunctata rivolta , 1874 , and thysaniezia ovilla rivolta , 1878 , with a prevalence ranging from 6 to 92% [ 1019 ] . in senegal , these species have been confirmed , but their monthly and seasonal dynamics in ruminants have not yet been elucidated .
the aims of present study were to determine the prevalence of the anoplocephalid cestodes infecting sheep and goats in senegal throughout the year and after performing scanning electron microscopic ( sem ) observations of specimens to put into evidence the characteristic of these species .
small intestines of a total of 462 sheep ( ovis aries ) and 48 goats ( capra hircus ) were collected from the main slaughterhouse of dakar , senegal , from january to december , 2013 .
sampling periods were characterized by a rainy season ( 500900 mm / year ) and a dry season extending from june to october and november to may , respectively .
ruminant hosts were mostly between 2 and 5 years old , and they came from different localities in senegal .
the cestodes were collected carefully from the intestines of small ruminants ( sheep / goats ) and identified after staining , using keys [ 2124 ] .
staining was with iron hydrochloric carmine , destained in acid ethanol ( 100 ml 70% ethanol + 2 ml concentrated hcl ) , dehydrated in a graded ethanol series , cleared with eugenol ( clove oil ) , and mounted in canada balsam .
stained specimens were examined and photographed under a leitz photo research microscope . to confirm detailed surface morphology , sem observations were performed . for sem , worms were isolated carefully from intestines and placed into a small amount of saline buffer . for living tapeworms , scolex ,
mature , and gravid proglottids of many specimens of each species were fixed overnight in cold 2.5% glutaraldehyde in a 0.1 m sodium cacodylate buffer at ph 7.4 .
then , they were dehydrated in a graded ethanol series and dried using co2 in an emitech k850 critical point dryer . after being mounted on metal stubs
, specimens were coated with gold / palladium in a quorum technologies sc7640 sputter coater and examined with a hitachi s-3400n scanning electron microscope at acceleration voltages between 3 and 20 kv in the service d'etude et de recherche en microscopie electronique de l'universit de corse .
the prevalence was calculated by dividing the number of animals harboring parasites by total number of examined animals .
mean intensity was assessed by dividing the total number of parasites by the number of infected hosts .
percentages ( % ) measure prevalence and chi - square ( ) association between prevalence of the parasite and species . in all the analyses ,
among the examined animals , 219 sheep and three goats were infected , corresponding to a prevalence of 47.4% and 6.2% , respectively . among sheep ,
the total number of parasites was 1304 and mean intensity was 5.9 ( range 140 ) . among goats
total number of parasites was 7 , with a mean intensity of 2.3 ( range 15 ) .
four species of anoplocephalid cestodes were recorded : avitellina centripunctata , moniezia expansa , stilesia globipunctata , and thysaniezia ovilla . in this study ,
38.7% of sheep were infected by a. centripunctata , 15.4% by m. expansa , 16.7% by s. globipunctata , and 0.4% by t. ovilla . for goats ,
monthly and seasonal dynamics of prevalence were shown in figures 1 and 2 , respectively .
the highest infection rates were recorded during may for a. centripunctata ( 54.5% ) , january for m. expansa ( 21.4% ) , and may for s. globipunctata ( 36.4% ) . the lowest value was noted in december for a. centripunctata ( 25.4% ) , april for m. expansa ( 7% ) , and march for s. globipunctata ( 6.5% ) . for each species ,
the differences on prevalence were not statistically significant between dry season and rainy season : p value = 0.94 for a. centripunctata , 0.64 for m. expansa , 0.28 for s. globipunctata , and 0.93 for t. ovilla .
the mean intensity of infection for the tapeworms was 5.1 , 4.2 , 3.4 , and 1.5 for s. globipunctata , m. expansa , a. centripunctata , and t. ovilla , respectively , and was not statically different between dry and rainy season ( table 1 ) .
single and associated species infections were evaluated among both sheep and goats . among sheep , the infections due to one , two , or three species were 56.2% , 37.4% , and 6.4% , respectively .
for the infections caused by one species , a. centripunctata was the most prevalent with 39.3% followed by m. expansa ( 13.2% ) and s. globipunctata ( 3.6% ) .
for the infections caused by two species , a. centripunctata and s. globipunctata were more prevalent ( 24.2% ) than a. centripunctata and m. expansa ( 11.4% ) , m. expansa and s. globipunctata ( 1.4% ) , and a. centripunctata and t. ovilla ( 0.5% ) .
as for the infections due to three species , a. centripunctata , m. expansa , and s. globipunctata were more prevalent with 5.9% than a. centripunctata , m. expansa , and t. ovilla ( 0.5% ) . among goats , two of infected animals harbored a single species m. expansa , and the remaining harbored m. expansa and t. ovilla .
a total of ten a. centripunctata , six s. globipunctata , six m. expansa , and six t. ovilla were examined .
all avitellina and stilesia and four moniezia and thysaniezia specimens were collected from sheep ; two moniezia and thysaniezia specimens were collected from goats .
the scolex is unarmed , spherical in shape , 1000 to 1600 m in diameter , and provided with four suckers located at its peripheral margin ( figure 3(a ) ) .
each sucker shows a triangular opening from which the point of the triangle is directed towards the center of the scolex ( figure 3(b ) ) .
the tegument of the scolex , suckers , and strobila of species are covered with acicular and capilliform filitriches ( figures 3(c ) and 3(d ) ) .
the strobila has a threadlike appearance and is acraspedote , showing unilateral genital pores irregularly alternating .
proglottids are wider than longer and weakly distinguished even in mature proglottids ( figures 3(e ) and 3(f ) ) .
the scolex is unarmed , globular in shape , 400 to 600 m in diameter , and provided with four suckers located at its peripheral margin ( figure 4(a ) ) .
each sucker shows a circular or subcircular opening ( figures 4(b ) and 4(c ) ) .
the genital pores are paired in each proglottid and disposed laterally ( figures 4(e ) and 4(f ) ) .
the tegument of the scolex , suckers , and strobila are covered with acicular and capilliform filitriches ( figure 4(d ) ) .
the cirrus is entirely covered by capilliform filitriches ( figures 4(f ) and 4(g ) ) .
the scolex is unarmed and is club - shaped , 300 to 600 m in diameter , and provided with four well - developed suckers ( figure 5(a ) ) .
each sucker shows a circular opening ( figure 5(b ) ) . at the center of the scolex ,
the tegument of scolex , suckers , and strobila is covered with acicular and capilliform filitriches ( figure 5(c ) ) .
strobila is craspedote , threadlike , and very spiral ( figures 5(d ) and 5(e ) ) .
the scolex is unarmed and round , 300 to 450 m in diameter , and provided with four well - developed suckers ( figure 6(a ) ) .
the tegument of the scolex , suckers , and strobila is covered with capilliform filitriches ( figure 6(c ) ) .
the proglottids are wider than longer with unilateral genital pores irregularly alternating ( figure 6(d ) ) . from the genital pores , a rounded cirrus protrudes , which exhibits a cavity in the center ( figure 6(f ) ) .
infections by anoplocephalid cestodes are common among sheep and goats [ 17 , 19 , 2629 ] . in senegal ,
five species of anoplocephalidae , a. centripunctata , m. expansa , m. benedeni , s. globipunctata , and t. ovilla , have already been described among ruminants [ 11 , 14 , 26 , 30 ] . in our study , all except m. benedeni were found among sheep , but among goats only m. expansa and t. ovilla have been observed .
the overall prevalence of anoplocephalids was significantly higher among sheep than among goats , as previously reported in senegal by ba et al . and ndao et al .
other studies in west african countries such as gambia , burkina faso , and cte d'ivoire showed similar prevalence to those obtained in our study and confirmed that sheep were more frequently infected than goats [ 15 , 17 , 27 ] . the behavior of goats in pasture seems to limit their risk of infections and could explain also their low infestation intensity .
goats consume longer pasture than sheep , making intermediate oribatid mite hosts less available ; another fact is that goats consume more ligneous food , rich in tannins , which are known natural anthelmintics and probably limit the infection .
our study and others showed that infection in sheep is generally higher in western african areas compared to others : in north africa such as egypt ( 11% ) and in some european areas such as turkey ( 4.4% ) [ 19 , 32 ] .
the prevalence among goats was slightly lower in turkey ( 6.3% ) compared to previous reports in senegal [ 14 , 31 ] .
these differences can be attributed to climatic factors that are often highly variable from one area to another , such as the duration of the rainy season , which may affect the dynamics of intermediate hosts ( oribatid mites ) .
also the soil kind , as oribatids , prefers acid soils and the type of farming in the different localities may therefore affect the prevalence of anoplocephalids . in the previous studies of prevalence of anoplocephalids among small ruminants , m. expansa was the most prevalent [ 26 , 31 ] . nevertheless in our work ,
these facts can be probably explained by the different age - group of ruminants examined in the studies , because it is known that a. centripunctata is the more common among adult ruminants and m. expansa is the highest among one - year - old ruminants [ 13 , 34 ] .
another fact is that a. centripunctata better resists the immune system of hosts than m. expansa .
sharkhuu reported in mongolia that moniezia is common in goats and avitellina and thysaniezia are common in sheep . in present study ,
the prevalence of m. expansa among sheep and goats are similar to those reported in western ethiopia , but lower than in senegal [ 30 , 31 ] , cte d'ivoire , eastern ethiopia , and guinea . for a. centripunctata ,
the prevalence is lower than those reported in senegal , similar to cte d'ivoire , and higher than in senegal and in cte d'ivoire . in our study ,
a. centripunctata was not found among goats as previously reported in cte d'ivoire [ 16 , 17 ] and mongolia but in senegal ba et al .
the prevalence in sheep in our study was similar to that in cte d'ivoire , but higher than in senegal .
the same result was reported previously in cte d'ivoire , guinea , and western ethiopia . for t. ovilla ,
the prevalence was low , similar to the data from sharkhuu , but different from reports by nadge who found t. ovilla from sheep in senegal , with a prevalence of 84% .
. this reduction may be due to different control practices . since 1996 with the national plan for the development of animal husbandry ( npdah ) and its strategy of contributing to improving health and zootechnical conditions of livestock ,
a strategic deworming plan was generally proposed during the rainy season because of the heavy infestation of ruminants and at the end of the dry season for their weakness .
other investigations have shown that climate is an important factor in determining levels of infection by anoplocephalids .
generally anoplocephalids are more prevalent in the rainy season than the dry season [ 15 , 26 ] .
oribatids are susceptible to desiccation and this fact may be a reason for little infection during the warmer months . however , our study revealed two peaks of prevalence both in the dry and in the rainy seasons unlike the previous reports . except for t. ovilla , anoplocephalids were present in small ruminants all the year .
this lack of difference between seasons can be explained by deworming control methods applied in both rainy and dry seasons . also the mean intensity was generally low , due probably to the large size of these tapeworms exceeding generally one meter and/or control methods by veterinaries services and breeders .
no significant difference between dry and rainy season can be attributed to the longevity of the tapeworms , which exceed a year .
in the present study , the infection with a. centripunctata and s. globipunctata was the most prevalent ( 24.2% ) , followed by a. centripunctata and m. expansa ( 11.4% ) .
the multiple infestations play an important role in growth retardation and weight loss of small ruminants .
more , it was reported that coinfections can be lethal among small ruminants [ 33 , 3639 ] .
only a few scanning studies have been made on a. centripunctata , t. ovilla , and m. expansa , and these previous studies focused only on the scolex [ 19 , 40 ] .
suckers have circular , oval , or linear openings , depending on the level of contraction .
the proglottids are wider than longer ; they are craspedote for m. expansa , s. globipunctata , and t. ovilla and acraspedote for a. centripunctata .
m. expansa and t. ovilla are large worms compared to a. centripunctata and s. globipunctata .
they differ in the presence of paired genitalia in m. expansa and single and irregularly alternating genital pores in t. ovilla .
the body of cestodes is fully covered by microtriches , presenting different forms and size among specimens .
then standardized terminology was adopted to define the different kinds of microtriches . in our study ,
the microtriches observed among a. centripunctata , m. expansa , and s. globipunctata are acicular and capilliform filitriches . on the other hand ,
previous isoenzyme electrophoretic studies have shown that a. centripunctata , m. expansa , and t. ovilla are species complex [ 14 , 30 ] .
but recently for moniezia expansa , a genetic analysis showed an intraspecific variation other than speciation , and further molecular investigation is necessary , in order to identify species in all of these genera .
in small ruminants from senegal , four cestode species a. centripunctata , m. expansa , s. globipunctata , and t. ovilla were found among sheep .
there were no statistically significant differences between dry and rainy season prevalence of anoplocephalidae among sheep .
multiple infections of anoplocephalids were recorded and the double infection a. centripunctata and s. globipunctata was most prevalent with 24.2% . | this study was undertaken to determine the prevalence of anoplocephalid cestodes in sheep and goats in senegal .
intestines of 462 sheep and 48 goats were examined ; 47.4% of sheep and 6.2% of goats were infected .
the species identified and their prevalence were , among sheep , avitellina centripunctata 38.7% , moniezia expansa 15.4% , stilesia globipunctata 16.7% , and thysaniezia ovilla 0.4% . among goats , they were m. expansa 6.2% and t. ovilla 2.1% .
the prevalence of all species was not statistically different between dry and rainy seasons .
the infections were single or multiple .
indeed , 56.2% of sheep were infected by a single species , 37.4% by two species , and 6.4% by three species . for goats ,
66.7% were infected by m. expansa and 33.3% by both m. expansa and t. ovilla .
scanning electron microscopic ( sem ) observations of tapeworms show the general diagnosis characters of these species . |
odontogenic keratocyst ( okc ) is a developmental odontogenic cyst with specific histopathological features and clinical behaviors , high recurrence rate , and aggressive behavior compared to other
developmental odontogenic cysts . due to its specific clinico - pathologic characteristics ,
its name has been changed to keratocystic
odontogenic tumor in the new classification of the world health organization ( who).[1 - 2 ] dentigerous ( follicular ) cyst is the most common odontogenic developmental cyst , which instigates from separation of dental follicle from crown of an unerupted tooth .
the reactions between the odontogenic epithelium and its ectomesenchymal components have a role in the formation of odontogenic lesions , including odontogenic
cysts .
blood vessels present in the connective stroma of odontogenic cysts help the growth of their epithelium by supplying oxygen and
nutrients.3 neoplastic tissues require angiogenesis in order to continue their growth , development and progression , which can also contribute to their invasion
and metastasis.[3 - 4 ] angiogenesis is defined as the formation of new blood vessels from the existing
blood vessels and has been evaluated in various lesions including breast cancer , melanoma , endometrial carcinoma , squamous cell carcinoma , lichen planus , ameloblastoma ,
and some odontogenic cysts.[5 - 11 ]
one of the techniques used for the quantitative analysis of angiogenesis is mean vascular density ( mvd ) , which has been evaluated using various molecules ,
including vascular endothelial growth factor ( vegf ) , von willebrand factor , factor viii , cd31 , cd34 , and cd105 ( endoglein).[12 - 13 ]
cd34 ( qbend 10 ) is a pan - endothelial marker and trans - membranous monomeric glycoprotein of the cell surface and is expressed in the normal and neoplastic endothelial cells of blood vessels.[14 - 18 ]
cd105 ( endoglein ) is a hypoxia - dependent membrane hemodimer disulfide glycoprotein , associated with cell proliferation , and expressed on activated vascular endothelial cells .
since the expression of this protein increases in tumoral tissues compared with normal tissues , it can be inferred that expression of cd105 is related to tumor angiogenesis .
several studies have shown that cd105 antibody has higher accuracy and specificity to bind to newly formed blood vessels in tumoral tissues compared to other pan - endothelial markers such as factor viii , von willebrand factor , cd31 and cd34 .
[ 21 - 22 ]
only a limited number of studies have investigated stromal factors such as role of angiogenesis in odontogenic lesions reported by seifi et al
and de andrade santos et al .
hence , the aim of this study was to determine mvd by using cd34 and cd105 markers in okcs and dentigerous cysts and evaluating their relationship with the biologic behaviors of these lesions .
in this retrospective cross - sectional analytical study , the archive files of hamadan faculty of dentistry were evaluated from 1997 to 2010 .
h&e slides were
evaluated and confirmed by two oral pathologists and 15 samples from each lesion with sufficient tissues and proper fixation were selected .
inflammation was considered one of the factors affecting angiogenesis and therefore , as a confounding factor for the results .
the files of patients with
the diagnosis of odontogenic lesions , including dentigerous cysts and okc were retrieved .
their clinical and demographic data including age , gender , and locations of the lesions were recorded in tables .
slices with 4- thickness were prepared from paraffin blocks for immunohistochemical staining .
the slides were deparaffinized in xylene and then hydrated in graded alcohol series . for cd34endogenous
peroxidase activity
was blocked by incubating the slides in hydrogen peroxide ( 3% ) in phosphate buffer and for cd105 incubating the slides with 3% h2o2 in methanol for 30 minutes .
antigen retrieval
for cd105 was performed by treating sections with proteinase - k for 5 minutes .
antigen retrieval for cd34 was performed in a microwave oven for 10 minutes at 120 degree centigrade . to prevent nonspecic
reactions ,
sections were incubated with 10% serum for 10min.then monoclonal mouse anti - human cd105 antibody ( clone : sn6h ; product code : m3527 , dako , north america inc . ) and monoclonal mouse anti - human
cd34 antibody ( clone : qbend 10 , product code : m7165 a / s , glostrup , dako , denmark ) were applied as primary antibody .
cd105 antibody was incubated at room temperature in a humidifying chamber for 90 minutes .
for cd34
, the sections were incubated with anti- cd34 antibody for 30 minutes at room temperature with a working dilution of 1:50 , followed by incubation with secondary biotinylated antibody and streptavidin
for 15 min each .
diaminobenzidine was used to produce brown staining followed by counterstaining with mayer s hematoxylin . after each step
the human
tonsillar tissue was used as the positive control and the primary antibody was eliminated and replaced with phosphate buffer saline for the negative control .
the technique described by weidner et al .
the stained slides were evaluated under a light microscope ( olympus bx41 , japan )
by two oral pathologists at 100400 magnification . in order to determine the mvd ,
three areas with the highest amount of vascularization , known as hot spots , were selected under low magnification ( 100 ) .
microvessels were counted in each of the three elds at 400 magnication and the mean density was reported .
positive staining for cd105 and cd34 was defined as each brown - colored endothelial cell separately ( or in aggregates ) was distinctively separated from the adjacent micro - vessels .
the objects which seemed
to be originated from one blood vessel were counted if completely separated from it .
blood vessels with muscular walls were excluded and the mean number
of blood vessels in the three selected was considered as mean vascular density ( mvd ) .
the data were analyzed by pss16 statistical software with setting p value at 0.05 .
descriptive statistics of clinical data in study groups
cd34 and cd105 were expressed in all samples .
( figure 1 ) the mean ( sd ) mvds with cd34 marker in dentigerous cysts and okcs were 8.482.8 and 12.245.8 respectively which shows mvd in
okc was higher than in dentigerous cysts .
independent samples t - test revealed significant statistical difference between groups ( p= 0.032 ) .
in addition , mean ( sd ) mvds with
cd105 marker in dentigerous cysts and okcs were 5.942.4 and 9.22.94 respectively , so mvd in okc was higher than that in dentigerous cysts .
( table 2 ) the evaluation of mvds in okc ( by cd105 and cd34 markers ) showed a statistical significant differences between these markers ( 9.22.94 and 12.245.8 respectively ) .
the results showed mvd with the use of cd34 marker was significantly higher than that with the use of cd105 marker .
independent samples t - test revealed significant difference between groups ( p= 0.017 ) .
a : cd 105 positive immunoreactivity in dentigerous cyst ( 400 ) b : cd 34 positive immunoreactivity in dentigerous cyst ( 400 ) c : cd 34 positive immunoreactivity in
odontogenic keratocyst ( 400 ) d : cd 105 positive immunoreactivity in odontogenic keratocyst ( 400 )
cd34 and cd105 expression and mvd in studied groups
the mvds in dentigerous cysts with the use of cd105 and cd34 markers were 5.942.4 and 8.482.8 respectively .
the mean number of stained vessels in dentigerous cysts with the use of cd34
marker was significantly higher than that with the use of cd105 marker .
okc has aggressive behavior and a high recurrence rate compared to other developmental odontogenic cysts . in recent years
, various factors such as p53 , mdm2 , ki67 , bcl2 , mmp , il-1 ,
and tgf- markers which were supposed to be related to greater aggressive behavior of okc ( compared to other odontogenic cysts ) have been evaluated in various studies.[24 - 31 ] the growth mechanism of okc is different from dentigerous cyst .
some investigators suggest the increase in some matrix metalloproteinase of connective tissue would affect the
okc growth .
one of the effective factors that probably increase the angiogenesis in okc in comparison to dentigerous cysts would be the escalation of matrix metalloproteinase .
browne was one of the pioneers to propose that the connective tissue wall of okc may have an important role in the pathogenesis of this lesion .
angiogenesis has been previously studied in okc but the number of studies is limited .
among the many angiogenic markers , cd105 has higher accuracy and specificity to bind newly formed blood vessels and cd34 is a pan endothelial marker that shows stronger staining with endothelial cells with lower error rate in comparison to cd31 .
[ 15,21 - 22 ]
cd34 can not distinguish between the newly formed blood vessels and the old host ones but it has important role in evaluation of mvd .
these points would justify why the current study evaluated the expression of these markers in odontogenic lesions . in this study cd34 and cd105
were expressed in all samples but mvd in okc was significantly higher than dentigerous cysts .
several other studies , have also confirmed the role of angiogenesis as an important
factor in the aggressive behavior of okc compared to dentigerous cysts .
alaeddini et al . determined mvd by using cd34 marker and reported that mvd was signicantly higher in okcs than in dentigerous cysts .
they suggested that angiogenesis could
be one of the mechanisms possibly responsible for the different biological behaviors of these lesions .
rubini et al .
observed a significant different expression of vascular endothelial
growth factor ( vegf ) in keratocysts compared to follicular cysts .
they found that in all cases of okc , there was a statistically significantly higher expression of vegf whilst most follicular cysts were
negative for vegf expression .
they concluded angiogenesis could be an active mechanism in the invasive growth of the okc .
.
found cd105 to be strongly expressed in microvessels of okc compared with that in dentigerous cyst and normal oral mucosa .
they suggested that tumor angiogenesis may be associated with locally aggressive
biologic behavior of odontogenic lesions and angiogenesis can show the prognosis and clinical behavior of odontogenic lesions .
unlike the first three studies that evaluated only
one marker of angiogenesis ( cd34 or cd105 ) , we studied two markers of angiogenesis and the marker used in the rubini et al .
the results of all above mentioned studies and our study showed that angiogenesis was higher in okc compared with dentigerous cyst .
angiogenesis is an important step in the majority of
physiologic and pathologic processes and is necessary for the cells to access to oxygen and nutrients in distances more than 12 mm from the blood vessels .
therefore , more angiogenesis in okc compared to dentigerous cysts can indicate higher tissue metabolism .
mvd which is considered as a quantitative analysis of angiogenesis in a lesion can predict the growth rate ,
metastasis , patient survival , and the aggressive behavior .
therefore
, more angiogenesis and higher mvd in okc compared to dentigerous cysts can indicate more aggressive biologic behavior , higher recurrence rate and more growth and proliferation
. it might be possible to control the aggressive behavior of this cyst with anti - angiogenic treatments .
the result of this study was different from result of the study performed by de andrade santos et al .
.
studied the expression of cd105 in okcs , dentigerous cysts , and radicular cysts .
they showed that micro vessel count was higher in dentigerous cyst than in okcs and the differences in the biologic behavior of these lesions do not appear to be associated with the angiogenic index .
our results based on cd105 are not in agreement with the results of the study of de andrade santos et al .
this difference might be attributed to the differences in methodology such as counting method and number of samples .
in the present study ,
mvds based on cd105 marker in okc and dentigerous cysts were significantly less than those with cd 34 .
,
miyata et al . , kumagai et al.,[39
and czekierdowski which compared the expression of cd105 and cd34 antibodies in different lesions have shown a lower mvd based on cd105 marker compared to cd34 marker .
the results of the current study were consistent with their results since the expression of cd105 is a prominent feature of newly formed vessels ; moreover , its expression in previously formed vessels and also in the endothelium of the vessels of normal tissues is little or even negative .
nevertheless , cd34 is expressed in the vascular endothelial cells of normal and neoplastic tissues and can not distinguish the newly formed blood vessels from the old blood vessels of host .
compared to cd34 , these findings suggest that cd105 is a more specific and sensitive marker for angiogenesis
.
the results of this study would probably contribute in better understanding of the role of angiogenesis in the biological behavior of okc and dentigerous cyst .
further studies with larger number of cases are required to elucidate the role of angiogenesis in the aggressive behavior of okc .
in the present study , mvd in okc was higher than that in dentigerous cysts , which can indicate that angiogenesis is one of the possible mechanisms involved in the greater
aggressive biologic behavior and higher recurrence rate of okc compared to dentigerous cysts . | statement of the problem : odontogenic keratocyst ( okc ) is a developmental odontogenic cyst with specific histopathological features , high recurrence rate , and aggressive clinical behavior .
angiogenesis might be considered as an important factor for the growth , expansion , and distribution of this lesion .
purpose : the aim of the present study was to determine the mean vascular densities ( mvd ) of okcs and dentigerous cysts to evaluate their relationship with the biologic behavior of these lesions .
materials and method :
in this cross - sectional analytical study , angiogenesis was assessed in okc and dentigerous cyst by measuring the mvd .
immunohistochemistry was carried out using cd34 and cd105 .
the results were analyzed with independent samples t - test .
the data were analyzed , setting p value at 0.05 .
results : the mvds with the use of cd34 and cd105 markers were significantly higher in okc compared to dentigerous cyst ( p < 0.05 ) . in addition ,
mvds obtained by cd105 in dentigerous cysts and okc were significantly less than those based on cd34 ( p < 0.05 ) .
conclusion : based on the results of the present study , it can be suggested that angiogenesis might be one of the possible mechanisms involved in higher aggressive biologic behavior and greater recurrence rate of okc compared to dentigerous cysts . |
this is a cross - sectional study based on the primary health care clinics of qatar .
the survey was conducted among qatari nationals between 25 and 65 years of age during a period from january to july 2007 .
a multistage stratified sampling design was developed using the administrative divisions of the primary health centres in qatar that had approximately equal numbers of inhabitants .
the sample size was determined on a priori presumption that the prevalence rate of somatisation in qatar would be more or less similar to rates found for several other countries in the arabian gulf and eastern mediterranean ( 13 , 15 ) , where the reported prevalence of somatisation among adults ranged from 15% to 20% . assuming the prevalence of somatisation to be 17% , with the 99% confidence interval for an error of 2% at the level of significance , a sample size of 2,320 would be required to meet the specific objectives of the study . of the total 22 primary health care clinics available ,
this study was approved by research and ethical committee of hamad medical corporation , qatar . during the study period ,
a consecutive series of 2,320 patients were selected from the register of the recruited health centres .
only patients attending the primary health care centres for scheduled routine follow - up appointments or new complaints were approached .
of the approached 2,320 patients , 1,689 responded to the 12-item general health questionnaire ( ghq-12 ) with a response rate of 72.8% .
the research assistants conducted a face - to - face interview with the patients and completed questionnaires until they reached the required sample size .
also , patients who were not willing to disclose their personal information and medical history were not included in this study ( 631 patients ) . a consultant and
training held two sessions for administering the ghq-12 , the socio - demographic questionnaire , cis and the procedure as a whole . after dealing with the presenting complaint ,
the individuals were considered to be probable cases for clinical interview on the bases of scores above the cut - off point on ghq-12 items .
a sample of 404 out of 1,689 ( 23.9% ) scored more than 13 in ghq were planned for the clinical interview schedule ( cis ) using cis questionnaire .
identified cases were assigned diagnoses according to the icd-10 classification of mental illness ( 16 ) .
the severity of their illness was measured according to the total weighted score ( tws ) scores .
( 17 ) as a self - administered questionnaire that would identify non - psychotic psychiatric cases .
the ghq-12 is the shortest version of the ghq and it is concerned mainly with psychoemotional disturbance .
the arabic version of the ghq-12 has already proven to be a valid instrument for screening psychiatry morbidity among arab primary health care patients in qatar ( 1 ) .
it consists of questions designed to elicit the presence or absence of 10 defined psychiatric symptoms and 12 manifest abnormalities ( 17 ) .
each item of psychiatric symptoms and manifest abnormality was graded on 5 points ( 04 ) , 0 = absent , 1 = mild , 2 = moderate , 3 = severe , 4 = very severe ) .
the tws is calculated by adding the sum of the 10 reported symptoms scores plus twice the sum of the 12 manifest abnormality scores . for the purpose of this study the criteria used to define a psychiatric case was a tws of 20 or more .
this threshold was found appropriate when previously used in uae ( 13 , 18 , 19 ) . to avoid ambiguity and overgeneralisation in using the term
somatisation in this sample , we adopted the restrictive operational criteria set by bridges and goldberg ( 20 ) and el rufaie et al .
the bridges and goldberg criteria for somatisation include two terms which are subject to rating bias .
first is the decision required of the interviewer as to whether the physical symptoms can be explained by the psychiatric disorder , and second is the interviewer 's opinion on whether these symptoms could be alleviated by suitable treatment .
the classification of the pgs and sts is as follows : sts : patients with psychiatric disorder who presented to their gp with physical complaints , but in whom a functional diagnosis was recorded.pgs : patients with psychiatric disorder who presented to their gp with psychological complaints.among the 404 psychiatric cases identified , 210 patients were classified as sts and 194 patients were pgs according to bridges and goldberg 's operational criteria ( 20 ) .
sts : patients with psychiatric disorder who presented to their gp with physical complaints , but in whom a functional diagnosis was recorded .
student 's t - test was used to ascertain the significance of differences between mean values of two continuous variables and confirmed by non - parametric mann
multivariate stepwise linear regression analysis was performed to determine the predictors of sts and pgs .
( 17 ) as a self - administered questionnaire that would identify non - psychotic psychiatric cases .
the ghq-12 is the shortest version of the ghq and it is concerned mainly with psychoemotional disturbance .
the arabic version of the ghq-12 has already proven to be a valid instrument for screening psychiatry morbidity among arab primary health care patients in qatar ( 1 ) .
it consists of questions designed to elicit the presence or absence of 10 defined psychiatric symptoms and 12 manifest abnormalities ( 17 ) .
each item of psychiatric symptoms and manifest abnormality was graded on 5 points ( 04 ) , 0 = absent , 1 = mild , 2 = moderate , 3 = severe , 4 = very severe ) .
the tws is calculated by adding the sum of the 10 reported symptoms scores plus twice the sum of the 12 manifest abnormality scores . for the purpose of this study the criteria used to define a psychiatric case was a tws of 20 or more .
this threshold was found appropriate when previously used in uae ( 13 , 18 , 19 ) . to avoid ambiguity and overgeneralisation in using the term
somatisation in this sample , we adopted the restrictive operational criteria set by bridges and goldberg ( 20 ) and el rufaie et al .
the bridges and goldberg criteria for somatisation include two terms which are subject to rating bias .
first is the decision required of the interviewer as to whether the physical symptoms can be explained by the psychiatric disorder , and second is the interviewer 's opinion on whether these symptoms could be alleviated by suitable treatment .
the classification of the pgs and sts is as follows : sts : patients with psychiatric disorder who presented to their gp with physical complaints , but in whom a functional diagnosis was recorded.pgs : patients with psychiatric disorder who presented to their gp with psychological complaints.among the 404 psychiatric cases identified , 210 patients were classified as sts and 194 patients were pgs according to bridges and goldberg 's operational criteria ( 20 ) .
sts : patients with psychiatric disorder who presented to their gp with physical complaints , but in whom a functional diagnosis was recorded .
student 's t - test was used to ascertain the significance of differences between mean values of two continuous variables and confirmed by non - parametric mann
multivariate stepwise linear regression analysis was performed to determine the predictors of sts and pgs .
from the total of 1,689 patients studied , 404 were identified for cis ( 23.9% ) .
the socio - demographic characteristics of sts and pgs are shown in table 1 . among the 404 identified psychiatric cases , 210 patients were sts ( 52% ) and 194 were pgs ( 48% ) , a ratio of 1.1:1 .
the prevalence of somatisation was 12.4% of the total studied sample , while for psychologisation it was 11.5% .
somatisation and psycholgisation were significantly more frequent among the middle - aged patients , especially in the age group of 3039 years ( 40.5% vs. 44.3% ; p=0.01 ) .
somatisation was higher in female patients ( 51.4% ) , whereas psychologisation was more frequent in male patients ( 56.2% ) .
there were no significant differences observed in their gender , marital status , education and occupation .
the socio - demographic characteristics of somatisers and psychologisers a total of 1,689 patients were the study patients who responded to the 12-item general health questionnaire ( ghq ) .
the severity of psychiatric illness as measured by the tws of the patients suffering from different types of disorders is given in table 2 .
the severity of the psychiatric illness ; generalised anxiety disorder , depressive episode , recurrent depressive disorder , dysthymia , prolonged depressive reaction were significantly more in pgs ( p<0.001 ) .
the severity of psychiatric illness in both somatisers and psychologisers note : ns , not significant ; tws , total weighted score .
results of the comparison of postulated determinants in both sts and pgs are shown in table 3 .
family history of psychiatric disorder ( p=0.014 ) and physical illness during adulthood were higher in pgs than sts .
significant physical illness during adulthood before the onset of presenting symptoms was significantly higher in pgs ( 23.2% ; p=0.007 ) .
dissatisfaction at work was significantly higher in sts ( 63.8% ; p=0.007 ) , while dissatisfaction with social life was significantly higher in pgs ( 77.8% ; p=0.007 ) .
results of the comparison of postulated determinants in both somatisers and psychologisers chi - square test was performed .
the factors associated with sts and pgs using multivariate stepwise linear regression analysis is listed in table 4 .
most episodes of depression and anxiety the common mental disorders are contained and managed in primary care .
common somatic symptoms are the main reason for the primary health care visits ; they are also concomitants of anxiety and depression ( 14 ) . many patients with psychological disorders present to their gp with common somatic symptoms
this combination has been referred to as somtisation and is associated with lower rates of diagnosis of depression and anxiety .
most studies in other countries have looked at patients ' beliefs about their symptoms that have focused on the dichotomy between somatising and psychologising . in the arab region
, very few studies have been conducted on somatisation and this study is the first attempt to document the differences between sts and pgs in primary care qatari patients .
a who cross - cultural study reported the prevalence of somatisation ( 19.7% ) with a range of 937% in 15 primary care centres ( 21 ) .
the prevalence rate of somatisation in qatar ( 12.4% ) was lower than the international prevalence of somatisation ( 19.7% ) , but similar to the proportion of uae ( 12% ) ( 19 ) . in saudi arabia ( 22 ) , psychiatric morbidity in primary care is estimated at 3046% with a 19.3% prevalence of somatisation and 20% of depression which is higher than the prevalence found in qatar .
also , the present study revealed a higher prevalence of somatisation ( 12.4% ) than psychologisation ( 11.5% ) .
our results are comparable to uae , but inconsistent with the study in saudi arabia which demonstrated a higher prevalence of psychologisation than somatisation .
our prevalence rates are comparable in relation to somatisation , but not to psychologisation which is consistent with another study that over half of their patients with diagnostic illness were sts , whereas only about one - fifth were pgs ( 14 ) .
ratio of sts : pgs in the present study was 1.1:1 which is similar to the ratio reported in a study on somatised mental disorders among primary care arab patients conducted in uae ( 13 ) ( 1.1:1 ) .
but the results of the spanish study revealed that somatic patients were three times more prevalent than pgs in primary care ( 21 ) . among the identified psychiatric cases in the present study , 52% of them were sts and 48% of them were pgs .
our ratio is consistent with reports in non - western cultures claiming higher somatisation rates . in conformity with other reports
, there was no significant difference between the sts and pgs groups in relation to gender , educational level and marital status ( 2224 ) .
but , a study ( 5 ) reported that the group of sts had a significantly lower level of education and tended to be older ( p=0.05 ) which is consistent with the results of the study ( 14 ) that st would have lower level of education and would belong to a lower social class .
the reason for this finding is that patients who have received less education may be less able to elaborate and interpret their psychological distress . in the present study ,
the diagnostic categories , mixed anxiety and depressive disorder and prolonged depressive reaction , were common in both pgs and sts in the current study . however , prevalence of generalised anxiety disorder , recurrent depressive disorder , dysthymia , depressive episode and brief depressive reaction were more prevalent among the pgs than the sts .
the uae study also reported that the severity of most psychiatric illness was estimated to be greater among the pg group than the sts ( 19 ) .
it is important that primary care physicians acquire specific skills for the recognition of depression .
our study revealed that stressful life events was significantly more among sts than pgs which was comparable to the findings of another study ( 23 ) .
for sts , general health and adverse childhood experience were strong positive predictors , whereas for pgs general health and impairing life or social life were positive predictors .
the erroneous diagnosis of depression in patients leads to the risk of unnecessary and potentially dangerous medication . in conclusion
, the present study revealed that the prevalence of somatised mental disorder was little higher than the psychologised mental disorder .
the prevalence of somatisation and psychologisation is comparable with other reported studies from the middle - east and western countries .
dissatisfaction at work and stressful life events within the 12 months before the onset of the presenting symptoms were significantly more among sts than pgs .
for sts , general health and adverse childhood experience were strong predictors for sts , whereas general health and impairing family or social life were significant predictors for pgs .
it is important to have educational programmes that raise awareness of the full spectrum of somatic symptoms as well as aiming to improve attitudes and consulting skills in primary care .
the authors have not received any funding or benefits from industry to conduct this study . | backgroundmedically unexplained somatic complaints are among the most common clinical presentations in primary care in developing countries and they are considerable burden for patients and health care system.objectivesthe aim of this study was to determine the prevalence of somatisation in comparison to psychologisation among a sample of qatari patients who were visiting primary health care ( phc ) centres and to investigate the clinical and socio - demographic characteristics of somatisers ( sts ) and psychologisers ( pgs).methodthe survey was conducted among phc qatari patients during the period from january to july 2007 .
about 2,320 patients were approached , of whom 1,689 agreed to participate and responded to the questionnaire . among the studied qatari patients ,
404 patients were identified for clinical interview .
the first stage of the study was conducted with the help of general practitioners , using the 12-item general health questionnaire .
the second stage was carried out by a consultant using the clinical interview schedule .
a specific operational criterion was used to identify sts and pgs.resultsthe prevalence rate of sts among the total studied sample was 12.4% , while the pgs were 11.5% . among the identified psychiatric cases , the proportion of sts ( 52% ) was higher than pgs ( 48% ) .
most of the diagnostic categories were more prevalent among pgs .
the dissatisfaction at work and stressful life events within 12 months before the onset of the presenting symptoms were the three postulated determinants which were significantly more among sts than pgs.conclusionthe prevalence of somatised mental disorder was little higher than the psychologised mental disorder .
the prevalence of somatisation and psychologisation is comparable with other reported studies from the middle - east and western countries .
dissatisfaction at work and stressful life events were significantly higher among sts than pgs . |
rms is the most common solid primary tumor in children , but only a few cases of breast metastases have been described .
we present the case of a young female with primary pelvic alveolar rhabdomyosarcoma ( rms ) which metastasized to the breast .
in april 2000 , a 14-year - old female presented with a lump in her right breast affecting the mammary surface and appearing as a violaceus mass .
iv right pelvic alveolar rms [ figure 1 ] with metastatic lymphadenopathies in the inguinal , iliac , retroperitoneal , mediastinal , and left supraclavicular regions .
this protocol included : iva ( ifosfamide , vincristine , actinomycin d ) 1 cycle , vincaepi 1 cycle ( vincristine , carboplatin , epiadriamycin ) , followed by a high - dose sequential monotherapy schedule incorporating cyclophosphamide , etoposide , and carboplatin with autologous stem cell rescue . magnetic resonance and t1-weighted images showing heterogeneous lesion with early enhancement and fast wash out in the right breast she achieved complete response ( cr ) in primary and metastatic regions and then received maintenance chemotherapy with vac 9 cycles ( vincristine , actinomicina d , and cyclophosphamide ) .
concomitant irradiation was administered to the initial affected fields ( 41 , 4 gy , 180 cgy / day ) and boost to the primary tumor ( 14 gy , 200 cgy / day ) no surgery was performed , due to the inaccessible primary localization . at the time of the breast lump , our patient was off treatment for 5 months .
considering her previous history , a metastatic relapse was suspected . magnetic resonance imaging ( mri ) showed bilateral heterogeneous lesions as well as lymph nodes in the right axillary region [ figures 1 and 2 ] .
magnetic resonance and t1-weighted images showing enhance lymph nodes in the right axillary region she then commenced second line chemotherapy with alternating cycles of topotecan ( 0.75 mg / m for 5 days ) and cyclophosphamide ( 250 mg / m for 5 days ) in 21-day intervals .
after 2 cycles , partial response was noted in both breast masses , with cr in axillary region . at this point ,
the multidisciplinary breast cancer board strongly suggested local radiotherapy based on the argument that all the fields previously irradiated were free of disease at relapse .
however , this proposal was rejected by the radiotherapy service who argued about the overlap of the previously irradiated fields and the potential risk of breast irradiation in such a young female . instead of radiotherapy , the board decided a radical subcutaneous bilateral mastectomy with dissection of axillary lymph nodes followed by an immediate breast reconstruction as a local treatment .
after surgery , she was given four cycles of the previous chemotherapeutic regimen as a maintenance therapy , which was discontinued after the fourth cycle because of acute hemorrhagic cystitis .
three months after finishing treatment , she noticed once again , a new subcutaneous mass in her right breast .
she was diagnosed as having a second isolated metastatic relapse in the right breast . at this point ,
the prognosis was considered very poor and in agreement with the family , we decided to start palliative treatment .
consolidation with local radiotherapy was again considered and agreed , at a total of 50 gy to the right mammary volume .
the assessment with mri and positron emission tomography at the end of treatment showed cr .
the patient remains disease - free for 12 years after finishing treatment with a good quality of life .
children presenting breast metastases from rms are usually females in postmenarcheal age due to the greater vascularity of the breast , being the limbs and the buttock the most common primary sites with alveolar phenotype .
although the occurrence of breast metastases is a rare phenomenon , early diagnosis and treatment may have a major impact on prognosis / outcome .
therefore , it seems appropriate to place special attention on the subgroup of risk patients for breast metastases , not only at the time of diagnosis but also during the follow - up .
physical examination and ultrasound may be useful as a first approach , but mri is considered to be the gold standard .
the overall survival is generally < 30% of patients with stage iv rms ; but patients having unfavorable prognostic factors at initial diagnosis like alveolar or undifferentiated histology , multiple metastases , age more than 10 years , unfavorable primary location ( defined as pelvis , extremities , spine and bone marrow involvement ) , have an even worse outcome . among the predictive survival factors after recurrence , the alveolar pattern associated with irs groups 2 , 3 , and 4 showed the worst prognosis with a 5 years survival rate approaching 3% .
although rms is generally a highly chemosensitive tumor , local treatment plays an essential role in achieving the cure , because local progression or relapse is the major cause of treatment failure .
mutilating surgery is not usually recommended as an initial approach , but can be useful as salvage treatment when other procedures have failed .
radiotherapy has been long considered as having an important role in local control of childhood rms .
early evidence suggests that the newer methods of radiation therapy , including intensity - modulated radiotherapy , proton beam radiotherapy , and brachytherapy , may reduce long - term sequelae without affecting efficacy , in comparison with three - dimensional conformal radiotherapy .
therapy of breast metastases of rms in children is a challenge , mainly because it is unclear if the criteria commonly used for breast tumors in adults , may be adopted in the pediatric population .
particularly , there is no consensus regarding conservative surgery , and there is also a concern about irradiation of mammary area in young women due to the risk of a secondary tumor . we conclude that although breast metastases in rms are very rare , we must consider them in female patients with risk factors .
although treatment is a challenge for the clinician , multimodal strategies might achieve prolonged or complete remission .
newer methods of delivering radiotherapy which potentially minimize side effects must be taken into account .
| metastatic breast disease is a very rare condition in children .
rhabdomyosarcoma ( rms ) is the most common solid primary tumor in children , but only a few cases of breast metastases have been described .
we present the case of a young female with a primary pelvic metastatic alveolar rms , which metastasized to the breast twice and achieved prolonged complete remission with a multimodal approach . |
the type 5 g protein subunit ( g5 ) can form complexes with members of the regulator of g protein signaling 7 ( rgs7 ) family , but the relevance to neuronal g protein signaling is unclear .
we report that mouse rgs7/g5 complexes bind to g protein gated potassium channels and facilitate their functional coupling to gabab receptors in neurons .
these findings identify a novel compartmentalization mechanism critical for ensuring high temporal resolution of neuronal g protein signaling . | |
the bar - code symbol represents general data such as name , age , sex , address , occupation , phone number , and nationality of the patient , which can be scanned with a specific scanning device to transfer information to the computer . after scanning the bar code ,
labeling of dentures can be important in early identification of the dead and injured people in the event of major disasters such as earthquakes , floods , plane crashes , and accidents ; for patients suffering from dementia ; or the denture itself being inadvertently misplaced on admission of the patient in the hospital.[16 ] denture identification is important for hospitalized patients or patients in geriatric institutions .
loss of dentures or confusion in identification of denture is not uncommon , particularly during cleaning by the nursing staff .
the loss is of greater consequence to the elderly patient who may have difficulty in learning to control new dentures .
as a bar - code system can incorporate large amount of data , data pertaining to the treatment procedure can be stored as a record .
the same bar code can be accessed , modified , or updated with the scanning device .
bar codes are more efficient and provide a method to track and store information about individuals .
they serve an important role and provide advantages as compared to manual entry of the information .
manual tracking leads to many human errors , whereas bar codes are nearly 10,000 times more accurate than are the manual entries .
some of the basic requirements of bar coding are as follows :
must be easy to incorporatemust give definite information in every situationmust not alter the structure or properties of the material of the denturesmust not affect adaptation of the dentures to the supporting tissuesmust not affect aestheticsmust resist high temperaturesmust be inexpensive .
must be easy to incorporate must give definite information in every situation must not alter the structure or properties of the material of the dentures must not affect adaptation of the dentures to the supporting tissues must not affect aesthetics must resist high temperatures the various sites where bar codes can be incorporated are palatal portion of the maxillary denture , external posterior buccal surface of the denture , and lingual flange of the mandibular denture .
labeling can be made up of metallic or nonmetallic materials , such as microlabels , chips , acrylic resin strips , disks , or printed papers , that are incorporated into the denture base during the flasking and/or the packing process.[816 ] at times , the nonmetallic labels can not withstand high temperatures , and for this reason a newer technique of ceramic marking system can be used that can endure high temperatures and are aesthetically pleasing as well .
bar coding can be done in complete dentures , removable partial dentures , fixed partial dentures , and maxillofacial prosthesis .
bar coding is done on a printed paper of 15 25 mm , which is further laminated and then incorporated in a maxillary complete denture .
the major advantage of the bar - coding technique is that it can incorporate large amount of data .
various types of software are available that can facilitate encoding of patient 's information and generate a corresponding bar code . with the help of appropriate software ,
the encoded information can be retrieved by scanning the bar code with the precise laser staff [ figure 1 ] .
information retrieved after scanning the bar code the procedure involves a compression molding technique of denture fabrication .
first , a heat - cured clear acrylic resin dental products of india ( dpi ) is applied on the palatal surface ( near the first or second molar region ) of the maxillary mold , followed by placing the laminated bar code in the inverted fashion [ figure 2 ] . over the bar code ,
a heat - cured pink acrylic resin dpi is applied and packed in the usual manner .
the palatal surface of the maxillary denture was chosen for incorporating the bar code because of its larger surface area and to avoid distortion [ figure 4 ] .
palatal surface of the maxillary mold showing the site for bar - code incorporation finished and polished denture intra - oral photograph following are the limitations of this technique :
patients need to carry their dentures wherever they go.denture incorporated with a bar code if not in use should be thoroughly discarded on medicolegal grounds .
denture incorporated with a bar code if not in use should be thoroughly discarded on medicolegal grounds .
labeling of dentures can be important in early identification of the dead and injured people in the event of major disasters such as earthquakes , floods , plane crashes , and accidents ; for patients suffering from dementia ; or the denture itself being inadvertently misplaced on admission of the patient in the hospital.[16 ] denture identification is important for hospitalized patients or patients in geriatric institutions .
loss of dentures or confusion in identification of denture is not uncommon , particularly during cleaning by the nursing staff .
the loss is of greater consequence to the elderly patient who may have difficulty in learning to control new dentures .
as a bar - code system can incorporate large amount of data , data pertaining to the treatment procedure can be stored as a record .
the same bar code can be accessed , modified , or updated with the scanning device .
bar codes are more efficient and provide a method to track and store information about individuals .
they serve an important role and provide advantages as compared to manual entry of the information .
manual tracking leads to many human errors , whereas bar codes are nearly 10,000 times more accurate than are the manual entries .
some of the basic requirements of bar coding are as follows :
must be easy to incorporatemust give definite information in every situationmust not alter the structure or properties of the material of the denturesmust not affect adaptation of the dentures to the supporting tissuesmust not affect aestheticsmust resist high temperaturesmust be inexpensive .
must be easy to incorporate must give definite information in every situation must not alter the structure or properties of the material of the dentures
must not affect adaptation of the dentures to the supporting tissues must not affect aesthetics must resist high temperatures the various sites where bar codes can be incorporated are palatal portion of the maxillary denture , external posterior buccal surface of the denture , and lingual flange of the mandibular denture .
labeling can be made up of metallic or nonmetallic materials , such as microlabels , chips , acrylic resin strips , disks , or printed papers , that are incorporated into the denture base during the flasking and/or the packing process.[816 ] at times , the nonmetallic labels can not withstand high temperatures , and for this reason a newer technique of ceramic marking system can be used that can endure high temperatures and are aesthetically pleasing as well .
bar coding can be done in complete dentures , removable partial dentures , fixed partial dentures , and maxillofacial prosthesis .
bar coding is done on a printed paper of 15 25 mm , which is further laminated and then incorporated in a maxillary complete denture .
the major advantage of the bar - coding technique is that it can incorporate large amount of data .
various types of software are available that can facilitate encoding of patient 's information and generate a corresponding bar code . with the help of appropriate software ,
the encoded information can be retrieved by scanning the bar code with the precise laser staff [ figure 1 ] .
information retrieved after scanning the bar code the procedure involves a compression molding technique of denture fabrication .
first , a heat - cured clear acrylic resin dental products of india ( dpi ) is applied on the palatal surface ( near the first or second molar region ) of the maxillary mold , followed by placing the laminated bar code in the inverted fashion [ figure 2 ] . over the bar code ,
a heat - cured pink acrylic resin dpi is applied and packed in the usual manner .
then , the retrieved denture is finished and polished [ figure 3 ] . the palatal surface of the maxillary denture was chosen for incorporating the bar code because of its larger surface area and to avoid distortion [ figure 4 ] .
palatal surface of the maxillary mold showing the site for bar - code incorporation finished and polished denture intra - oral photograph following are the limitations of this technique :
patients need to carry their dentures wherever they go.denture incorporated with a bar code if not in use should be thoroughly discarded on medicolegal grounds .
denture incorporated with a bar code if not in use should be thoroughly discarded on medicolegal grounds .
heath and stevenson proposed different techniques for writing names on removable dentures in 1980s . in the 1990s , researchers such as ling , furst , ibrahim , and berry wrote personal information on different materials that were then buried in acrylic.[181719 ] with people becoming more techno savvy at the dawn of twenty - first century , i d labels became more prevalent by making use of microchips and radiofrequency devices . in 2002 , rajan and julian reported a method based on electronic microchip manufacturing technology . to maximize the amount of data that could be written on a metallic i d label , ling et al . used a high - power copper vapor laser in 2003 . in 2004 , millet and jeannin implanted a radiofrequency i d transponder in a complete upper denture .
i d labels incorporated on the prostheses should not be affected by daily routines followed by the patient .
in addition , the surfaces of the labels should be smooth and have high polish to prevent plaque formation and facilitate hygiene maintenance .
hence , in future , researches should focus on developing the bar - code technique so as to increase the domain of its usage . | a number of commercial methods for identifying dentures are available
. they can be either invasive or noninvasive techniques .
the less sophisticated procedures include simple engraving with bur , and more sophisticated procedures use labels or chips . bar coding system is a way of transferring data to the computer and huge data can be stored as a record .
bar coding can be easily incorporated during acrylization of the denture and thus could be used in individual identification . |
alopecia areata ( aa ) is a nonscarring alopecia that genetic and immunological causes have been suggested to be affective in the pathogenesis of the disease .
prevalence of aa in the united states is 0.10.2% with an estimated lifetime risk of 2% .
aa is characterized by sudden onset of hair loss in one or more regions , generally head , and some other parts of the body covered with hair that can be patchy , totalis ( 100% terminal hair loss on the scalp ) , universalis ( 100% terminal hair loss on the scalp and body ) , and ophiasis ( hair lose around the periphery of the scalp ) .
about 766% of the patients have nail involvement , and the most common form of nail involvement is pitting .
various studies revealed that aa can accompany with anxiety disorders , depression , and social phobia .
the psychological and social role of hair , particularly scalp hair , is much more important , in comparison to the biological aspect .
hair can be a social characteristic of sex , age , values , and even being the member of a group . beyond the social meaning of hair
, it plays an important role in the identity and self - imagination of the body .
hair also plays a role in physical attraction and is an important factor in interpersonal relationships . in some studies ,
aa is reported accompanied with other autoimmune illnesses such as hashimoto 's thyroiditis , vitiligo , diabetes , lichen planus , and also in a few reports , celiac disease ( cd ) ( gluten - sensitive enteropathy ) .
although diagnosis of aa is usually facile , treatment is not . curative treatment does not exist yet
cd is a small intestinal disease characterized by villous atrophy , mucosal inflammation , and crypt hyperplasia , which occurs upon exposure to dietary gluten and which exhibits improvement after the removal of gluten from the diet . for the diagnosis of cd , initially , serological tests are recommended .
other diagnostic procedures include endoscopy , biopsy of the small intestine , oral tolerance tests ( xylose and lactulose ) , and radiographic studies of small intestine .
the prevalence of gliadin antibody in patients with aa is estimated to be about 1 in 116 . in another study ,
prevalence of anti - gliadin in patients with aa is estimated to be about 18 in 100 .
the chronic and recurrent nature of aa and its stressful effect on patients make this study important .
unfortunately , there is no cure for aa . according to most of the reports , hair regrew in patients with aa and cd after they accepted a gluten - free diet .
aa is a skin disease and can be easily diagnosed , but can be a manifestation of an internal disease such as celiac , so cd can be treated before any complication . in previous studies ,
assessment of these three antibodies at the same time can help to understand the relationship between these two diseases .
it was a case control study conducted at one of the biggest university of medical sciences of center of iran , in which patients with aa referred to dermatology clinics were analyzed and studied .
thirty - five patients were selected through simple sampling in patients with aa and control group .
data such as age , sex , duration of disease ( classified according to the olsen 's study ) , family history of aa ( first degree ) , and nail involvement were collected in questionnaires .
all patients with aa were considered to be the case group and all healthy participants without a past history and family history of aa , cd , and other autoimmune diseases ( vitiligo , hashimoto 's hypothyroidism , etc . ) were considered as the control group .
exclusion criteria included patients who were treated with a gluten - free diet , pregnant women , nursing mothers , and people who did not cooperate with the investigation .
after an explanation of how the process works , a written informed consent was signed by the participants . ethical approval for our study was obtained from the isfahan university of medical sciences ( ethical code : 393232 ) .
both groups were referred to an accredited laboratory for the following tests : anti - gliadin iga , anti - gliadin igg , and anti - tissue transglutaminase iga . for this purpose , first , taking a small sample of blood ( 5 ml clot blood without anticoagulant ) from a vein in arm was necessary and then it was centrifuged for 10 min at around 25003000 . finally , the laboratory examined the obtained serum samples from centrifugation in elisa method with binding site 's kits of the uk . a normal range of tests was defined according to the laboratory standards and finally was reported as positive / negative .
the statistical analyses of the results were performed using chi - square test ( for qualitative data ) and t - test ( to compare the means of two groups and for quantitative data ) in spss 16 for windows ( ibm corporation , ny , usa ) . in this study , the significance level was set at p < 0.05 .
it was a case control study conducted at one of the biggest university of medical sciences of center of iran , in which patients with aa referred to dermatology clinics were analyzed and studied .
thirty - five patients were selected through simple sampling in patients with aa and control group .
data such as age , sex , duration of disease ( classified according to the olsen 's study ) , family history of aa ( first degree ) , and nail involvement were collected in questionnaires .
all patients with aa were considered to be the case group and all healthy participants without a past history and family history of aa , cd , and other autoimmune diseases ( vitiligo , hashimoto 's hypothyroidism , etc . ) were considered as the control group .
exclusion criteria included patients who were treated with a gluten - free diet , pregnant women , nursing mothers , and people who did not cooperate with the investigation .
after an explanation of how the process works , a written informed consent was signed by the participants . ethical approval for our study was obtained from the isfahan university of medical sciences ( ethical code : 393232 ) .
both groups were referred to an accredited laboratory for the following tests : anti - gliadin iga , anti - gliadin igg , and anti - tissue transglutaminase iga . for this purpose ,
first , taking a small sample of blood ( 5 ml clot blood without anticoagulant ) from a vein in arm was necessary and then it was centrifuged for 10 min at around 25003000 .
finally , the laboratory examined the obtained serum samples from centrifugation in elisa method with binding site 's kits of the uk . a normal range of tests was defined according to the laboratory standards and finally was reported as positive / negative .
the statistical analyses of the results were performed using chi - square test ( for qualitative data ) and t - test ( to compare the means of two groups and for quantitative data ) in spss 16 for windows ( ibm corporation , ny , usa ) . in this study , the significance level was set at p < 0.05 .
the average age in the case and control group was 28.11 7.1 and 27.6 7.2 years , respectively ( p = 0.62 ) . nearly , 58.8% and 41.2% of the patients of the case and control groups were males , respectively ( p = 0.15 ) . regarding the type of aa , 88.6% of the patients had patchy aa , 8.6% and 2.8% of them had totalis and universalis aa , respectively .
concerning the nail involvement , 88.5% of the patients had no involvement , but 5.7% , 2.9% , and 2.9% of them had pitting , ridging , and onycholysis , respectively .
most of the participants in the case group did not have any positive familial history of aa ( 82.9% ) .
forty percent of the patients suffered from the disease for <3 months and 8.6% of the patients suffered from aa over 5 years [ table 1 ] .
frequency and duration of alopecia areata in the case group the serum of one aa patient ( 2.8% ) was positive for antibodies .
there was no significant difference between the two groups based on chi - square test ( p = 0.31 ) .
in the current study , the serum of one aa patient ( 2.8% ) was positive for antibodies .
the control group was seronegative for these antibodies . until now , the results of studies about the relationship between aa and cd are controversial .
several studies declare an association between aa and cd . in 1995 , one of the first research reports connecting aa with cd was published .
they reported a 14-year - old boy with universalis alopecia , completely regrew after he accepted a gluten - free diet .
this case report , and a few others , alerted the physicians to monitor a large group of patients with alopecia for cd .
in fact , they did determine a relatively high rate of cd in their patients , much greater than accidental .
on the basis of this analysis , they suggested that cd antibody testing should be carried out in all patients with aa .
a few case reports proposed an association between cd and aa . in a prospective trial of 256 aa patients , 6 had positive tissue transglutaminase and endomysial with positive biopsy .
in another study , volta et al . estimated that the prevalence of gliadin antibody in patients with aa was 1 in 116 , which was about 2.5 times more than the prevalence of this antibody in normal people .
estimated the prevalence of anti - gliadin antibodies in patients with aa to be about 18100 .
however , results of this study showed that in this population , there was no significant difference between the two groups in terms of celiac autoantibodies ( anti - gliadin iga , anti - gliadin igg , and anti - tissue transglutaminase iga ) , which could be due to different demographic compositions . in this study ,
the prevalence of antibodies had not been determined , but compared between the case and control groups . on the other hand , in this study ,
all these three antibodies had been checked at the same time , which seems to increase the accuracy of this study .
if the previous studies had checked these three autoantibodies at the same time , they could not find statistically significant positive results .
however , it seems that evaluating the prevalence of antibodies instead of frequency distribution of the antibodies was the advantage of other studies . in general
, aa may occur at any age , but the disease was more common in younger age and under 20 years . in seirafi 's study ,
it can be explained through the fact that children 's clinics were separated from adult 's clinics . in one study ,
more than 66% of the patients were younger than 30 years old and only 20% were over 40 years old .
aa affected both males and females . in one study , the affected men were more than women . in this study ,
in halaji 's study , the most frequent form of aa was patchy which was consonant with this study .
the prevalence of nail involvement in aa patients was estimated at a wide range of 7% to 66% in different studies .
, most patients ( 88.5% ) did not have nail involvement in the study group .
perhaps , the reason was that over 50% of our patients suffered from the disease less than a year , and still there had not been enough time for nail involvement .
reported that 72% of the patients had nail changes and the most common form was pitting , which was similar to this study .
, the frequency of positive familial history had been reported about 327% . in another study ,
the prevalence of positive familial history of aa was estimated to be about 1422% . in this study
, 17.1% of the cases had a positive familial history , which was in the range of other studies .
forty percent of the patients suffered from aa for <3 months , and 6.8% of the patients suffered from aa over 5 years . in some studies , the average duration of disease was 10.3 months with a range within 1 week to 8 years which was similar to this study .
unlike many studies , this study shows that the frequency distribution of celiac autoantibodies ) anti - gliadin iga , anti - gliadin igg , and anti - tissue transglutaminase iga ) in patients with aa does not differ from the normal population .
thus , we show here that the biological tests to search for cd do not bring information and proof enough , and it is why we recommend another approach to disclose gluten intolerance in aa patients .
| background : alopecia areata ( aa ) is a noncicatricial ( nonscarring ) alopecia .
the association between aa and celiac disease ( cd ) is debatable .
several studies declare the relationship between aa and cd as measurement of celiac autoantibodies ( anti - gliadin iga and anti - gliadin igg ) , but a few studies consider anti - tissue transglutaminase iga .
the aim of this study was to evaluate the frequency distribution of celiac autoantibodies ( all of them ) in patients with aa compared with controls.methods:this study is a case control study .
thirty - five patients entered in each group .
anti - gliadin iga , anti - gliadin igg , and anti - tissue transglutaminase iga were tested in all patients .
samples were examined in elisa method with binding site 's kits , and the result was reported as positive / negative .
finally , the frequency distribution of autoantibodies was examined.results:the age average did not show a significant difference between two groups ( p = 0.62 ) .
in addition , there was no significant difference between the two groups based on gender ( p = 0.15 ) .
the prevalence of antibody in case and control groups was 2.85% and 0% , respectively .
there was no significant difference between the two groups ( p = 0.31).conclusions : there may be a relationship between cd and aa , but the absence of statistical association between aa and cd does not mean that there is no relationship between gluten and aa in certain patients .
thus , we have shown here that the biological tests to search for cd do not bring information and proof enough , and it is why we recommend another approach to disclose gluten intolerance in aa patients . |
nanocontainers that
can bind and hold hydrophobic molecules have
attracted significant interest due to implications in areas such as
self - healing materials and drug delivery . among the factors
that are taken into account while arriving at a molecular design for
these containers , encapsulation stability is often considered a critical
one .
encapsulation itself is often defined by
the loading capacity of the host , i.e. , the amount of guest molecule
that a host assembly can hold .
this capacity is dictated by the thermodynamic
distribution coefficient of the guest molecule between the host and
the solvent .
a feature that has not received much attention involves
the guest - exchange dynamics .
this is important , as this is a direct
and arguably the most rigorous indicator of encapsulation stability
of a host assembly . considering the diversity
of the environments that a supramolecular assembly encounters in a
typical biological system ,
it is important that we understand the
influence of these external environmental factors upon encapsulation
stability . in this paper
, we show that the mechanism of guest - exchange
dynamics and thus the factors that affect encapsulation stability
can greatly vary with environmental changes .
three limiting
scenarios exist for the mechanism of guest exchange :
( i ) collision exchange separation mechanism , ( ii ) exit re - entry
mechanism , and ( iii ) fission recombination mechanism .
the first mechanism is collision - based , where the guest exchange
occurs only because of a collision between two host assemblies .
the second pathway is diffusion - based , where the guest - exchange rate
depends on the ability of guest to exit and enter from the host assemblies .
the third mechanism involves fragmentation of the host assembly into
two smaller entities as the first step , followed by a recombination
to regain the original host assembly ( figure s1 , supporting information ) . to simplify the possibilities , we
chose to use host assemblies that are based on cross - linked polymer
nanogels , which do not lend themselves to the third possibility . the
cross - linking feature of nanogels obviates the fission possibility ,
and
the two
viable pathways for the host assembly are illustrated in figure 1 . between these pathways , although the kinetic orders
of these mechanisms are different , it is difficult to quantify the
order of a molecular exchange process , as this is not a chemical reaction .
here , we design a series of experiments to probe the possible pathways
for guest exchange in these polymeric nanogels .
schematic representation
of two possible mechanisms for guest exchange
between cross - linked nanogels : ( a ) the collision exchange separation
mechanism , also called the collision - based mechanism , and ( b ) the
exit re - entry mechanism , also called the diffusion - based mechanism .
we use the recently reported fluorescence
resonance energy transfer
( fret ) as the tool to interrogate the guest - exchange process , which has proved to be quite a robust method
for a variety of host guest assemblies .
briefly , a hydrophobic fret pair , 3,3-dioctadecyloxacarbocyamine
perchlorate ( donor ) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine
perchlorate ( acceptor ) , were independently encapsulated in otherwise
similar nanogel hosts . when these solutions were mixed
, the emission
spectrum of the mixed solution in response to donor excitation at
450 nm was monitored over time .
no fret would be observed if the two
dye molecules are stably encapsulated and continue to be in their
separate nanocontainers , since the distance between the two dye molecules
is much higher than their frster radii . on the other hand ,
if the guest exchange between the host and its surroundings was significant ,
the extent of fret would evolve , as the guest composition in the nanogel
changes over time .
2,2-dithiodipyridine , 2-mercaptoethanol ,
poly(ethylene glycol ) monomethyl ether methacrylate ( pegma , mw 450 ) , 2-(diisopropylamino)ethyl methacrylate
( dpa ) , d , l - dithiothreitol ( dtt ) , 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine
perchlorate ( dii ) , 3,3-dioctadecyloxacarbocyanine perchlorate
( dio ) , 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid , and other
conventional reagents were obtained from commercial sources and were
used as received without further purification .
pyridyl disulfide
ethyl methacrylate ( pdsema ) was prepared using a previously reported
procedure .
the ph value was determined
via the accumet ab 15/15 + benchtop ph meter .
h nmr spectra
were recorded on a 400 mhz bruker nmr spectrometer using the residual
proton resonance of the solvent as the internal standard .
molecular weights
of the polymers were estimated by gel permeation chromatography ( gpc )
using pmma standard with a refractive index detector .
dynamic light
scattering ( dls ) measurements were performed using a malvern nanozetasizer .
uv visible absorption spectra were recorded on a varian ( model
el 01125047 ) spectrophotometer .
transmission electron microscopy
( tem ) images were taken using a jeol 2000fx at 200 kv .
random copolymer p2 , containing dpa groups , was synthesized according to a
previously reported procedure .
a mixture
of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid ( 12.0 mg , 0.043
mmol ) , pdsma ( 150.0 mg , 0.588 mmol ) , pegma ( 215.0 mg , 0.453 mmol ) ,
dpama ( 130.0 mg , 0.609 mmol ) , and aibn ( 1.5 mg , 0.010 mmol ) was dissolved
in thf ( 1.0 ml ) and degassed by performing three freeze pump thaw
cycles . the reaction mixture was sealed and then transferred into
a preheated oil bath at 65 c and stirred for 10 h. to remove
unreacted monomers , the resultant mixture was precipitated in cold
diethyl ether ( 20 ml ) and redispersed in thf three times to yield
the random copolymer p2 as a waxy solid .
h nmr ( 400 mhz , cdcl3 ) : 8.46 , 7.67 , 7.10 ,
4.354.09 , 3.943.37 , 3.02 , 2.62 , 2.041.64 ,
1.430.87 .
the molar ratio ( pds / peg / dpa ) was determined to
be 34%/28%/38% by integrating the methoxy protons in the polyethylene
glycol unit ( hd = 3.37 ppm ) , the aromatic protons
in the pyridine ( ha = 8.46 , hb = 7.67 , hc = 7.10 ) , and the methylene protons
next to the nitrogen in dpa units ( he = 2.62 , hf = 3.02 ) .
polymer ( 10
mg ) was dissolved in 1 ml of water , and the ph was adjusted to around
10 through the addition of naoh aqueous solution ( 1 m ) . then 0.04
ml of dii in acetone stock solution ( 5 mg / ml ) or 0.08 ml of dio in
acetone stock solution ( 2.5 mg / ml ) was added into the polymer solution
( 0.2 mg of each dye for 10 mg of polymer in 1 ml of water ) .
the mixed solution was stirred
overnight at room temperature , open to the atmosphere to allow the
organic solvent to evaporate .
then a measured amount of dtt ( 0.2 mol
for 15 mol % against pds groups ) was added . after stirring for 4 h ,
insoluble dii / dio was removed by filtration and the pyridothione byproduct
was removed from the nanogel solution by extensive dialysis using
a membrane with a molecular weight cutoff of 7000 g / mol . finally ,
nanogel stock solutions with a concentration of 1 mg / ml were prepared
for further studies .
nanogel containing
dii and nanogel containing dio were mixed in phosphate buffer solution
at ambient temperature and at a certain ph value such that the total
volume was 1 ml .
at the
same time , absorption spectra were also measured for all samples to
ensure that there is no loss of dye molecules due to precipitation .
the fret ratio ia/(ia + i d ) , where ia and i d are the fluorescence
intensities of the acceptor ( dii ) and the donor ( dio ) , respectively ,
was plotted against time to show the dynamics of the dye exchange
in nanogel solution .
the leakage coefficients ( ) were calculated
on the basis of the slopes from four earlier points in the linear
regime . in the case of very fast guest exchange ,
two cross - linked polymeric nanogels were used in this study . the
first nanogel ( ng - c )
is based on a random copolymer ( p1 ) , formed from a hydrophilic oligoethylene glycol ( oeg )
functionalized methacrylate monomer and a hydrophobic pyridyl disulfide
( pds ) functionalized methacrylate monomer ( scheme 1 ) .
the second nanogel ( ng ) and its precursor
( p2 ) contain a diisopropylamine ( dpa ) functionalized
methacrylate as a comonomer in addition to the oeg- and the pds - functionalized
monomers .
the size of these two types
of nanogels at neutral ph was found to be very similar ( figure 2 ) .
size distribution of nanogel at different ph values via dls measurements :
( a ) ng with 30% cross - linking density and ( b ) ng - c with 20% cross - linking density .
first , we were interested in understanding the mechanism
of guest
exchange in the control nanogel ng - c , which is not sensitive
to the external environment , as this provides the baseline for our
study . among the collision- and diffusion - based pathways ,
one would expect strong and positive correlations between the collision
frequency and the concentration of nanogels in solution .
we used the
fret - based method , which provided a measure of the dye exchange rate
in the form of the leakage coefficient ( ) , which can be obtained
as the initial slope of the linear fit ( earlier four points in the
linear regime ) by plotting the fret ratio ia/(ia + i d ) against time , where ia and i d are the fluorescence intensities of the acceptor
( dii ) and the donor ( dio ) respectively .
the concentrations of the
nanogels were varied as 0.05 , 0.10 , and 0.15 mg / ml for both ng - c containing dii and ng - c containing dio at
ph 7.1 .
figure 3c shows that there was indeed
a faster evolution of fret with time in the nanogels with a concentration
of 0.15 mg / ml , compared to those with a concentration of 0.05 mg / ml
( figure 3a ) and 0.10 mg / ml ( figure 3b ) .
values of 0.0192 , 0.0284 , and 0.0315
min were observed for the concentrations of 0.05 ,
0.1 , and 0.15 mg / ml , respectively , indicating that the guest exchange
between nanogels indeed increased with increasing concentration ( figure 3d ) .
these data provide the initial evidence that
the guest exchange in these control nanogels happens via the collision - based
mechanism as the dominant pathway .
fluorescence evolution when ng - c respectively containing
dii and dio are mixed in solutions at ph 7.1 : ( a ) 0.05 mg / ml , ( b )
0.10 mg / ml , and ( c ) 0.15 mg / ml .
( d ) comparison of the dynamics of
guest exchange between ng - c with different concentrations
at ph 7.1 . additionally , the size of the
nanogel would also impact guest exchange ,
if the collision - based mechanism was the dominant mechanism for guest
exchange , since the collision frequency would be affected by the size
of nanogels
. the ph - sensitive nature of the dpa moieties endows ng with ph - dependent size variations in this nanogel .
the
size of ng increases with decreasing ph , while the size
of ng - c does not change with ph ( figure 2 ) .
figure 2a shows that the sizes of ng increased from 10 to 18 nm when the ph changed from 8.0
to 5.1 . the observed size increase in ng
is attributed
to the protonation of the dpa units , which presumably results in the
swelling of the nanogels due to electrostatic repulsion between these
positively charged dpa groups inside . as the size of ng varies with ph
, we expected that the dye - exchange rate would also
change with the ph .
figure 4a shows that
there is a rapid evolution
of fret with time for ng at ph 8.0 . on the other hand , ng at ph 6.4 exhibits little change in fret ratio with time
and
thus , the exchange rate indeed decreased from a value
of 0.080 min at ph 8.0 to 0.004 min at ph 6.4 ( figure 4d ) .
these results are
consistent with the collision - based mechanism , as the size of ng indeed affected the rate of guest exchange , providing further
evidence that the dominant guest exchange in nanogels might happen
via the collision - based mechanism .
it should be noted , however , that
while the collision frequency is expected to decrease with increase
in size , because of the slower diffusion ( via the stokes einstein
equlation ) , it is also known that the collision frequency can increase
with an increase in size .
one possible
explanation is that the number of effective collisions , i.e. , collisions
that result in guest exchange , decrease with an increase in size .
this is reasonable , as the effect of the reduced diffusion could potentially
reduce the effective collisions .
this explanation is consistent with
the results but remains provisional , as there are no established quantitative
relationships to this effect at this point .
fluorescence evolution
when two ng solutions independently
containing dii and dio dye molecule as guest are mixed in solutions
at different ph values : ( a ) ph 8.0 , ( b ) ph 6.4 , and ( c ) ph 5.1 .
( d )
guest - exchange rates between ng at different ph values .
the concentration of each nanogel was 0.10 mg / ml . interestingly , however , an unexpected change in the fret
evolution
occurs below ph 6.4 , even though the size - increase is observed through
the entire ph range from 8.0 to 5.1 . from ph 6.4 to 5.1 , the fret
evolution was found to increase ( figure 4c ) .
figure 4d shows that the exchange rate systematically
increases with decreasing ph from 6.4 to 5.1 .
the value of
0.004 min at ph 6.4 increased back to 0.083 min at ph 5.1 . since the size of the nanogel increases
with decreasing ph , including at this ph range , these results are
not consistent with the collision - based mechanism
therefore , it is
possible that the diffusion - based pathway is the dominant operating
mechanism in this scenario , providing the first indication that there
might be an environment - dependent change in mechanism .
however , a
few alternating possibilities need to be considered prior to reaching
this conclusion .
first , it is possible that the dye molecules
themselves have a
certain ph - dependent solubility and thus is affecting the guest molecule
exchange in the collision state . to test this possibility , we investigated
the ph - dependence of guest - exchange dynamics in ng - c ,
where the size of the nanogel does not change with ph . figure 5a , b shows that there was indeed similar evolution
of fret with time in the nanogels at ph 8.0 and 7.1 , while a slightly
faster fret evolution was observed at ph 6.1 ( figure 5 ) .
we attribute the small ph - dependence to the possibility
that the solubility of dii and dio molecules may vary at different
ph values .
the solubility may be improved a bit at low ph due to the
presence of tertiary amine ( scheme 1 ) , leading
to easier dye exchange during the collision .
note that we do not observe
any significant dye loss in the nanogel during our fret experiments ,
which suggests that the repulsion between these positively charged
dyes and protonated ng at lower ph might be very small .
two features are noteworthy in these results : first , the magnitude
of difference in exchange dynamics is too small compared to those
observed with ng and , therefore , does not account for
the results observed with ng .
second , the guest - exchange
kinetics continues to increase with ph and there is no change in the
trend after ph 6.4 .
these results suggest that the results observed
in ng are not due to the inherent difference in dye molecule
behavior at different ph values .
comparison of the dynamics of guest exchange
between ng - c with 0.10 mg / ml at different ph values .
we hypothesized next that the
observed change in ph - dependence
trend at ph 6.4 could perhaps be explained by a change in the hydrophobicity
of the nanogel interior , i.e. , the host properties .
it is reasonable
to anticipate that the protonation of dpa groups would decrease the
hydrophobicity of the nanogel .
specifically , the ratio of the intensities between the first and
the third peaks ( i1/i3 ) in the pyrene emission spectrum can be used to determine
the polarity of the dye s microenvironment .
the value of this
ratio can range from 1.9 in polar solvents to 0.6 in certain hydrocarbon
solvents .
we utilized this dye as the guest molecule to probe the
hydrophobicity of the microenvironment within the nanogel interiors .
aqueous buffer solutions of ng containing 2 wt % pyrene
were prepared for this purpose at different ph values .
the hydrophobicity of nanogel at different ph values was studied by
calculating the intensity ratio of the first and third emission peaks
of pyrene , commonly referred to as the i1/i3 ratio .
figure 6b shows the variations in i1/i3 of pyrene encapsulated in ng at
different ph values .
when the ph was reduced from 8.0 to about 6.4 ,
the i1/i3 remained
constant around 1.25 .
however , when the ph was further decreased ,
a steady increase in the i1/i3 value was observed , suggesting that the microenvironment
of the dye is becoming increasingly polar . the i1/i3 ratio at ph 5.1 was found
to be about 1.37 . on the contrary ,
pyrene in the control nanogel ng - c ( that lacks the dpa units ) did not exhibit any discernible
change in the i1/i3 ratio over the same ph range of 5.18.0 , suggesting
that the hydrophobicity of these nanogels was not ph - dependent .
( a ) fluorescence emission spectrum
measured for nanogel ng - c loading 2 wt % pyrene and ( b )
calculated i1/i3 ratios for nanogels at different ph values ( black , ng ; red , ng - c ) . while the environment inside ng was quite hydrophobic
between ph 8.1 and 6.4 , it became less hydrophobic when the ph value
decreased from 6.4 to 5.1 , which indicates that the ability of ng at these ph values to encapsulate the dii or dio molecules
could be greatly depleted .
our studies with the control nanogel ng - c also suggest that the solubility of these dye molecules
can be slightly improved at lower ph .
therefore , the dye might be
able to exit one nanogel and diffuse through the solvent to re - enter
another ; i.e. , the diffusion - based mechanism is possible in this case .
thus , these results suggest that the guest exchange is primarily based
on the collision - based mechanism from ph 8.0 to 6.4 , but changes to
a diffusion - based mechanism below ph 6.4 .
comparison of the dynamics
of guest exchange between ng of different concentrations
( 0.05 , 0.10 , and 0.15 mg / ml ) at ( a )
ph 8.0 , ( b ) ph 7.1 , ( c ) ph 6.1 , and ( d ) ph 5.1 . to further test this possibility , we investigated the effect
of
nanogel concentration on the guest - exchange rate at different ph values
in ng .
we varied the concentrations of ng samples ( 0.05 , 0.1 , and 0.15 mg / ml ) and studied their fret evolution
at ph 5.1 , 6.1 , 7.1 , and 8.0 .
figure 7a , b shows
that the exchange rates increase with nanogel concentration at both
ph 7.1 and 8.0 .
this suggests that the guest exchange at these ph
values indeed occurred mostly via the collision - based mechanism .
however ,
while there is no clear trend at ph 6.1 ( figure 7c ) , the guest - exchange rate became slower with increasing concentration
at ph 5.1 , as values of 0.098 , 0.083 , and 0.046 min were observed for nanogel concentrations of 0.05 , 0.1 , and 0.15
mg / ml , respectively ( figure 7d ) . note that
in a diffusion - based mechanism , the diffusion rate of dye out from
the nanogel should depend on the concentration of nanogels in solution .
at higher concentrations ,
the concentration of the host nanogel is
higher , while the concentration of the dye is very low at all these
concentrations . in this case , the driving force for the guest molecules
to diffuse into the solvent from the host nanogel is lower at higher
host concentrations .
therefore , the observed decrease in guest - exchange
rate with the increasing nanogel concentration supports the assertion
that a diffusion - based mechanism is dominant at ph 5.1 .
if our
hypotheses were correct , it should also follow that there
should not be any change in the mechanism of guest exchange in ng - c , as the hydrophobicity of its interior does not change
with ph . to perform this control experiment
, we investigated the ph - dependent
trends at different concentrations of ng - c .
the concentration
dependence of guest exchange was similar for this nanogel at all ph
values ( figure s6s8 , supporting information ) .
indeed , the fret evolution increased with concentration at all
four ph values ( figure s9 , supporting information ) , indicating that the guest - exchange rate indeed increased with
the increasing concentration .
this suggests that the operating mechanism
is collision - based and is not dependent on ph in the ph - insensitive
nanogel ng - c .
the results from the control nanogel are
consistent with our conclusions for the ph - sensitive nanogels ng .
in summary , we have designed a series of
experiments to study the
guest - exchange mechanism in ph - sensitive nanogels along with ph - insensitive
nanogels as a control .
we have shown that the dominant mechanism for
guest exchange in these ph - insensitive nanogel hosts is collision - based .
perhaps , the most important take - home message of this work is that
the mechanism of guest exchange in the ph - sensitive host guest
assemblies can change on the basis of the microenvironment of the
host . when the nanogel interior is hydrophobic , the collision - based
mechanism is the dominant pathway .
however , when the interior is sufficiently
hydrophilic , the dominant mechanism changes to a diffusion - based one .
from an even broader perspective
, it is important to recognize that
both the intrinsic factors , such as size and interior environment
of the host assemblies , and the extrinsic factors , such as ph and
concentration , can have significant impact on encapsulation stability .
from the application standpoint , these findings could have implications
in many areas .
for example , in drug delivery applications , the drug - loaded
nanocarrier experiences both concentration and environmental changes
upon biodistribution to diseased tissues .
the mechanistic variations
in two different environments provide insights into molecular designs
that can afford stable encapsulation in one environment and release
of the molecules in another .
similarly , the results
from this study may also provide new opportunities for designing nanoreactors in which catalysts encapsulated in a host can be used to reversibly
turn a chemical reaction on or off , because of the environment - dependent
diffusion of reactant molecules . | dynamic exchange of guest molecules ,
encapsulated in host assemblies ,
is a phenomenon in supramolecular chemistry that has important implications
in several applications .
while the mechanism of exchange in micellar
assemblies has been previously investigated , the effect of host and
guest environment upon the guest - exchange dynamics has received little
attention , if any . in this paper , we study the guest - exchange mechanism
in ph - sensitive nanogels along with ph - insensitive nanogels as a control .
by systematically comparing the behavior of these nanogels ,
we show
that size , concentration , and hydrophobicity can all play a critical
role in guest - exchange dynamics .
more importantly , these studies reveal
that the dominant mechanism of guest exchange can intimately depend
on environmental factors . |
the
occurrence of primary sex cord - stromal tumors at extraovarian sites is
extremely rare .
these tumors are predominantly granulosa cell tumors .
to our knowledge , this is the first case of primary mesenteric sertoli - leydig
cell tumor .
a new case of a
78-year - old woman who
presented with bowel
obstruction is reported .
three years previously , she underwent bilateral salpingo - oophorectomy and total
abdominal hysterectomy for bilateral mucinous cystadenoma and vaginal prolapse .
ultrasonography showed a solid mesenteric tumor ; exploratory laparotomy showed
a nodular solid tumor at the mesenteric border of the distal ileum , measuring
11 8 cm .
macroscopically , the tumor was nodular and well circumscribed measuring 11 8 4 cm .
the cut surface was composed of pale yellow - grey soft tissue with foci of haemorrhage .
microscopic examination revealed cellular lobules with nests and poorly
developed tubules of sertoli cells ( figure 1 ) that showed moderate atypia and
mitotic figures that average 510 per high - power field ( figure 2 ) .
leydig cells are found at the periphery of the cellular
lobules ( figure 3 ) . by immunohistochemistry
, the neoplastic cells showed
positive staining for antibodies against inhibin ( figure 4 ) and vimentin .
tumor
cells were negative for ema ( epithelial membrane antigen ) , calretinin ,
synaptophysin , chromogranin a , and cd117 ( c - kit ) .
the diagnosis of
sertoli - leydig cell tumor with intermediate differentiation of the mesentery was
established .
the patient has not received any additional therapy , yet she
remains free of disease after five years .
the occurrence
of primary sex cord - stromal tumor at extraovarian sites is extremely rare ,
such that in the english literature only 13 cases have been reported [ 113 ] .
eight were classified as granulosa cell tumors [ 16 , 11 , 12 ] , two as thecomas
[ 7 , 8 ] , two as sex cord - stromal tumors , and one as an unclassified form of
stromal sex cord tumor . the sites of origin were usually within the pelvis
: six arose in the broad ligament , two in the retroperitoneum [ 2 , 12 , 13 ] ,
one in the fallopian tube , one in an umbilical herniae sac , one in the
adrenal gland , and one in the pelvic sidewall .
these previous reported
cases are summarized in table 1 . to our
knowledge , this is the first case of a primary mesenteric sertoli - leydig cell
tumor .
however , in
our case , no biologic assay for estrogen content of the patient 's urine or
blood was performed , because the nature of the tumor was not suspected until it
was removed .
the histogenesis
of extraovarian sex cord - stromal tumors has been reviewed in the literature . in recent
years
, several investigators have claimed that the sex cords may originate from
the mesonephros .
a dualistic theory of both coelomic epithelium and mesonephros
in the origin of the pregranulosa cells has also been proposed .
accordingly , the mesonephros itself or its functional influence seems to be
necessary for creating the sex cords .
this is consistent with gonad formation
being limited to the gonadal ridge and may explain why the sites of origin of
extraovarian sex cord - stromal tumors are limited to the broad ligament ,
retroperitoneum , mesentery , and adrenal gland , all of which differentiate close
to the mesonephros and mesonephric duct .
sex cord - stromal tumors can be
difficult to distinguish from several other neoplasms including
undifferentiated carcinoma , gastrointestinal stromal tumors ( gist ) or
metastatic melanoma .
the prognosis
for extraovarian sex cord - stromal tumors seems to be favorable ; however , reported
cases and clinical experiences are limited . | the occurrence of primary sex cord - stromal tumors at extraovarian sites is exceedingly rare .
we report a new case of sertoli - leydig cell tumor in the mesentery of a 78-year - old woman who presented with occlusive syndrome and reviewed the previously reported cases of extraovarian sex cord - stromal tumors in the english literature . |
approximately 30,000 new cases of cancer in the oral cavity and oropharynx are diagnosed in the united states each year , corresponding to about 3% of all malignant tumors .
although visibly detectable due to the accessibility of the oral cavity , oral cancer has a high morbidity and mortality because it is typically at an advanced stage when it is finally clinically visible .
accordingly , the five - year survival rate for all stages combined is only 51% . of the cancers involving the oral cavity and oropharynx ,
more than 90% are squamous cell carcinomas [ 13 ] . up to 40% of those diagnosed with squamous cell carcinoma
will develop a metastatic tumor or a second primary tumor to a nearby organ at a later time .
early diagnosis of oral and oropharyngeal squamous cell carcinoma is crucial for a more favorable prognosis .
the oral cavity and oropharynx are easily accessible for routine screening for squamous cell carcinoma , but reliable detection in early stages will require robust markers that are easy to assay .
methods for testing oral epithelial cells for malignancy traditionally involve an invasive and often painful biopsy . whether as a diagnostic tool or prognostic marker , the potential to develop a simple , less invasive method for oral cancer screening would be of great value .
human calmodulin - like protein calml3 ( synonyms : clp , calmodulin - related protein nb-1 ) is a 148-amino - acid - residue calcium sensor protein closely related to the ubiquitous calmodulin [ 4 , 5 ]
. however , in contrast to calmodulin , calml3 is tissue specific and seems to be expressed almost exclusively in normally differentiating epithelia such as those of breast , thyroid , prostate , kidney , and skin . in a study comparing normal reduction mammoplasty specimens to archival breast cancer specimens , rogers et al
. found that calml3 was expressed at high levels in normal breast epithelium and that significant calml3 downregulation occurred in 79% to 88% of invasive ductal carcinoma and lobular carcinoma specimens .
the authors concluded that calml3 downregulation is common in breast tumorigenesis and that the status of calml3 expression can serve as a marker for the nonmalignant state .
we have previously found that calml3 is expressed at the mrna transcript level and can be detected by immunohistochemistry in normal oral mucosa tissue .
by contrast , there was a notable reduction in immuno staining in areas of malignant transformation .
here we performed immunohistochemical analysis to compare calml3 expression in tissue samples representing the various phases of oral tumor progression to see if there is a statistically significant trend of changes in calml3 expression during disease progression .
in addition , direct comparisons in calml3 expression were made between the individual categories of benign , dysplasia , carcinoma in situ , and invasive squamous cell carcinoma .
under irb - approved protocol 754 - 04 , the mayo clinic tissue registry was searched for patients that had undergone surgical biopsy of the oral cavity .
a database was compiled placing the tissue specimens into 4 categories : benign squamous mucosa ( including mild reactivity and hyperplasia ) , squamous dysplasia , squamous cell carcinoma in - situ ( cis ) , and invasive squamous cell carcinoma .
stained slides for the surgical procedures were evaluated and the most appropriate tissue block for each case was processed for calml3 immunostaining . a staff surgical pathologist ( thomas j. sebo ) confirmed the diagnosis of each sample .
samples were eliminated if there were any questionable diagnoses or if the quality of the section was poor .
a total of 90 tissue specimens were derived from 52 patients with 62 surgical procedures . for the purpose of this study
specimens from the same patient were generally from different lesion sites taken during more than one date of surgery . among the 52 patients , 31 ( 60% ) were male and the overall mean age at the time of diagnosis was 64 years ( sd , 15 ; median , 67 ; range , 6 months87 years ) . among the 90 tissue specimens , 30 ( 33% ) were diagnosed as benign , 6 ( 7% ) as dysplastic , 12 ( 13% ) as carcinoma in situ , and 42 ( 47% ) as invasive squamous cell carcinoma .
of the 42 samples diagnosed as squamous cell carcinoma , 2 ( 5% ) were grade 1 , 11 ( 26% ) were grade 2 , 27 ( 64% ) were grade 3 , and 2 ( 5% ) were grade 4 .
other data recorded included location of specimen ( tongue , floor of mouth , right , left , etc . ) and type of surgery ( excision versus biopsy ) .
the generation , affinity purification , and characterization of a rabbit polyclonal antibody ( tg7 ) against human calml3 have been described .
reamstown ) recognize a peptide corresponding to the c - terminal residues 127 to 148 of calml3 ( the most divergent region between calml3 and calmodulin ) .
affinity - purified antibodies showed excellent sensitivity and specificity for calml3 [ 5 , 7 ] .
the paraffin - embedded tissue specimens were cut at a thickness of 6 m , mounted on positively charged slides , deparaffinized , and treated with h2o2/methanol to block endogenous peroxidase activity essentially as described [ 10 , 11 ] .
the specimens received heat - induced epitope retrieval in 1 mm edta at a ph of 8.0 for 20 minutes .
nonspecific protein - binding sites were blocked in 5% normal goat serum in phosphate - buffered saline solution ( pbs)/0.05% tween-20 for 1 h , and the sections were immunostained by sequential incubations ( 1 - 2 h at room temperature each ) in affinity - purified calml3 antibody tg7 ( 40 g / ml in pbs/0.05% tween-20/1% normal goat serum ) , biotinylated goat anti - rabbit igg ( 1 : 200 in pbs/0.05% tween-20/1% normal goat serum , dako , carpinteria , ca ) , and horseradish peroxidase - conjugated streptavidin ( 1 : 300 , dako , carpinteria ) essentially as described .
the sections were then incubated in 3-amino-9-ethylcarbazole in the presence of h2o2 and counterstained with hematoxylin and mounted with coverslips .
two observers ( a staff surgical pathologist ( thomas j. sebo ) and a maxillofacial prosthodontist ( michael d. brooks ) ) evaluated the specimens for expression of calml3 .
staining patterns and intensity were observed and recorded for all categories of samples ( benign , dysplasia / carcinoma in - situ , and invasive squamous cell carcinoma ) .
35 separate fields of view were analyzed in 2 mucosal cell layers : superficial cells and basal cells .
analysis of staining in cellular compartments was further subdivided into cytoplasmic membrane ( cm ) , cytoplasm ( c ) , and nucleus ( n ) . staining
was graded on a 4-point scale : 0 : absent ( no staining above background of a negative control incubated with anti - calml3 igg preabsorbed with calml3 ) , 1 : weak ( barely above background and no strong staining in specific cellular regions ) , 2 : intermediate ( moderate staining with varying degree of subcellular staining ) , 3 : strong ( intense staining with distinct cellular localization ) .
the cytoplasmic membrane , cytoplasm , and nuclei for each mucosal cell region were individually assigned a grade . assigning a grade to
first , the squamous cells were initially evaluated using two low magnification lenses ( 2.5x and 5.0x for a total magnification strength of 25x and 50x ) to obtain a general sense of staining intensity of all the squamous cells on the slide .
second , for cytoplasmic membrane staining , cells either exhibited circumferential membrane staining or no membrane staining .
third , membrane staining , apart from cytoplasmic staining , was typically most readily apparent in the intermediate layer of the squamous mucosal cells .
as the cells reach the actual superficial layer of , for example , benign mucosa and become flattened , the ability to distinguish cytoplasmic staining from cytoplasmic membrane staining was limited .
invariably , tissue samples in which no calml3 staining in the squamous cells was detected could very quickly be assigned a grade of 0 ( no staining ) . in the situation of superficial squamous cell staining ,
conversely , strong ( 3 + ) calml3 immunostaining could be readily ascertained using the same approach in 5%30% of the case material . in evaluating basally positioned squamous cells
, no staining could be detected very quickly in 50% to almost 100% of the case material , whereas strong ( 3 + ) staining was very rare ( about 2% ) .
the challenge in this study , and all studies in which immunostain intensity is visually graded , came in grading squamous cells as displaying either weak ( 1 + ) or intermediate ( 2 + ) calml3 expression . in these instances , an overall assessment of staining intensity at low magnification showed that the degree of staining could not clearly be categorized as 0 or 3 + , and a close analysis of all squamous cells in the tissue sample was performed using all magnification lenses ( 2.5x , 5x , 10x , 20x , and 40x for overall magnification of 25x , 50x , 100x , 200x , and 400x ) to arrive at a grade reflecting the staining intensity for all squamous cells .
thus , a grade of 1 + ( weak ) indicates that the overall expression of the squamous cells was weak , showing only barely visible staining above 0 . for an intermediate grade ( 2 + ) , the degree of calml3 staining was felt to be unquestionably present but not as visually strong as a grade of 3 + . on occasion , rare cells within any given tissue specimen graded as 1 + or 2 + showed strong ( 3 + ) or no ( 0 ) expression .
however , no effort was made to further stratify case material on the basis of the percentage of cells showing a range of calml3 expressions .
as such , our grading system reflects an overall score of calml3 staining intensity . in those instances in which the two reviewers ( thomas j. sebo and michael d. brooks ) recorded different grades for a given case , rereview of the case material was undertaken to arrive at a consensus grade .
this occurred in roughly 10% of the case material . for analysis purposes , grades 0 and 1 were collapsed and reported together as no / weak staining and grades 2 and 3 were collapsed and reported together as intermediate / strong stain .
this is because visually weak staining most closely approximates no ( 0 ) staining and intermediate ( 2 + ) staining most closely approximates strong ( 3 + ) staining .
total stain scores were derived by summing the stain grades for the three cellular compartments . the cochran - armitage test for trend
was used to evaluate whether the proportion of specimens with intermediate / strong expression significantly changed with increasing disease severity .
in addition , the chi - square test ( or the fisher 's exact test , as appropriate ) was used to compare the proportion of specimens with intermediate / strong expression between the benign , dysplasia / cis , and invasive tissue groups .
the kruskal - wallis test was used to compare the total stain scores between the three groups .
if the p value for the overall test of group differences was < 0.05 , then pair - wise comparisons among the three groups were performed .
all calculated p values were two - sided and p values less than 0.05 were considered statistically significant .
a bonferroni correction could be applied by assessing the pair - wise comparisons between the three tissue types using an alpha level of 0.0167 to account for the three comparisons .
statistical analysis was performed using the sas software package ( sas institute , cary , nc ) .
as we expected from previous studies [ 6 , 8 ] , benign mucosa displayed robust immunostaining ( figures 1(a ) and 1(b ) ) .
distinct staining was noted in the periphery ( cytoplasmic membrane ) and nuclei of the more superficially located epithelial cells ( figure 1(b ) ) . on the other hand , in samples with dysplasia
( figures 1(c ) and 1(d ) ) and carcinoma in - situ ( not shown ) , a marked decrease was seen in strength of stain . a strong reduction ( or complete lack ) of calml3 immunostaining
was seen in invasive squamous cell carcinoma compared to both benign tissue and dysplasia / cis tissue ( figures 2 and 3 ) , although in low - grade invasive squamous cell carcinoma , keratin pearls did illustrate mild immunoreactivity .
the basal cell layers in general did not stain as intensely as the more superficial cell layers , and no statistical difference was seen with basal nuclear and cytoplasmic staining among the groups .
table 1 summarizes the calml3 expression results at the superficial and basal mucosa levels , separately for the tissue types .
because similar results were found for the dysplastic and cis specimens , these two tissue types were combined for the analysis . in addition , we found that aside from the superficial cytoplasmic membranes and nuclei of the benign specimens , only a small percentage of the other tissue regions exhibited strong expression .
therefore to evaluate whether there was a trend in the expression level with tissue disease severity , the specimens with weak or no staining were combined , as were those with intermediate or strong staining ( see table 1 ) .
however , the detailed summary of all staining results broken down by individual tissue type is available upon request .
based on the cochran - armitage test for trend , the calml3 expression level significantly decreased with increasing disease severity for the following sites : superficial cytoplasmic membrane ( p < 0.001 ) , superficial nucleus ( p
< 0.001 ) , and basal cytoplasmic membrane ( p = 0.009 ) . on the other hand ,
a statistically significant trend was not observed for superficial cytoplasm ( p = 0.19 ) and basal cytoplasm ( p = 0.19 ) , in which several dysplastic / cis specimens had stronger expression than the benign tissue .
none of the basal nucleus sites had a stronger than weak staining score for any of the tissue types . the chi - square test ( or the fisher 's exact test , where appropriate ) was used to compare the proportion of specimens with an intermediate or strong stain between the benign , dysplasia / cis , and invasive tissue groups ( table 1 ) .
benign tissue specimens had significantly more calml3 expression in the superficial mucosa compared to invasive specimens ( p < 0.001 ) and dysplasia / cis specimens ( p = 0.013 ) , as revealed by a comparison of the total stain scores ( table 1 and figure 4 ) .
this difference in calml3 staining was most pronounced for the cytoplasmic membrane and the nucleus .
dysplasia / cis tissue also had significantly ( p = 0.003 ) more expression than invasive tissue in the superficial mucosa ( table 1 and figure 4 ) , and this difference was most clearly demonstrated in the cytoplasm ( 50% versus 7.1% , p
< 0.001 ) and in the nuclei ( 38.9% versus 2.4% , p < 0.001 ) .
all of the previously outlined comparisons of expression in the superficial layer between the three tissue groups retained statistical significance if a bonferroni correction was applied and significance is assessed using an alpha level of 0.0167 instead of 0.05 .
expression was not strong in any of the sites in the basal layer ( table 1 ) , though there was evidence suggesting a slight increase in expression in the cytoplasmic membrane of benign squamous cells compared to invasive carcinoma ( 13.3% versus 0% , p = 0.027 ) . in analyzing the 42 specimens of squamous cell carcinoma separately by grade ,
previously , loss of calml3 immunoreactivity has been linked to early breast cancer development . here
, we show that while calml3 is strongly expressed in the superficial layers of benign oral mucosa , different oral mucosa tissue types representing the various stages of carcinogenesis exhibit a reduction of calml3 expression as squamous cells progress from benign , to dysplastic , to carcinoma in situ , to invasive squamous cell carcinoma . in general , a trend is seen that as disease severity increases , calml3 expression decreases .
many of the benign specimens showed an increase in staining intensity the further the cells moved away from the basal cell layer .
as the cells mature , they differentiate and migrate to the upper layers as underlying cells develop .
the more intense calml3 staining towards the outer layers of mucosa is consistent with previous reports that noted markedly increased staining in more differentiated layers of stratified epithelia .
therefore , calml3 may play a role in terminal differentiation of oral keratinocytes , as has been proposed for the basal and suprabasal keratinocytes of the epidermis during wound healing .
calml3 is a calcium - sensor protein related to calmodulin and as such is thought to exert its function by regulating specific target proteins . among these ,
the unconventional myosin - x is known to be involved in directional cell migration and cell adhesion , both are important events during terminal keratinocyte differentiation . the staining for calml3 in the cytoplasmic membrane of superficial cells in benign oral mucosa may thus reflect its function in regulating myox concentrated at the cell periphery . like calmodulin , calml3 has multiple targets , one of which ( iq motif containing protein e or iqce ) was recently identified in a yeast two - hybrid screen and may be a protein involved in dna metabolism ( richard d. bennett and emanuel e. strehler , unpublished ) .
such a putative nuclear calml3 target protein(s ) could help explain the nuclear staining for calml3 that we observed in the superficial cells of the normal oral mucosa .
however , additional studies are needed to determine the role of calml3 in the nucleus and its relevance to normal epithelial differentiation
techniques being implemented for diagnostic tests include transepithelial brush biopsy / exfoliative cytology , fine needle aspiration , and toluidine blue [ 1523 ] .
these techniques all rely on cytological markers to indicate the presence of malignant or premalignant cells .
although these tests are preferred over conventional biopsy confirmation because they are less invasive and more tolerable for the patient , their reliability and efficacy have not been validated [ 2426 ] . because the specific expression and cellular localization of calml3 indicate normal cell function , the potential for development of a simple diagnostic test for oral cancer using calml3 as a marker is promising .
currently diagnostic screening tools are being utilized to visualize autofluorescence in normal oral tissue with loss of autofluorescence indicative of tissue change or abnormality .
noninvasive screening could be implemented by the use of exfoliative brushing cytology , minibiopsy , or a swish - and - spit procedure .
this would allow a clinician who observes a suspicious area in the mouth to routinely test for calml3 levels .
a change in calml3 expression could indicate the presence or the early developing stage of oral cancer . | oral cancer is often diagnosed only at advanced stages due to a lack of reliable disease markers .
the purpose of this study was to determine if the epithelial - specific human calmodulin - like protein ( calml3 ) could be used as marker for the various phases of oral tumor progression .
immunohistochemical analysis using an affinity - purified calml3 antibody was performed on biopsy - confirmed oral tissue samples representing these phases .
a total of 90 tissue specimens were derived from 52 patients .
each specimen was analyzed in the superficial and basal mucosal cell layers for overall staining and staining of cellular subcompartments .
calml3 was strongly expressed in benign oral mucosal cells with downregulation of expression as squamous cells progress to invasive carcinoma .
based on the cochran - armitage test for trend , expression in the nucleus and at the cytoplasmic membrane significantly decreased with increasing disease severity .
chi - square test showed that benign tissue specimens had significantly more expression compared to dysplasia / cis and invasive specimens .
dysplasia / cis tissue had significantly more expression than invasive tissue .
we conclude that calml3 is expressed in benign oral mucosal cells with a statistically significant trend in downregulation as tumorigenesis occurs .
calml3 may thus be a sensitive new marker for oral cancer screening . |
the stress arising from first child birth is classified even as severe stress among the psychosocial stresses . according to the global statistics , 1015% of pregnant women somehow experience different degrees of anxiety disorders and depression .
antenatal anxiety and depression are so prevalent and serious problems in pregnancy that more than half of the pregnant women have anxiety antenatally and results of studies show that there is an association between mental health of women during the prenatal period and the outcomes of pregnancy the above percentage depends on the gestational age , as in the second trimester and third trimester of pregnancy , the incidence was reported as 1215% and 14% , respectively .
psychological pressures during pregnancy , delivery , and breastfeeding may lead to adverse outcomes including abortion , vomiting , diarrhea , preeclampsia , weight loss , premature delivery , immune suppression , and subsequently , increase in episiotomy and neonatal infections and also degrees of postpartum psychological disorders .
a significant correlation has been also reported between reduced quality of life ( qol ) and the above factors such as depression or general health problems .
it is a new method that probably constitutes the core of treatments for anxiety and mental stress and a disciplinary method that is internalized with practice .
relaxation techniques that include muscle relaxation and breathing exercises are among the physiological mechanisms that link the mind and the body , and as a non - medicinal method , they have positive effects on stress management in physical , mental , and social dimensions of general health and also on anxiety .
this technique is widely used in research , and reduces stress , and learning to sense and control muscle tension constitutes its basis of treatment .
different studies have examined the effects of the method on various groups ; however , only a few studies have examined those effects on pregnant women who are among the vulnerable groups in the society . in urech
's study on the effects of relaxation on mental health during pregnancy , the results showed useful effects of relaxation methods on physical and psychological system . during pregnancy ,
many studies on the psychological effectiveness of progressive muscle relaxation also found similar results . in this regard ,
a quasi - experimental study with the objective of examining the effect of progressive muscle relaxation on anxiety of nursing students showed a significant difference between anxiety mean scores of the experimental and control groups after muscle relaxation training , as the anxiety in the experimental group was reduced .
furthermore , results of the studies on the effects of progressive muscle relaxation in women with bronchial asthma showed that progressive muscle relaxation was an effective method for improvement of blood pressure , pulmonary parameters , and heart rate , and reduction of state
trait anger ; consequently , the qol related to the health of pregnant women with bronchial asthma was improved
. results of many studies have shown the effectiveness of progressive muscle relaxation on many illnesses such as reduction of pain in multiple sclerosis patients , anxiety in pulmonary disease , and anxiety and depression , nausea , and vomiting in cancer patients .
also , some studies reported that progressive muscle relaxation training could improve the qol and decrease pain in pregnant women with low back pain and reduce stress in pregnant women . but
no study has reported exactly about the effects of progressive muscle relaxation on prenatal mental health .
community - based programs that promote healthy coping can optimize maternal and newborn health . borcherding ( 2009 )
quoted lowenkron as saying that nurses and the other health professionals have many opportunities to impact coping styles during the prenatal period .
a stronger effort could be made to assess coping during the prenatal period and incorporate healthy ways of coping into the plan of care .
results of many studies have shown that severe stress during pregnancy can affect the pregnancy outcome .
considering the mutual relationship between body and psyche , excessive vulnerability of pregnant women , the direct impact of mental stress on the mother 's and fetus 's health , the emphasis on the effectiveness of social support and care for women during pregnancy on mental health after child birth , and lack of similar studies in this regard , the present study was conducted to determine the effect of progressive muscle relaxation as a non - pharmacological method on pregnant women 's general health .
sixty primigravida women meeting the inclusion criteria of the study , who referred to the prenatal clinic of a selected hospital in tehran , iran , constituted the population of this clinical trial .
they were in the first trimester of pregnancy ( up to 12 weeks ) and in the age range of 1835 years .
the studied samples were selected with the following inclusion criteria : primigravida in the first trimester , single pregnancy , no high - risk pregnancy such as history of diabetes , hypertension , mental disorders , and use of antidepressants . then , the participants were randomly divided into control ( n = 30 ) and experimental ( n = 30 ) groups . at first , the participants level of general health was measured with general health questionnaire-28 ( ghq-28 ) ; next , the experimental group subjects were given training in progressive muscle relaxation technique ( pmrt ) , which has been in use since its introduction in 1938 by edmond jacobson .
the training for pmrt was administered as three sessions conducted theoretically and practically every other day in a week , with duration of 1.52 h in each session . in the sessions
, the pregnant women were familiarized with the concept of relaxation technique and the role and importance of performing the relaxation technique ; following this , and the research units were requested to practice the technique step by step .
after teaching and making sure that the participants have learnt the practical training , the training cd was prepared and the participants were provided with the contents of the above - mentioned method , so that they listen to it during practicing at home .
the participants performed the practices for 8 weeks , at least twice a day at home , and recorded the results of the practice in a daily report checklist .
meanwhile , during these 8 weeks , the researcher was in contact with the participants by telephone to answer their questions and follow them to ensure that they were doing the practice . in this study
, we had 18 cases as sample attrition based on exclusion criteria included : dissatisfaction of the pregnant women to continue participating in the study , being absent for more than one session of the training class , failure to do the practices regularly during 8 weeks , and failure to complete the checklist . during this period ,
finally , 8 weeks after intervention , general health of both groups was measured using ghq-28 . in this study ,
total score of each participant is the sum of scores of the four subscales , which ranges from 0 to 84 . and
this questionnaire has been used as a standard instrument in numerous studies and its scientific reliability and validity have been measured .
data were entered into spss-15 software . to interpret and analyze the data , descriptive statistics of mean and standard deviation , and inferential statistics of paired and independent t - tests and chi - square test were used ( p < 0.05 ) . for the research ethics ,
all participants signed the informed written consent forms after receiving sufficient information about the research and its objectives , and those who did not want to continue taking part in the study were excluded .
the pregnant women were informed about the aims of the study and also about being free to withdraw from the study at any time .
also , in this study , the control group merely benefited from routine education and care . for ethical considerations , by the end of the study , necessary training for relaxation as well as educational notes
were provided , and the training cd was also given to the control group after the test .
regarding demographic information , the results showed that most of the study subjects had educational degree of high school diploma or higher and were housewives .
also , they had been regularly referring to the prenatal clinic for getting routine prenatal care and there was no significant difference between the characteristics of the control and the experimental groups [ table 1 ] .
distribution of control and experimental groups based on demographic information before the intervention , total score of general health of the experimental group and the control group was 35.83 ( 6.92 ) and 29.46 ( 8.3 ) , respectively , and after the intervention , it was 20.2 ( 5.61 ) and 27.85 ( 8.24 ) , respectively .
furthermore , paired t - test showed a significant difference on comparing the mean scores of women 's general health before and after intervention in the experimental group ( p < 0.001 ) [ table 2 ] , but it showed no significant difference in the control group [ table 3 ] .
comparison of mean scores before and after the intervention in the experimental group comparison of mean scores before and after the intervention in the control group comparison of the mean difference in the four subscales of physical symptoms , anxiety and insomnia , social functioning disorder , and depression by paired t - test showed a significant difference ( p < 0.001 ) before and after intervention in the experimental group , but in the control group , this difference was not significant . also , the mean difference of general health in the experimental group before and after intervention was obtained as 15.63 ( 5.73 ) , whereas it was 4.77 ( 1.96 ) in the control group , and paired t - test showed a significant difference on comparing this mean difference ( p < 0.001 ) [ table 4 ] .
comparison of the mean difference scores of general health in the control and experimental groups before and after intervention
the main objective of this study was to determine the effect of progressive muscle relaxation on the general health of pregnant women referring to the prenatal clinic of the selected hospital in tehran .
the results of the present study on the effects of relaxation training on different aspects of pregnant women 's general health , including anxiety and insomnia , showed a significant difference between the general health scores of pregnant women before and after intervention .
pregnancy , as an important event in the life of a woman and her family , causes major changes in the woman physically ( physiologic and anatomic ) and psychologically .
however , studies on the psychological aspect of the changes are lesser than those on the physical aspect of changes . due to the abundant different stresses during pregnancy and the physical and mental adverse effects of stress on pregnant women and their fetuses , learning the methods
although there is no similar study , the above result agreed with those of related studies , including marc et al . 's study which examined the effects of relaxation on mental health during pregnancy and showed useful effects of relaxation on physical and psychological systems .
other similar studies examined the effect of progressive muscle relaxation in other conditions ; nickel et al . reported the effectiveness of progressive relaxation training in improving qol and reducing anger signs in women with bronchial asthma .
body interventions on pregnant women 's temperament , perceived stress , and pregnancy outcomes . in the above studies , progressive muscle relaxation was the most common intervention .
the results show effects such as increased birth weight , decreased duration of delivery , lesser use of instruments for delivery , and reduction of perceived anxiety and stress .
's study conducted in iran on the effect of relaxation training based on self - efficacy theory on pregnant women 's mental health showed that the training program reduced the three dependent variables ( state anxiety , trait anxiety , and perceived stress ) in the experimental group significantly .
also , use of progressive muscle relaxation in various diseases has been shown to improve mental health .
also , in a review study , brenes et al . emphasized the effectiveness of progressive relaxation training in reducing stress of patients with chronic obstructive pulmonary disease ( copd ) . in another study ,
yoo et al . examined the effect of muscle relaxation and guided imagery on the symptoms of depression , anxiety , and anger and life quality of women with breast cancer in seoul .
the results showed that both the level of depression and anxiety and qol in patients of the experimental groups were significantly lesser than those in the control group .
therefore , the researchers mentioned that these methods were useful in improvement of mental symptoms and qol in patients with breast cancer .
time consumption because of the high number of samples of pregnant women followed up after training and high sample attrition following obstetrical intervention or unwillingness to continue participation in the study were the limitations of this study .
another limitation was individual differences in participants'mental and emotional aspects in doing the practice , that may be affect on muscle relaxation . also , absence of study subjects in training sessions , requiring individual repeat training , and lack of response to 8-week follow - up after training led to exclusion and replacement of some subjects .
existence of only few relevant studies , due to which there was limited chance of comparison of results with other studies , and lack of generalizability of results were among the other limitations of this study , which necessitates more comprehensive studies to be conducted in future .
the study results demonstrated significant difference between the general health scores of women in the experimental group and the control group in terms of physical , psychological , and social aspects .
in general , the results confirmed the hypothesis of the study on the effectiveness of relaxation on pregnant women 's general health .
thus , considering the effectiveness of the method , lack of side effects , especially during pregnancy , and its cost - effectiveness , it is recommended to be used for pregnant women . | background : pregnancy may be accompanied by serious physiological and psychological changes as it is a stressful period in a woman 's life .
so , this study was conducted to determine the effect of progressive muscle relaxation on pregnant women 's general health.materials and methods : in this clinical trial , 60 primigravida women admitted to the prenatal clinic of selected hospitals in iran constituted the study population . using purposive sampling method ,
the level of general health of the women was measured with general health questionnaire-28 ( ghq-28 ) .
then , the samples were randomly divided into control and experimental groups .
three 1.52 h relaxation training sessions were held for the experimental group .
after 8 weeks , the level of general health of both groups was measured again .
finally , the collected data were analyzed using chi - square and paired t - test ( p < 0.05).results : total mean score of general health of the experimental group and the control group before the intervention was 35.83 ( 6.92 ) and 29.46 ( 8.3 ) , respectively , and after the intervention , the respective scores were 20.2 ( 5.61 ) and 27.85 ( 8.24 ) . although after the intervention both groups showed an increased level of general health , the difference in general health between before and after intervention was significant in the experimental group ( p < 0.001 )
.
furthermore , comparison of variations in mean scores of general health level before and after intervention in the two groups showed a significant difference ( p < 0.001).conclusions : given that the results showed the effectiveness of progressive muscle relaxation on pregnant women 's general health , the prenatal clinics can include a training program for progressive muscle relaxation in the routine training programs for pregnant women . |
cells : hek293 t , human embryonic kidney 293 cell line expressing sv40 tag ,
cells were maintained under an atmosphere of 5% co2 at 37c in dulbecco s minimum
essential medium supplemented with 10% fbs ( fetal bovine serum ) , 2 mm l - glutamine ,
penicillin ( 100 u ml ) and streptomycin ( 0.1 mg
ml ) .
all experiments using hek293 t cells were performed in
collagen - coated dishes ( iwaki , chiba , japan ) .
plasmids and transfection : for expression of vmpyv vp1 , the full - length
vmpyv vp1 gene was amplified from the vmpyv genomic dna ( genbank accession number : ab767298 )
with xhoi and noti restriction sites added to the 5 and
3 ends , respectively .
the pcr product was cloned into the
xhoi - noti restriction sites of a pcmv - flag vector , so
that a flag tag was added to the n - terminal of expressed vp1 .
the c - terminal deletion mutant
of vmpyv vp1 was also cloned into the vector .
these plasmids were transfected into hek293 t
cells individually using the lipofectamine 2000 according to the manufacturer s instructions
( invitrogen , carlsbad , ca , u.s.a . ) .
immunocytochemical and immunoblot analyses : hek293 t cells transfected with
the plasmids were collected at 48 hr post transfection .
the cells were washed with pbs ,
fixed in 100% methanol for 5 min at 30c and blocked with 1% bsa ( bovine serum albumin ) in
pbs containing 0.5% triton x-100 , followed by incubation with an anti - sv40 vp1 antibody
overnight at 4c .
the cells were visualized with
secondary antibodies ( alexa fluor 488-conjugated goat anti - rabbit igg ; invitrogen ) and 4 ,
6-diamidino-2-phenylindole dihydrochloride ( dapi ; invitrogen ) for 1 hr at room temperature .
all the fluorescent images were captured and analyzed using a microscope ( ix70 ; olympus ,
tokyo , japan ) , a charge - coupled device camera ( dp30bw ; olympus ) and dp controller software
( olympus ) . for immunoblot analysis ,
the cells were harvested in ripa buffer [ 10 mm tris - hcl ( ph 7.5 ) ,
5 mm edta , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1% deoxycholic acid , 0.1% sds and 50
mm naf ] , supplemented with complete protease inhibitor cocktail ( roche diagnostics ,
indianapolis , in , u.s.a . ) .
cell lysates were
centrifuged at 20,400 g for 15 min at 4c , and the resulting supernatants
were subjected to sodium dodecyl sulphate - polyacrylamide gel electrophoresis ( sds - page ) and
immunoblotting with the following primary antibodies overnight at 4c ; an anti - sv40 vp1
antibody and an anti - actin antibody ( mab1501 ;
millipore , bedford , ma , u.s.a . ) .
actin was used as a loading control . after washing the
membrane with tbst (
tris - buffered saline containing 0.05% tween 20 ) , the membrane was
incubated with the following secondary antibodies for 1 hr at room temperature ; a
horseradish peroxidase ( hrp)-conjugated anti - rabbit igg ( biosource international , camarillo ,
ca , u.s.a . ) and hrp - conjugated anti - mouse igg ( biosource international ) .
the
chemiluminescence signals were visualized using a versadoc 5000mp ( bio - rad , hercules , ca ,
u.s.a . ) , and images were analyzed using quantity one software ( bio - rad ) .
electron microscopy : ultra - thin - section electron microscopy was performed
as described previously . in brief , 48 hr post
transfection cells were fixed with 2.5% glutaraldehyde in 0.1 m cacodylate buffer ( ph 7.3 )
for 20 min at 4c .
the cells were scraped from the dish and fixed with 2% osmium tetroxide
in the same buffer for 1 hr at 4c .
pellets were dehydrated with a series of ethanol
gradients ( 50% , 70% , 90% and 99.5% ) followed by propylene oxide , embedded in epon 812 resin
mixture ( taab laboratories equipment , berkshire , u.k . ) and polymerized for 3 days at 60c .
thin - sections ( 70 nm ) were stained with uranyl acetate and lead citrate . for negative staining
, purified fractions fixed with 0.25% glutaraldehyde were adsorbed to
collodion - carbon - coated copper grids ( nisshin em corporation , tokyo , japan ) and negatively
stained with 2% phosphotungstic acid solution ( ph 5.8 ) .
all samples were examined with an
h-7650 electron microscope at 80 kv ( hitachi , kyoto , japan ) .
sucrose gradient sedimentation analysis : sucrose gradient sedimentation
analysis was performed as described previously .
briefly , 72 hr post transfection cells were harvested in 10 mm tris - hcl ( ph 7.5 ) , 2 mm
mgcl2 and 0.25% brij 58 ( sigma , st .
the cellular lysates
were subjected to three cycles of freezing and thawing , cellular debris was removed via
centrifugation at 500 g for 10 min at 4c , and the resulting supernatants
overlaid onto a preformed 3050% sucrose gradient in 20 mm tris - hcl ( ph 8.0 ) .
samples were
centrifuged at 192,000 g for 1 hr at 4c ( sw55 ti rotor , beckman coulter ,
brea , ca , u.s.a . ) , and each 400 l fraction was taken from the top for 12
fractions .
each fraction was subjected to sds - page and immunoblotting with the anti - sv40 vp1
antibody overnight at 4c .
after washing the
membrane with tbst , the membrane was incubated with hrp - conjugated anti - rabbit igg for 1 hr
at room temperature ( biosource international ) .
( de3 ) plyss competent cells ( stratagene , la jolla , ca , u.s.a . ) were
transformed with the pet15b plasmid ( novagen , madison , wi , u.s.a . ) encoding the full - length
jcv vp1 gene , and vlps were purified .
expression of vmpyv vp1 in hek293 t cells : the full - length of vmpyv vp1
gene was amplified and cloned into the pcmv - flag vector .
in addition , we synthesized a
c - terminal deletion mutant of vmpyv vp1 ( c vp1 ) . the deleted region was determined based on
the amino acid sequences alignment with the reported pyv vp1s ( fig . 1fig .
the vp1 sequences of pyvs were obtained from genbank
( abbreviations and accession numbers are indicated in table 1 ) .
amino acid identities are shaded as follows : black shading indicates
that all amino acid sequences were conserved , whereas grey shading indicates that more
than 51% of them were conserved . ) .
we synthesized the c vp1 of 1387 amino acid residues of wild - type vmpyv vp1 ( wt
vp1 ) .
the c - terminal deletion mutant vp1 gene with a stop codon ( taa ) was also amplified and
cloned into the pcmv - flag vector .
these plasmids were transfected into hek293 t cells
individually and incubated for 48 hr . to detect the vp1 protein expression
both wt and c vp1s were mainly detected in the nuclei and
sometimes in the cytoplasm in transfected cells with encoding wt or c vp1 plasmids ( fig .
immunocytochemical and immunoblot
analyses ( ic and ib ) of vmpyv vp1 with the anti - sv40 vp1 antibody .
the hek293 t cells
were transfected with wt vp1 , c vp1 or the corresponding empty vector ( mock ) as a
negative control .
( b ) wt and c vp1 signals were
detected in cellular lysates from hek293 t cells at the expected molecular weight
positions in immunoblotting .
we also performed immunoblotting analysis to confirm the expression levels of wt
and c vp1s .
we detected both wt and c vp1s in the cells using the anti - sv40 vp1 antibody
at the expected molecular weights of 56 and 44 kda , respectively ( fig .
2b ) . because c vp1 is devoid of 388503 amino acid residues
( 116 a.a .
the expression
levels of wt and c vp1s were almost similar relative to the expression levels of internal
control protein actin ( fig .
these results
suggested that the intracellular localization and expression levels of wt and c vp1s were
similar in the transfected cells .
the vp1 sequences of pyvs were obtained from genbank
( abbreviations and accession numbers are indicated in table 1 ) .
amino acid identities are shaded as follows : black shading indicates
that all amino acid sequences were conserved , whereas grey shading indicates that more
than 51% of them were conserved .
immunocytochemical and immunoblot
analyses ( ic and ib ) of vmpyv vp1 with the anti - sv40 vp1 antibody .
the hek293 t cells
were transfected with wt vp1 , c vp1 or the corresponding empty vector ( mock ) as a
negative control .
( b ) wt and c vp1 signals were
detected in cellular lysates from hek293 t cells at the expected molecular weight
positions in immunoblotting .
expression of vlps using electron microscopy : because the vmpyv vp1 was
detected in transfected hek293 t cells , we confirmed the formation of vlps using transmission
electron microscopy ( tem ) . at 48 hr post transfection of wt and c vp1 encoding plasmids ,
ultra - thin - sections with a
thickness of 70 nm were stained with uranyl acetate and lead citrate .
tem revealed a large
number of vlps with a diameter of approximately 50 nm in the nuclei of wt vp1-expressing
cells ( wt vlps ; fig . 3a3cfig .
electron micrographs of
hek293 t cells expressing wt vp1 ( a c ) and c vp1 ( d f ) .
( b , c , e and f ) higher
magnification of the regions indicated in panels a , b , d and e , respectively .
we were also able to confirm vlps with a diameter of approximately 4550 nm in the
nuclei of c vp1-expressing cells ( c vlps ; fig .
3d3f ) ; however , the number of c vp1 vlps was much lower than that of wt vp1
vlps .
electron micrographs of
hek293 t cells expressing wt vp1 ( a c ) and c vp1 ( d f ) .
( b , c , e and f ) higher
magnification of the regions indicated in panels a , b , d and e , respectively .
sucrose gradient sedimentation analysis : to confirm that vlps were formed
by vmpyv vp1 in transfected cells , we also performed the sucrose gradient sedimentation
analysis , which can distinguish vlps from vp1 pentamers . as a positive control in the analysis
the vp1 signal in jcv vlps was mainly detected in fractions 6 to
9 ( fig .
( a c ) immunoblot analyses of vp1 in
fractionated samples after sucrose gradient sedimentation of jcv vlps and cellular
lysates from hek293 t cells expressing wt vp1 or c vp1 .
cellular lysates were
separated by 3050% sucrose gradient sedimentation and fractionated into 12 fractions
from the tops of the tubes .
the 12 fractions were separated by sds - page and subjected
to immunoblotting with the anti - sv40 vp1 antibody .
( b ) hek293 t cell lysates transfected with wt vp1 and ( c )
c vp1 .
( d ) electron micrograph of negative staining of fractions 6 to 9 from hek293 t
cells transfected with wt vp1 .
the vp1 signal in cellular lysates from the wt vp1-expressing cells was also mainly
detected in fractions 6 to 9 ( fig .
however ,
the vp1 signal in cellular lysates from c vp1-expressing cells was mainly detected in
fractions 1 to 4 and slightly detected in fractions 5 to 8 ( fig .
4c ) . to confirm the formation of wt vlps in fractions 6 to 9 , we
collected these fractions and verified them with negative - stained tem .
we observed a large
number of wt vlps with a diameter of approximately 50 nm ( fig .
we also confirmed the presence of jcv vlps in fractions 6 to 9 ( data not
shown ) .
( a c ) immunoblot analyses of vp1 in
fractionated samples after sucrose gradient sedimentation of jcv vlps and cellular
lysates from hek293 t cells expressing wt vp1 or c vp1 .
cellular lysates were
separated by 3050% sucrose gradient sedimentation and fractionated into 12 fractions
from the tops of the tubes .
the 12 fractions were separated by sds - page and subjected
to immunoblotting with the anti - sv40 vp1 antibody .
( b ) hek293 t cell lysates transfected with wt vp1 and ( c )
c vp1 .
( d ) electron micrograph of negative staining of fractions 6 to 9 from hek293 t
cells transfected with wt vp1 .
vmpyv was originally detected in a vm spleen using nested broad - spectrum pcr techniques .
it
has a longer vp1 orf in the c - terminus region compared with the sequences of other known
pyvs vp1s , whereas its functions are still unclear . in general
, the pyv capsid contains 360 molecules of vp1 formed with 72
pentamers , 5 molecules of vp1 and 1 molecule of vp2/vp3 [ 13 , 22 ] . in the current study , to examine
the role of c - terminal of vmpyv vp1 in virion formation , vmpyv vlps consisting of wt vp1 or
c vp1 were generated in mammalian hek293 t cells .
immunocytochemical analysis revealed that wt and c vp1s were expressed in the transfected
hek293 t cells ( fig .
both wt and c vp1s were
also detected at the expected molecular weights by immunoblot analysis ( fig .
furthermore , there was no difference in the expression
level between wt and c vp1s ( fig .
although
vlps were observed in wt and c vp1-expressing cells by using electron microscopy ( fig .
3 ) , the number of wt vlps was higher than that of
c vlps .
in addition , the number of cells with wt vlps was higher than that of cells with c
vlps ( data not shown ) .
and c
vlps were observed in the nuclei ; however , no vlps were confirmed in the cytoplasm ( data not
shown ) .
immunoblot analysis revealed that the signal of jcv vlps was mainly
detected in fractions 6 to 9 ( fig .
the vp1
signal in cellular lysates from wt vp1-expressing cells was also mainly detected in
fractions 6 to 9 ( fig .
we confirmed the
presence of jcv vlps and wt vlps in fractions 6 to 9 with negative - stained tem ( fig .
however , the vp1 signal in cellular lysates
from c vp1-expressing cells was mainly detected in fractions 1 to 4 ( fig .
because the protein density in fractions 1 to 4 is lower
than that in fractions 6 to 9 , it is supposed that the vp1 signal in fractions 1 to 4 may
have represented the pentamers instead of vlps .
in addition , the faint vp1 signal in
fractions 5 to 8 of cellular lysates from c vp1-expressing cells may have represented vlps .
this result is convincing in light of the tem results showing fewer vlps in c
vp1-expressing cells ( fig .
taken together ,
the results showed that c vp1 formed vlps ; however , the efficiency of vlp formation was
lower than that of wt vp1 .
it has been reported that the c - terminal arm of pyvs vp1 can be subdivided into three
segments ; c helix ,
c insert and c loop [ 13 ,
22 ] . the c helix of sv40 ( sfllsdlinrrtq ; 305317
a.a . ) mediates contacts between pentamers as described previously [ 13 , 22 ] . as demonstrated in fig .
1 , the c helix of sv40 is predicted to correspond
to the 295307 amino acid residues ( sflltdlinrrtp ) of jcv and the 325337 amino acid
residues ( tsllgslftglmp ) of vmpyv . in comparison with tripartite ,
the homology was 85% for
sv40-jcv , 23% for sv40-vmpyv and 31% for jcv - vmpyv in the c helix of vp1s .
it revealed that
the c - helix of jcv may mediate contacts between pentamers , because of the high homology with
that of sv40 .
however , the c helix of vmpyv has low homology with that of sv40 and jcv . as
shown in fig .
3 , we observed that the number of wt
vp1 vlps was much higher than that of c vp1 vlps .
thus , the deleted c - terminal 116 amino
acid residues ( 388503 a.a . ) of vmpyv vp1 affect the efficiency of its vlp formation .
chpyv also encodes a unique extended c - terminal vp1 protein of 497 a.a . , and
these results suggest that the length of vp1 amino acid
residues has no effect on the size ( i.e. , diameter ) of vlps .
we observed that
vmpyv vlps have a typical shape of pyv virions , and their diameters were approximately 50 nm
in size , suggesting that native vmpyv virions also have morphology similar to that of its
vlps . in conclusion , we demonstrated that vmpyv vlps were formed in mammalian cells expressing
vp1 and found the extra c - terminal region of vp1 does not affect the size and morphology of
vlps , whereas the c - terminal of vmpyv vp1 may have some function for efficient vlp
formation .
the study to investigate function ( s ) of the extra c - terminal region of vmpyv vp1
needs to be continued . | abstractrecently , we detected novel vervet monkey polyomavirus 1 ( vmpyv ) in a vervet monkey .
among amino acid sequences of major capsid protein vp1s of other polyomaviruses ,
vmpyv vp1
is the longest with additional amino acid residues in the c - terminal region . to examine
the role of vmpyv vp1 in virion formation , we generated virus - like particles ( vlps ) of
vmpyv vp1 , because vlp is a useful tool for the investigation of the morphological
characters of polyomavirus virions .
after the full - length vmpyv vp1 was subcloned into a
mammalian expression plasmid , the plasmid was transfected into human embryonic kidney 293 t
( hek293 t ) cells .
thereafter , vmpyv vlps were purified from the cell lysates of the
transfected cells via sucrose gradient sedimentation .
electron microscopic analyses
revealed that vmpyv vp1 forms vlps with a diameter of approximately 50 nm that are
exclusively localized in cell nuclei .
furthermore , we generated vlps consisting of the
deletion mutant vmpyv vp1 ( c vp1 ) lacking the c - terminal 116 amino acid residues and
compared its vlp formation efficiency and morphology to those of vlps from wild - type vmpyv
vp1 ( wt vp1 ) .
wt and c vp1 vlps were similar in size , but the number of c vp1 vlps was
much lower than that of wt vp1 vlps in vp1-expressing hek293 t cells .
these results suggest
that the length of vp1 is unrelated to virion morphology ; however , the c - terminal region
of vmpyv vp1 affects the efficiency of its vlp formation . |
this study was a historical cohort conducted at a 1,000 bed community , teaching hospital .
there was no funding received for the study , and all data collection and analysis was completed by the primary author .
patients who had received intravenous contrast in 2011 were screened , via use of a random number generator , for inclusion into the study .
patients who met inclusion criteria had at least two of the following characteristics : baseline serum creatinine 1.2 mg / dl or a creatinine clearance < 50 ml / min ( calculated via cockcroft gault equation ) , age > 75 years , diabetes mellitus noted in their past medical history , systolic heart failure with documented ejection fraction < 40% , and/or hypertension evidenced by their past medical history or active use of antihypertensives .
in addition , patients had to have a serum creatinine level drawn at baseline ( within 1 month prior to receiving contrast ) and within 1296 hours following contrast administration .
excluded patients were those < 18 years of age , those who were pregnant or breast feeding , and those who were receiving dialysis prior to or during the study period .
a list of all patients who received intravenous contrast during the defined study period was generated . from this list
, an additional filter was added to separate patients who had also received n - acetylcysteine from patients who had not received n - acetylcysteine .
patients from these two lists ( those who received n - acetylcysteine and those who did not ) were selected randomly .
data on patients included in these two groups were then collected and analyzed ( fig .
the primary outcome was the absolute difference in the proportion of patients who developed cin with and without the administration of n - acetylcysteine .
cin was defined as a 0.5 mg / dl increase in serum creatinine or a 25% increase in serum creatinine within 1296 hours post - exposure to contrast .
secondary outcomes included sub - analysis of the primary outcome according to patients with diabetes mellitus , patients aged > 75 years , patients with systolic heart failure , and patients with hypotension ( systolic blood pressure < 90 mmhg ) immediately prior to contrast administration ; absolute difference in blood urea nitrogen ( bun ) post - exposure to contrast ; and proportion of patients with an elevation in serum creatinine of at least 0.3 mg / dl .
on the basis of reported rates of cin in previous studies , the anticipated incidence of cin without n - acetylcysteine was 15% .
for the primary outcome , we determined that 302 patients would provide a power of 80% to detect a 10% absolute risk reduction ( arr ) with a two - sided alpha level of 0.05 .
this study was a historical cohort conducted at a 1,000 bed community , teaching hospital .
there was no funding received for the study , and all data collection and analysis was completed by the primary author .
patients who had received intravenous contrast in 2011 were screened , via use of a random number generator , for inclusion into the study .
patients who met inclusion criteria had at least two of the following characteristics : baseline serum creatinine 1.2 mg / dl or a creatinine clearance < 50 ml / min ( calculated via cockcroft gault equation ) , age > 75 years , diabetes mellitus noted in their past medical history , systolic heart failure with documented ejection fraction < 40% , and/or hypertension evidenced by their past medical history or active use of antihypertensives . in addition , patients had to have a serum creatinine level drawn at baseline ( within 1 month prior to receiving contrast ) and within 1296 hours following contrast administration .
excluded patients were those < 18 years of age , those who were pregnant or breast feeding , and those who were receiving dialysis prior to or during the study period .
patients were identified through electronic prescription numbers in the electronic medical record . a list of all patients who received intravenous contrast during the defined study period was generated . from this list
, an additional filter was added to separate patients who had also received n - acetylcysteine from patients who had not received n - acetylcysteine .
patients from these two lists ( those who received n - acetylcysteine and those who did not ) were selected randomly .
data on patients included in these two groups were then collected and analyzed ( fig .
the primary outcome was the absolute difference in the proportion of patients who developed cin with and without the administration of n - acetylcysteine .
cin was defined as a 0.5 mg / dl increase in serum creatinine or a 25% increase in serum creatinine within 1296 hours post - exposure to contrast .
secondary outcomes included sub - analysis of the primary outcome according to patients with diabetes mellitus , patients aged > 75 years , patients with systolic heart failure , and patients with hypotension ( systolic blood pressure < 90 mmhg ) immediately prior to contrast administration ; absolute difference in blood urea nitrogen ( bun ) post - exposure to contrast ; and proportion of patients with an elevation in serum creatinine of at least 0.3 mg / dl .
the primary hypothesis was that n - acetylcysteine would reduce the incidence of cin . on the basis of reported rates of cin in previous studies , the anticipated incidence of cin without n - acetylcysteine was 15% . for the primary outcome , we determined that 302 patients would provide a power of 80% to detect a 10% absolute risk reduction ( arr ) with a two - sided alpha level of 0.05 .
the majority of patients had co - morbidities of diabetes and/or hypertension , with more patients with diabetes present in the group not receiving n - acetylcysteine compared to the group that did receive n - acetylcysteine . when comparing baseline renal function , mean pre - contrast serum creatinine in patients receiving n - acetylcysteine was 1.41 mg / dl compared to 0.95 mg / dl in those not receiving n - acetylcysteine ( p=0.0001 ) .
baseline characteristics scr = serum creatinine , crcl = creatinine clearance ( as estimated by cockcroft gault ) , bun = blood urea nitrogen , ibw = ideal body weight .
cin occurred in 14 ( 9.3% ) patients who received n - acetylcysteine and in 27 ( 17.9% ) patients who did not ( arr 8.6% , p=0.0428 ) ( table 4 ) . the mean increase in serum creatinine post - contrast in patients receiving n - acetylcysteine and patients not receiving
of the 41 patients who developed cin , 16 met both criteria in the definition of cin .
twenty - four patients met the definition solely based on a relative increase in serum creatinine of 25% , and one patient met the definition solely based on an absolute increase in serum creatinine of 0.5 mg / dl .
cin = contrast - induced nephropathy , bun = blood urea nitrogen , scr = serum creatinine .
secondary outcomes are presented in table 4 . in a subgroup analysis of the primary outcome
, significant differences in the incidence of cin were seen in patients aged at least 75 years and in patients with a history of hypertension .
a significant difference was also noted in the percent change in bun . in the overall population ( n=302 ) ,
77% of patients received fluids either prior to or immediately following contrast administration ; however , the mean percentage was different between the two study groups . in the group of patients receiving n - acetylcysteine , 85% of patients received fluids compared to only 69% of patients in the group that did not receive n - acetylcysteine .
patients who received fluids had a lower incidence of cin than patients who did not receive fluids ( 10.8% vs. 22.9% , respectively ; p=0.0157 ) .
the effect of fluid administration in the overall population , in those receiving n - acetylcysteine ( p=0.0411 ) , and in those not receiving n - acetylcysteine ( p=0.2561 ) , is presented in fig .
the majority of patients had co - morbidities of diabetes and/or hypertension , with more patients with diabetes present in the group not receiving n - acetylcysteine compared to the group that did receive n - acetylcysteine . when comparing baseline renal function , mean pre - contrast serum creatinine in patients receiving n - acetylcysteine was 1.41 mg / dl compared to 0.95 mg / dl in those not receiving n - acetylcysteine ( p=0.0001 ) .
baseline characteristics scr = serum creatinine , crcl = creatinine clearance ( as estimated by cockcroft gault ) , bun = blood urea nitrogen , ibw = ideal body weight .
cin occurred in 14 ( 9.3% ) patients who received n - acetylcysteine and in 27 ( 17.9% ) patients who did not ( arr 8.6% , p=0.0428 ) ( table 4 ) . the mean increase in serum creatinine post - contrast in patients receiving n - acetylcysteine and patients not receiving
of the 41 patients who developed cin , 16 met both criteria in the definition of cin .
twenty - four patients met the definition solely based on a relative increase in serum creatinine of 25% , and one patient met the definition solely based on an absolute increase in serum creatinine of 0.5 mg / dl .
cin = contrast - induced nephropathy , bun = blood urea nitrogen , scr = serum creatinine .
secondary outcomes are presented in table 4 . in a subgroup analysis of the primary outcome
, significant differences in the incidence of cin were seen in patients aged at least 75 years and in patients with a history of hypertension .
in the overall population ( n=302 ) , 77% of patients received fluids either prior to or immediately following contrast administration ; however , the mean percentage was different between the two study groups . in the group of patients receiving n - acetylcysteine , 85% of patients received fluids compared to only 69% of patients in the group that did not receive n - acetylcysteine .
patients who received fluids had a lower incidence of cin than patients who did not receive fluids ( 10.8% vs. 22.9% , respectively ; p=0.0157 ) .
the effect of fluid administration in the overall population , in those receiving n - acetylcysteine ( p=0.0411 ) , and in those not receiving n - acetylcysteine ( p=0.2561 ) , is presented in fig .
in this historical cohort , we evaluated the use of n - acetylcysteine for the prevention of cin .
based on the primary outcome , n - acetylcysteine is likely associated with a lower incidence of cin .
patients included in this cohort had to have at least two risk factors for the development of cin .
( 2 ) , the majority of patients in this cohort would have been at a 7.514% risk of developing cin by our definition .
the incidence of cin in patients not receiving n - acetylcysteine was approximately 18% , which is just above the average incidence in previously reported studies ( 15.7% ) ( 4 ) .
similarly , in patients who did receive n - acetylcysteine , the incidence of cin in our study was similar to the average incidence reported in previous literature ( 4 ) ( 9.3% vs. 8.9% , respectively ) .
this suggests that patients reviewed in this study were at similar baseline risk to those patients reviewed by others previously .
in addition , this study analyzed the effect of fluids , both with regard to use and non - use of n - acetylcysteine , since adequate hydration has been shown to be effective in preventing cin .
the definition of cin and the risk factors for cin used in this study are well agreed upon in the literature .
in addition to the primary outcome being analyzed with a commonly accepted definition of cin , other important markers used to describe acute kidney injury and cin were included as secondary outcomes ( absolute change in serum creatinine and change in bun ) ( 14 , 15 ) .
first , the retrospective nature of the study does not allow for cause and effect relationships to be analyzed , and we are only able to support an association between the incidence of cin and n - acetylcysteine use .
second , the dose , route , and frequency of n - acetylcysteine were not analyzed in this study .
all patients who received n - acetylcysteine received at least 600 mg / dose and received at least a total of four doses surrounding contrast administration , but no consistent pattern of administration was enforced .
for example , some patients received one dose of n - acetylcysteine before contrast and three after , while others received two doses before and three after , and so on . this is a potential confounder as previous literature suggests that certain dosing strategies may be associated with better outcomes ( 16 , 17 ) .
selection bias is a factor due to patients in the group receiving n - acetylcysteine having worse renal function at baseline .
this is likely reflective of healthcare professionals being more likely to order n - acetylcysteine in patients with poor renal function at baseline due to the potential benefit seen in previous literature .
in addition , there was an observed difference in the percentage of patients in each group that received intravenous fluids . since fluids are considered the first - line preventative strategy for cin , the difference in use could impact the results of this study .
finally , while the study did meet power for the primary outcome , it is possible that the non - significant results noted in some subgroups is a result of type ii error . despite the limitations presented above
, this study may provide information that can be used for the prevention of cin . in this time of nationwide drug shortages ,
based on the subgroup analysis of the primary outcome , a protocol can be developed that takes into account the patients at the highest risk for cin development .
in addition to helping identify those at the highest risk , a protocol could help with the establishment of more consistent n - acetylcysteine dosing .
developing this type of protocol would likely reduce drug cost and allow n - acetylcysteine to be used more resourcefully .
this study conducted in a community , teaching hospital may provide information to healthcare professionals in numerous health - system settings . specifically
, other community - based hospitals can use the data presented here to tailor or develop n - acetylcysteine protocols . while this study was retrospective ,
future prospective studies , analyzing a larger number of patients with various risk factors , would help in the development of a protocol for the optimal use of n - acetylcysteine . ideally , these future studies would take into account confounding factors such as the use of nephrotoxic drugs and the dosing strategy of n - acetylcysteine .
in conclusion , n - acetylcysteine was likely associated with a lower rate of cin in patients at risk for cin development .
subgroup analyses reveal patients who may have the greatest benefit , specifically those aged 75 years and those with a history of hypertension .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | backgroundcontrast - induced nephropathy ( cin ) remains a leading cause of acute renal failure in hospitalized patients .
n - acetylcysteine has been studied previously for the prevention of cin , resulting in mixed findings.objectivethe objective of this study was to determine the impact of n - acetylcysteine on the development of cin in order to guide its use at community , teaching hospitals.methodspatients admitted between january 1 and december 31 , 2011 , receiving intravenous radiocontrast dye were included if they were compliant with two or more of the following conditions : baseline serum creatinine > 1.2 mg / dl or estimated creatinine clearance < 50 ml / min , age 75 years , diabetes mellitus , heart failure , or hypertension .
the primary outcome was the difference in the proportion of patients in each group ( n - acetylcysteine or no n - acetylcysteine ) who developed cin , which was defined as a 0.5 mg / dl increase in serum creatinine or a 25% increase in serum creatinine within 1296 hours post - exposure to contrast.resultsa total of 302 patients were included , 151 who received n - acetylcysteine and 151 who did not receive n - acetylcysteine .
patients who received n - acetylcysteine had significantly worse renal function at baseline than those who did not receive n - acetylcysteine ( mean pre - contrast serum creatinine , 1.41 vs. 0.95 mg / dl , p<0.0001 )
. a lower proportion of patients developing cin was observed between those who received n - acetylcysteine and those who did not receive n - acetylcysteine ( 10.2% vs. 21.8% , p=0.0428).conclusionsthe use of n - acetylcysteine was likely associated with a reduced incidence of cin in patients at risk for cin development .
based on these results , hospitals may benefit from the development of a protocol to guide the appropriate use of n - acetylcysteine . |
chinese postpartum care ( zuoyuezi ) has been regarded as a crucial rite - to - passage for the woman 's recovery and the transition to motherhood after childbirth .
the use of sheng - hua - tang , a well - known traditional chinese medicine ( tcm ) compound prescription , during the postpartum has been popular in chinese communities over a long period .
previous study has shown that sheng - hua - tang use during the first month of the postpartum period may have a positive effect on women 's health - related quality of life , especially in terms of role limitations due to physical health and emotional problems .
wu - jia sheng - hua capsule ( comprising radix et caulis acanthopanacis senticosi , radix angelicae sinensis , rhizoma chuanxiong , semen persicae , radix glycyrrhizae , and rhizoma zingiberis preparata ) , derived from sheng - hua - tang , has been used widely in chinese women to promote blood flow , resolve blood stasis , ease postchildbirth pain , reduce bleeding volume and shorten bleeding duration after induced abortion in chinese women [ 3 , 4 ] .
the material base , namely the bioactive components , of wu - jia sheng - hua ( wjsh ) capsule is still unclear , though the prostaglandin f2alpha ( pgf2 ) , prostaglandin e2 ( pge2 ) , endothelin ( et ) , and nitrogen monoxide ( no ) levels were found to be partially responsible for the curative effects and recovery benefits of wjsh capsule on induced abortion with vaginal haemorrhage .
tcm serum pharmacochemistry , based on hypothesis that active compounds should appear in blood after administration of tcm , was proposed by homma et al .
, it was proved to be helpful in recognizing the real active components in tcm and in gaining a better understanding of the mechanisms under the therapeutic effects of tcm [ 79 ] . in the present study ,
an ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry ( uplc / esi - ms ) method was developed and applied for investigating the serum pharmacochemistry of wjsh capsule .
the identification of wjsh 's multiple absorbed bioactive components and metabolites might be helpful for better understanding the mechanisms under its therapeutic effects .
syringin , protocatechuic acid , liguiritin , ferulic acid , isofraxidine , chlorogenic acid , and liqustilide ( standards ) were purchased from national institute of control of pharmaceutical and biological products ( beijing , china ) .
acetonitrile , methanol , formic acid , and ammonium formate ( hplc grade ) were obtained from merck ( darmstadt , germany ) and sigma - aldrich ( st . louis , mo , usa ) , respectively .
wu - jia - sheng - hua capsule and each single herb in the prescription of wu jia sheng hua capsule were gifts from duo duo pharmaceutical co. ltd .
the chromatographic separation of the absorbed components and metabolites were performed on a waters acquity uplc system ( waters corp . ,
milford , ma , usa ) equipped with an uv detector , an autosampler , an acquity uplc beh c18 column ( 2.1 50 mm , 1.7 m ) , and an automatic thermostatic column oven . the mobile phase consisted of ( a ) water buffered with ammonium acetate ( 10 mm ) and formic acid ( 0.1% , v / v ) and ( b ) acetonitrile .
the elution of the target compounds was conducted in a gradient mode with the volume percentage of b changed from 5% to 50% in the initial 10 minutes and from 50% to 80% in the next 6 minutes .
the flow rate was 0.4 ml / min , and the injection volume was 8 l . to obtain the ms and ms / ms data of the investigated compounds , a waters quattro premiere xe triple - quadrupole mass spectrometer ( micromass ms technologies ) was coupled to the abovementioned uplc system .
the mass range was set at m / z 1001000 da with the scan time being 0.5 s. the ionization parameters were as follows : interval time , 0.2 s ; capillary voltage , 3.5 kv ; cone voltage , 35 kv ; ionization resource temperature , 120c ; desolvation temperature , 380c , desolvation gas flow rate , 500 l / h ( n2 ) ; and cone gas flow rate , 50 l / h ( n2 ) . masslynx software ( version 4.1 ) and quanlynx software were used for system control and data processing , respectively . 5 ml methanol was added to 1 ml blank rat serum sample or wjsh capsule serum sample .
the mixture was vortexed for 3 min . and centrifuged for 15 min .
each residue was reconstituted in 1 ml methanol and centrifuged for 15 min .
the extracts of wjsh capsule and raw medicinal materials of wjsh capsule were prepared separately .
10 ml methanol was added to 0.4 g wjsh or each raw medicinal material .
centrifuged ( 3000 rpm ) for 15 min . , and filtered through 0.45 m micropore film ( millipore , usa ) .
male sprague - dawley rats ( 230 g 10 g , experimental animal center of heilongjiang university of traditional chinese medicine ) were randomly divided into eight groups ( six for each ) : group a to group h. all care and handling of animals were performed with the approval of institutional authority for laboratory animal care of heilongjiang university of traditional chinese medicine .
after 12 hours of fasting , normal saline ( blank control ) and extract of wjsh capsule were given intragastrically to rats in group a and group b , separately .
extracts of raw medicinal materials were given intragastrically to rats of the other 6 groups ( one extract per group ) .
the dosage was 1.5 ml/100 g body weight , and the administration frequency was once daily in three consecutive days in each group . at forty minutes after the last administration , 5 ml blood
the blood samples were centrifugated for 10 min . at 5000 rpm at 4c to separate serum samples .
to purify the serum sample , the protein precipitation , liquid - liquid extraction , and solid - phase extraction methods were tried .
finally , the simple protein precipitation method with methanol was chosen , since most investigated compounds were found in the purified samples .
as compared to methanol - water system , higher resolution , better peak shape , and faster elution of compounds were achieved using acetonitrile - water system buffered with ammonium acetate and formic acid .
the chromatographic separation of the absorbed components and metabolites was conducted in gradient elution mode to overall reduce the retention time of these compounds characterized by different polarities .
the timetable of gradient elution was listed in table 1 . for the esi - ms conditions ,
the stronger responses of all compounds were obtained in positive ionization mode than in negative mode .
thus , positive mode was employed in the total ion current ( tic ) chromatograms of wjsh capsule serum samples in the m / z range of 100 to 1000 da .
the multiple bioactive components in tcm could be simultaneously identified using the uplc - esi - ms technique .
it is generally helpful in better understanding the mechanisms underlying tcm 's therapeutic effects .
the serum pharmacochemistry study of wjsh capsule was conducted in rats orally administered with wjsh capsule .
then , the absorbed bioactive components including their metabolites were identified by the established uplc - esi / ms method .
the typical tic chromatograms of wjsh capsule extract , wjsh capsule serum sample , and blank serum sample were shown in figure 1 .
twelve chemicals of which chromatographic peaks appeared in the tic chromatograms of wjsh capsule serum sample and wjsh capsule extract , but not in that of the blank serum sample , were presumed to be the absorbed components .
similarly , four chemicals whose chromatographic peaks presented only in the tic chromatograms of wjsh capsule serum sample , but not in that of the wjsh capsule extract or the blank serum sample , were supposed to be metabolites . by comparing the ms / ms spectra ,
the chromatographic peaks in tic chromatogram of wjsh capsule serum sample versus the standards , respectively , seven absorbed components were identified unequivocally .
for instance , the ms / ms spectrum of peak 5 was compared with that of the isofraxidine standard .
these ms / ms spectra were shown in figure 2 . since the quasi - molecular ion at m / z 223 [ m + h ] , fragmentation ions at m / z
192 [ m + h - och3 ] , and at m / z 161 [ m + h - och3-och3 ] were found simultaneously in the ms / ms spectra of peak 5 and the standard of isofraxidine ; this absorbed component was confirmed to be isofraxidine .
the other five absorbed components and the four metabolites were not identified and need further investigation . by comparing the tic chromatograms of serum samples , wjsh capsule , wjsh capsule without radix et caulis acanthopanacis senticosi , and radix et caulis acanthopanacis senticosi alone ,
similarly , the original plant(s ) of the other absorbed components and metabolites were confirmed .
the retention times , ms / ms data , and original plants of the absorbed components and metabolites were listed in tables 2 and 3 , respectively . by revealing the bioactive components of tcm compound prescriptions
, serum pharmacochemistry studies might be significantly helpful in better understanding the mechanisms under the therapeutic effects of these prescriptions .
for instance , wjsh capsule and danggui - shaoyao - san have been used extensively in chinese women to enhance uterine involution after giving birth and alleviate dysmenorrhea , respectively [ 11 , 12 ] . in pharmacological experiments ,
the prostaglandins ( pg ) levels regulation was proved to be partially responsible for the myometrium contraction stimulation and inhibition effects of wjsh capsule and danggui - shaoyao - san , separately [ 4 , 12 ] . in previous and present serum pharmacochemistry investigations ,
identical ( ferulic acid and liqustilide ) and different ( syringin , protocatechuic acid , liguiritin , isofraxidine , and chlorogenic acid for wjsh capsule ; paeoniflorin sulfonate , albiflorin , paeoniflorin , butylidenephthalide , and senkyunolide i for danggui - shaoyao - san ) bioactive components in wjsh capsule and danggui - shaoyao - san were identified . by conducting comparative pharmacokinetic and pharmacological experiments , the major bioactive components in wjsh capsule and danggui - shaoyao - san related to the up- and downregulation effect on myometrium contraction
might be confirmed , and the influence of other coexisting components on these effected might be revealed .
thus , the mechanisms under wjsh capsule and danggui - shaoyao - san 's therapeutic effects might be elucidated .
in this study , an uplc - esi - ms method was established for investigating the serum pharmacochemistry of wjsh capsule . in this way
these results might be helpful for better understanding the mechanisms underlying the therapeutic effects of wjsh capsule . | to identify the compounds absorbed in rat serum after the oral administration of wu - jia sheng - hua ( wjsh ) capsule , a traditional chinese medicine ( tcm ) compound prescription , an ultraperformance liquid chromatography coupled with electrospray ionization mass spectrometry ( uplc / esi - ms ) method , was established .
the chromatographic separation of the absorbed compounds and metabolites was achieved with an acquity uplc beh c18 column ( 2.1 mm 50 mm , 1.7 m ) under a gradient elution .
the mobile phase was composed of acetonitrile and water buffered with ammonium acetate ( 10 mm ) and formic acid ( 0.1% , v / v ) .
twelve absorbed compounds and four metabolites were found .
seven of the absorbed compounds were identified by esi - ms .
the identification of absorbed compounds might be helpful for the better understanding of the mechanisms underlying the pharmacological effects of wjsh capsule . |
a 14-year - old female patient presented to our clinic with the symptom of hollowness that had been present in both gluteal areas for 6 months .
the patient , who did not have any history of topical medication , redness , pain or itching in the hollowed area and did not describe any signs , had been receiving corticosteroid injections due to urticaria for the last 6 months .
the patient did not have any particularities in her personal history , and there were no persons in her family with similar symptoms .
the patient 's dermatological physical examination revealed an atrophic , oval and plaque - like lesion symmetrically localized on both thighs with a diameter of approximately 3 4 cm linearly overlaid by two flesh - colored striae with a diameter of approximately 1 4 cm .
the patient 's laboratory values were normal , and the deep incisional biopsy collected from the patient was found to be consistent with acquired localized lipoatrophy ( all ) .
histopathological examination revealed partly necrotic - looking adipocytes embedded among fibrous septa in the subcutaneous tissue interspersed with a few inflammatory cells .
the option of spontaneous healing was taken into account , and control visits at the dermatology outpatient clinic once every 3 months were recommended for our patient .
lipoatrophy is characterized by a preceding inflammatory period and consequent fat loss in the subcutaneous tissue . as for lipodystrophy
, it refers to the absence of subcutaneous fat tissue without any signs of inflammation .
lipoatrophy may be congenital or acquired and localized or diffused depending on the area of involvement .
primary and idiopathic lipoatrophy has clinical forms such as lipoatrophia semicircularis , lipodystrophia centrifugalis abdominalis infantilis and involutional lipoatrophy .
secondary lipoatrophy develops due to reasons such as pyogenic abscesses , connective tissue diseases ( lupus erythematosus , morphea , dermatomyositis or overlap disease ) , neoplasm ( t - cell lymphoma ) and iatrogenic ( subcutaneous , intramuscular and dermal injections ) factors .
all frequently develops secondary to dermal or intramuscular drug injections of corticosteroids as seen with our case .
additionally , all has also been reported following insulin , vasopressin , recombinant growth hormone , adrenal cortex hormone and methotrexate injections [ 2 , 3 , 4 ] .
lipoatrophy that develops due to subcutaneous corticosteroid injection results in two different histopathological pictures . in the first histopathological picture , there are small , retracted , slightly acidophilic or albuminous fat tissues surrounding the septal connective tissue accompanied by sparse inflammatory cells and prominent blood vessels . in the second histopathological picture
, there are small , atrophic fat tissues around vascular structures . decreased adipocyte counts and small - sized lobules
all diagnosis is based on the basis of clinical signs , physical examination , including deep incisional biopsy , and anamnesis .
primary localized lipoatrophy is generally self - limiting and may not require treatment . if the clinical signs persist and the patient has cosmetic concerns , liposuction and localized fat transplantation can be performed in a combined fashion .
| lipoatrophy is characterized by inflammation and tissue loss in fatty tissue . this disease may be congenital or acquired , primary or secondary .
secondary lipoatrophy develops with infections , collagen tissue diseases , tumors and drug injections . in this report , we present the case of a 14-year - old female patient who developed lipoatrophy following intramuscular steroid injection to both buttocks . |
the cdna microarray method has been developed recently and has been successfully applied to genomewide analysis of gene expression stimulated by hormones and/or chemicals ( inoue and pennie 2002 ; watanabe and iguchi 2003 ) .
knowledge of the patterns in the expression of estrogen - responsive genes is essential to understanding the action mechanism of estrogenic chemicals on mouse reproductive organs .
a large number of genes affected by estrogen were selected from the mouse ( moggs et al .
2002a , 2002b , 2003a ) and rat uterus ( daston and naciff 2005 ) . for most of the selected genes ,
their expression was not altered following e2 treatment in er- knockout mice , thus confirming the dependency of these genes on er-. activation of these genes suggests a basis for the marked uterotrophic effect observed several days following estrogen administration .
characteristic gene expression patterns were observed for each environmental estrogenic chemical , and these patterns were distinct from that of e2 , thereby suggesting specific mechanisms of action for endocrine disruption that could be different from that induced by endogenous estrogen ( daston and naciff 2005 ; watanabe et al .
2004a ) . physiological estrogens ( e2 ) , nonphysiological estrogens ( des ) , and dioxin have distinct effects on uterine gene expression ( watanabe et al .
2003b , 2004b ) . in the liver , however , np and dioxin activated another set of genes that were distinct from estrogen - responsive genes ( watanabe et al .
thus , these results suggest that only a small number of genes are directly involved in the uterotrophic effects of estrogen treatment , and np has effects very similar to those of e2 on gene expression in uterus but not in hepatic tissue . tissue - specific effects , therefore , should be considered in order to elucidate the distinct effects of various edcs .
extensive studies on contaminant - exposed and reference populations of american alligator ( alligator mississippiensis ) have revealed altered steroidogenesis , abnormal circulating hormone levels , hepatic transformation of androgen and endocrine organ morphology in juvenile alligators living in polluted environments , and a number of contaminants in eggs , serum , and body tissues ( guillette and iguchi 2003 ) .
affinity of some compounds is relatively high for alligator ers , and many compounds can displace e2 from the er ( guillette et al . 2002 ) in all species of crocodilians , sex is determined not by a genetic mechanism alone but also by the temperature at which the egg is incubated . in the alligator ,
the thermosensitive period ( tsp ) for sex determination is the 7- to 10-day window within stages 2124 of development ( lang and andrews 1994 ) .
treating embryos with estrogen during the tsp produces female offspring even at male incubation temperatures .
. however , the mechanisms of estrogen action on sex determination in the alligator are still uncertain .
furthermore , studies of contaminant - exposed alligators have shown alterations in steroid action ( guillette and iguchi 2003 ; guillette et al .
1994 ) . whether these abnormalities are caused , in part , by alterations in steroid receptor expression
the cdna encoding the ers and the progesterone receptors ( pr ) was isolated ( katsu et al . 2004 ) .
the er- amino acid sequence is similar to that of chicken er- ( 91% ) .
the er- sequence of the nile crocodile is quite similar to that of the american alligator .
the turtle er- sequence is closer to that of alligators than to that of the chicken ( katsu et al .
several thousand expressed sequence tags ( ests ) from the cdna library of adult alligators and those of the gonads of embryos incubated at temperatures that produce all males or all females have been sequenced and clustered .
we are currently establishing an alligator microarray for the study of the molecular mechanism of sex determination and of the chemical effects on sex determination and the toxic effects of chemicals .
exogenous chemicals that can interfere with the thyroid hormone axis could pose a significant hazard to human and wildlife health ( colborn 2002 ; zoeller 2003 ) .
amphibians represent a suitable model for monitoring reproductive performance , advanced development including metamorphosis , and sexual maturation ( kloas 2002 ) .
the influence of np , bpa , and e2 on developing xenopus laevis embryos was analyzed .
embryos were exposed to these agents between 3 and 96 hr postfertilization ( p.f . ) .
short body length , microcephaly , flexure , edema , and abnormal gut coiling were induced by 4.4 mg / l np or bpa or by 2.7 mg / l e2 at 96 hr p.f .
interestingly , the stages of embryos sensitive to bpa and np were different ; bpa affected earlier stages , whereas np affected later stages ( sone et al .
bpa interferes with the assembly of microtubules ( metzler and pfeiffer 1995 ) and causes mitotic arrest and aberrant spindles ( ochi 1999 ) .
these bpa actions may affect the susceptibility of embryos , especially at the earliest stages .
insensitivity of x. laevis embryos to bpa after 12 hr p.f . may reflect the weak in vitro activity of bpa relative to np or e2 ( nishikawa et al .
transcriptional levels of aromatase and er genes increased from stage 56 in x. laevis ( miyashita et al .
2000 ) , although er mrna was detected at stage 8 ( nishimura et al .
1997 ) . considering the ability of estrogen treatment to induce ectopic expression of the er ligand binding domain of fused mrna ( kolm and sive 1995 ) , it is possible that genes under the regulation of the estrogen er pathway are inactive at the developmental stages tested but are capable of transcriptional activation in the presence of an exogenous ligand .
to establish a model system for studying the effects of edcs on marine fish , we examined the effects of e2 on the early development of fundulus heteroclitus .
e2 ( 2.72 g / l ) reduced hatching and survival rates , and induced malformations with incomplete ossification of bones and 100% females ( urushitani et al .
2002 ) . to clarify the mechanisms contributing to these developmental effects of exogenous estrogen , we cloned fundulus er- ( fher- ) , which shared 81% identity with medaka ( oryzias latipes ) er- .
a receptor binding assay using the fher- ligand - binding domain showed that alkylphenols bind to fher- 50 times more efficiently than to human er- ( urushitani et al .
we characterized the estrogenicity of 4-methylbenzylidene , camphor , octylmethoxycinnamate , and propyl paraben ( n - propyl - p - hydroxybenzoate ) using medaka vitellogenin ( vtg ) plasma concentration , vtg , and choriogenin mrna expressions ( inui et al .
we are currently establishing a medaka microarray containing known genes related to steroidogenesis , sex development , degradation of chemicals , and estrogen - responsive genes .
the occurrence of intersexuality has been reported in wild roach ( rutilus rutilus ) ( jobling et al .
1998 ) , gudgeon ( gobio gobio ) ( van aerle et al . 2001 ) , and flounder ( platichthys flesus ) ( allen et al . 1999 ) in the united kingdom and in flounder ( pleuronectes yokohamae ) in japan ( hashimoto et al .
endocrine disruption of roach is thought to be caused by estrogenic agents in sewage effluents . in intersex roach , sexual maturation ,
2002a , 2002b ) . to understand the molecular mechanisms of intersex in roach that are induced by substances in sewage effluents , we have cloned genes of er- , er- , ( androgen receptor ( ar ) , pr , aromatase brain type , aromatase gonad type , dmrt-1 , and other genes related to steroidogenesis .
we are now establishing a roach microarray system . a number of studies have documented endocrine disruption derived from estrogenic responses caused by exposure to pesticides ( wester 1991 ) , surfactants ( white et al .
induction of secondary sex characteristics such as malelike coloration in female guppy ( poecilia reticulata ) , development of malelike gonopodium , and altered reproductive behavior in female mosquitofish ( gambusia affinisi ) have been reported following exposure to pulp mill effluent ( drysdale and bortone 1989 ; howell et al .
these data indicate the existence of contaminants with androgenic activity , which mimic or block endogenous androgen by interacting with the receptor in the aquatic organisms ( durhan et al .
however , compared with our knowledge of estrogenic environmental chemicals , the substances and mechanisms of androgenic action remain unclear ( gray et al .
recent studies with wild fathead minnows ( pimephales promelas ) have suggested the presence of potent androgenic substances in feedlot effluent ( orlando et al .
together with other wastewater contaminants , feedlot effluent has become a major ecological health concern .
trenbolone acetate , an androgenic and anabolic steroid , is a potent agonist of ar , and it has been used extensively as a growth promoter for beef cattle in the united states .
we have cloned mosquitofish ( gambusia affinis affinis ) ar- and ar- , and studied the effects of 17-trenbolone ( tb ) , a hydoxylated active compound of trenbolone acetate , on adult and newborn mosquitofish .
tb induced masculinization of the anal fin , accompanied by a transient up - regulation of ar- and ar- in adult females .
tb also induced differentiation of the anal fin into the gonopodium in fry at 0.310 g / l and stimulated precocious spermatogenesis in males and the formation of ovotestis in females at 110 g / l ( sone et al .
small freshwater fish used widely for toxicology research include the japanese medaka , the fathead minnow , and the zebrafish .
to date , very little has been done to apply genomics technologies to ecological risk assessment of aquatic species such as fish .
current genome sequencing efforts for several fish models such as zebrafish , medaka , and fathead minnow make these fish potential candidates for large - scale efforts to incorporate genomics technologies in an effort to understand the mechanistic toxicity pathways for environmental stressors ( miracle and ankley 2005 ) . in addition ,
genome sequencings are under way in other species such as european flounder , sheepshead minnow ( cyprinodon variegates ) , large mouth bass ( micropterus salmoides ) , rainbow trout ( onchorhynchus mykis ) , and the three - spined stickleback ( gusterosteus aculeatus aculeatus ) .
therefore , it is likely that the dynamic picture of various biological systems will be understood in the near future .
in contrast to the relatively large effort to examine the estrogenic / antiestrogenic and androgenic / antiandrogenic action of various environmental chemicals in vertebrates , there has been relatively little research on the tremendous array of invertebrates that inhabit fresh and marine water environments .
detailed information concerning the effects and mechanisms of action of industrial chemicals in invertebrates has been obtained from only a few invertebrate species , although invertebrates represent more than 95% of the known species in the animal kingdom ( defur et al .
the masculinizing effects , known as imposex and characterized by development of a vas deferens and a penis in females , of organotin compounds , such as tributyltin ( tbt ) , on snails have been found in about 150 species of mollusks ( gibbs and bryan 1986 ; horiguchi et al .
the mechanism by which tbt induces imposex in marine snails is still unknown , although tbt has been shown to inhibit aromatase activity ( bettin et al .
pg / l , an environmentally relevant concentration , for 4 weeks induced imposex in rockshells ( thais clavigera ) ; however , injection of the aromatase inhibitor , fadrozole , alone or in combination with testosterone ( t ) did not induce imposex ( horiguchi t , katsu y , ohta y , iguchi t , unpublished data ) .
aromatization of [ h]-t to [ h]-e2 was encountered in the rockshell gonad extract ( katsu y , horiguchi t , iguchi t , unpublished data ) .
these results suggest that neither inhibition of aromatase by tbt nor androgen action by tbt is the principal cause of imposex in rockshells .
cells trans - fected with a rockshell er - like sequence showed ligand - independent reporter gene activation ( katsu y , horiguchi t , iguchi t , unpublished data ) , which suggests that the rockshell er - like sequence has a specific unknown function in the rockshell , but it is unlikely that it acts like vertebrate er . in the freshwater snail , marisa cornuarietis , bpa and octylphenol ( op ) at concentrations as low as 1 g / l induced development of an additional vagina , enlargement of the accessory pallial sex glands , and enhancement of oocyte production . in the marine prosobranch , nucella lapillus ,
the same concentrations of bpa and op reduced the length of the penis and the size of the prostate gland ( oehlmann et al .
such results suggest that these snails have a functional er and , thus , that estrogenic chemicals could have a negative impact on these snails .
rxr , one of the nuclear receptors , from humans and from xenopus has been activated by tbt in reporter gene assay systems ( grun f , watanabe h , zamanian z , maeda l , arima k , chubacha r , et al . , unpublished data ) .
tbt and 9-cis retinoic acid have been shown to activate the rockshell rxr , and 9-cis retinoic acid has been shown to induce imposex in the rockshell ( nishikawa et al .
these results suggest that future research to examine the mechanism of action of edcs in invertebrates needs to focus on other nuclear hormone receptors distinct from the er and the ar .
reproductive , acute , or chronic toxicity tests on daphnids have been used widely for aquatic toxicology .
conflicting results on the molting frequency of daphnia magna following exposure to estrogenic chemicals have been reported ( caspers 1998 ) . a xenoestrogen - induced reduction in the molting frequency of d. magna ( niederlehner et al . 1998 ; zou and fingerman 1997 ) could not be confirmed for bpa ( tatarazako et al .
we found that styrene dimers and trimers , leached from disposable polystyrene cups , reduced the number of offspring in ceriodaphnia dubia .
styrenes ( 0.041.7 g / l ) , ecdysones ( 0.11.08 g / l ) , and juvenile hormone agonists ( 1.05 g / l ) reduced fertility , whereas e2 and bpa had no effect on the reproduction of c. dubia .
np ( 280 g / l ) influenced daphnids via membrane damage ( tatarazako et al .
we have cloned a full - length sequence of an ecdysone receptor from d. magna and established an ecdysone reporter gene assay ( watanabe et al .
we and others have revealed that exposure of adult daphnids to juvenile hormones and their analogs induces parthenogenetically reproducing d. magna to produce male neonates ( olmstead and leblanc 2002 , 2003 ; tatarazako et al .
2003 ) . ten juvenoids [ pyriproxyfen , fenoxycarb , methylfarnesoate , juvenile hormone i ( jh i ) , jh ii , jh iii , methoprene , kinoprene , hydroprene , and epofenonane ] have induced male neonate production ( oda et al .
in addition , daphnids are susceptible to the male sex determining effect of juvenoids during early oogenesis ( olmstead and leblanc 2002 ; tatarazako et al .
although there was a wide range of sensitivity to fenoxycarb ( 0.69.3 g / l ) , the production of male neonates in all four species ( moina macrocopa , moina micrura , c. dubia , and c. reticulata ) demonstrates that this phenomenon is a common response to juvenoids ( oda et al .
these findings suggest that juvenile hormone agonists , including some insecticides , affect the chemical signaling responsible for inducing the production of male offspring .
we constructed a cdna library of d. magna and characterized the ests of over 7,000 clones ( watanabe et al .
2005 ) . to understand the molecular functional mechanism of juvenile hormone agonists in the induction of male offspring
, we are currently analyzing juvenile hormone binding protein and establishing a micoroarray system for d. magna .
much of the literature to date on the edc issue focuses on steroid hormone receptor mediated toxicity .
therefore , information on gene and protein expression mediated by hormone receptors is essential for understanding chemical effects .
species differences with respect to the interaction of various chemicals with ers and with regard to the metabolism of chemicals have been observed .
therefore , we are currently cloning receptors of various steroid hormones , and steroid and xenobiotic receptors ( sxr ) , from various animal species , including alligator , quail , and various fish species , in order to find species that are sensitive to edcs .
we are also focusing on orphan nuclear receptors that may provide new insights into the mechanisms of chemical action , as shown with rxr activation by tbt in gastropods and even in xenopus and mice .
analyses of transgenerational effects of xenobiotic agents are also required in order to estimate and confront potential dangers to human and wildlife populations .
there are also species differences in the response of er and sxr to chemicals , in degradation of chemicals , in critical sensitive windows , and in development .
to clarify the adverse effects of chemicals , we need to understand the timing of gene expression ( critical developmental window ) , the amount of gene expression ( amount of chemicals ) in specific organs , the degradation ability of chemicals , and the normal range of various biomarkers in each species . by the application of omic technologies ( genomics , transcriptomics , proteomics , and metabolomics ) in the study of edcs
, we will understand the detailed mechanisms of action of chemicals in the future . in this review , we have focused primarily on receptor - mediated gene expression ; however , it is critical to broaden the spectrum of hormonal disruption in the hypo - thalamic pituitary end gland axes , and to include the ability of animals to cope with stress or chemical communication ( propper 2005 ) .
further basic biological understanding of comparative molecular endocrinology , genomics , and toxicology in animal species is essential in order to be able to apply omics technologies to the study of wildlife species . | chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans . developing organisms are particularly sensitive to estrogenic chemicals .
exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in both reproductive and nonreproductive organs , including persistent molecular alterations .
estrogen - responsive genes and critical developmental windows of various animal species , therefore , need to be identified for investigators to understand the molecular basis of estrogenic activity during embryonic development .
for investigators to understand molecular mechanisms of toxicity in various species , toxicogenomics / ecotoxicogenomics , defined as the integration of genomics ( transcriptomics , proteomics , metabolomics ) into toxicology and ecotoxicology , need to be established as powerful tools for research . as the initial step toward using genomics to examine endocrine - disrupting chemicals , estrogen receptors and other steroid hormone receptors
have been cloned in various species , including reptiles , amphibians , and fish , and alterations in the expression of these genes in response to chemicals were investigated .
we are identifying estrogen - responsive genes in mouse reproductive tracts using cdna microarrays and trying to establish microarray systems in the american alligator , roach , medaka , and water fleas ( daphnia magna ) .
it is too early to define common estrogen - responsive genes in various animal species ; however , toxicogenomics and ectotoxicogenomics provide powerful tools to help us understand the molecular mechanism of chemical toxicities in various animal species . |
the technology has grown tremendously , and now there are numerous types of probes and target elements .
target elements can include genes , oligonucleotides , or bacterial artificial chromosomes ( bacs ) and new microarray chips can contain on the order of a hundred thousand probes .
due to the technology , the signal obtained is a combination of the biological signal and technological signal .
array - based comparative genomic hybridization ( acgh ) technology is similar to cdna arrays and is an extension from conventional cgh that is used to identify and quantify dna copy number changes across the genome in a single experiment .
the advantages of acgh include high - resolution and high - throughput measurement capability allowing for more quantitative analysis of the genomic aberrations . a thorough introduction to the design and manufacture of microarrays
is provided in while provides an introduction to the statistical issues in analyzing microarray datasets . in bac acgh ,
the probes corresponding to locations on a genome are cloned ( grown ) in a bacterial culture and then arrayed to a glass slide .
bac acgh technology can be employed to discover markers in diseases as in [ 58 ] and for detecting genomic imbalances in cancers as described in [ 920 ] . in bac acgh studies ,
the markers for cancer are often discovered by comparing the signal at a given chromosome loci between the tumor sample and a control sample .
specifically , researchers often examine the logarithm ( base 2 ) of the ratio of the tumor sample to the control sample ( log t / c )
. this value will allow researchers to determine the presence of an imbalance in copy number for a given marker between the tumor sample ( t ) and the control sample ( c ) .
it is necessary to normalize the raw log t / c values before subsequent analysis to determine regions of chromosomal imbalance .
normalization procedures have been recognized as necessary for microarray experiments , and a recent search on pubmed ( http://pubmed.org ) reveals over 500 references to articles on microarray normalization . for two - channel microarrays , some of the normalization algorithms similar to the proposed analysis in this paper are cited in [ 21 , 22 ] .
specifically , with regards to acgh datasets , recent normalization algorithms are proposed in [ 2327 ] and in r software packages [ 28 , 29 ] .
our normalization approach for the log t / c data will be similar to the approach in but will feature several important differences in the estimation procedures for the technical effects . specifically , the goal of this paper is to isolate the biological signal in the log t / c by removing the technological signal via the novel smootharray
the technological signal is composed of three major components : ( 1 ) signal due to the intensity of each scanning channel , ( 2 ) signal due to spatial ( array ) location , and ( 3 ) signal due to the spotting technology .
we compare our procedure against other normalization procedures and use several metrics to measure the improvements of our method . although our results can be applied to any print - tip microarray setting , our examples were obtained from the roswell park cancer institute ( rpci ) acgh microarray facility .
note our software is written in the r programming language and is freely available at .
in the rpci acgh microarray facility , differentially labeled total genomic dna from a test and a reference cell population are cohybridized to the bac clones .
after hybridization , a genepix axon scanner generates two images of the array at the wavelengths of light corresponding to the two dyes ( cy3 and cy5 ) .
the images are processed to generate a single number corresponding to each sample ( dye ) for each spot on the array . for the rpci facilities
the resulting ratio of the fluorescent intensities at a location on the chromosomes is approximately proportional to the ratio of the copy numbers of the corresponding dna sequences in the test and reference genomes .
a traditional experiment describing tumor extraction , preparation , and so on is described in . for our bac acgh studies ,
the rpci 19 k bac array was utilized containing 19,000 bac clones ( probes ) that were chosen by virtue of their sts content , paired bac end - sequence , and association with heritable disorders and cancer . reference and test sample genomic dna
( 1 g each ) were individually fluorescent labeled using the bioarray cgh labeling system ( enzo life sciences ) as described in .
the hybridized bac - based acgh slides were scanned using a genepix 4200al scanner ( molecular devices ) to generate high - resolution ( 5 m ) images for both cy3 ( test ) and cy5 ( control ) channels . in bacterial artificial chromosome ( bac )
acgh technology , the target dna elements are physically arrayed in a two - dimensional grid on a chemically modified glass slide . note that the bac clones are stored in freezers on a total of 51 plates ( 384 wells per plate ) .
another source of potential variation is due to the pin array process of printing the bac clones on the glass microarray slide . for our data ,
the 48 pins in the arrayer are arranged in a 12 4 matrix structure , approximately 4.5 mm on center , so that they transport the probes to the slide where each pin fills one region or
the spots are approximately 80 m in diameter , with respective centers 150 m apart from each other to ensure no overlap between spots .
the array has the spots laid out in a 116 348 array of 40368 spots .
more specifically , each of the grids within the array ( corresponding to pin number ) has dimensions 29 29 thus there are 841 spots per grid ( pin ) .
the array 's spot locations are consecutively labeled row - wise within each pin , first numbering within pin 1 ( 1841 ) , followed by the spots within pin 2 ( 8421682 ) , and so forth .
thus , the spot location values range from 1 to 40368 . due to this geometry
, each bac clone is repeated on the array ; in other words , each clone has two spots on each array .
further , note that there are 384/48 = 8 spots per grid per plate . since 8 51 2 = 816 and each grid has 841 spots , there are 841 816 = 25 blank spots in each grid .
each plate is used twice , as each spot is replicated within a grid on the array . put another way
, this procedure can produce the intensity levels of 40,368/2 25 48 = 18984 bac clones per array arranged in a two - dimensional array on the slide that accommodates up to 40368 spots .
the remaining 25 48 = 1200 spot locations remain unused and are , therefore , not considered in this analysis ( note that however , these unused spots may contain valuable information regarding the background and laser scanner settings ) .
the data summary gives the intensity readings from the cy3- and cy5- labeled genetic material for each spot , as produced by the image processing software .
since our dataset is related to oncology research , we will refer to the data in terms of the tumor channel ( t ) and the control channel ( c ) .
for spot i on the array , we will be interested in the logarithmic ratio of the tumor channel ( ti ) to the control channel ( ci ) . in other words , we will focus on mi log2ti / ci for a given spot i. we will define the vector m as the collection of mi values for a given sample .
from there are three major sources of possible systematic variation in m which are a consequence of the experimental procedure and do not contribute to differential expression .
this bias is evident from figure 2 and is a noted source of variation in two - channel microarrays . in the data shown in figure 2 , this bias predominately produces a curvature shape where the lower intensity probes tend to have a large log t / c value .
the second source of variation is the physical layout on the glass slide ; one can imagine that there are spatial effects across the slide ( caused , e.g. , by the way the dye - labeled material is hybridized to the slide ) which would manifest as a pattern of row and/or column effects if the data were analyzed as 348 116 array .
the third source of variation stems from the 384 well plates which are the source of the spots on the glass slide ; one can imagine that there are effects which are localized to one ( or more ) specific plates which would appear as localized effects on the glass slide .
as stated in [ 23 , 36 ] , this bias may be caused by the fact that different clones that are produced in the plates might have experienced slightly different physical conditions during the polymerase chain reaction ( pcr ) or in subsequent purification steps .
also there are potential effects due to the 384 well - plate rows ( 16 rows ) and 384 well plate columns ( 24 columns ) .
note that the localization is complicated because of the arrayer procedure described above ; recall the complex numbering scheme .
one can easily imagine that the pins vary in shape , head size , or some other property that causes the observations to vary from quadrant to quadrant on the array .
equally , one can imagine a serial ( in time ) correlation among the observations caused by , for example , the pins not being adequately cleaned between successive dips into the wells on the plates .
this is not intended to be an exhaustive list of possible sources of systematic variation , but rather simply a short list of obvious possibilities .
the key point here , and in all subsequent analysis , is that we assume a random spatial distribution of the probes on the microarray chip .
in other words , we assume that there is no correlation between a probe 's genomic location and its spatial coordinates on the array .
this random assumption is required in order for our normalization method to preserve the biological signal present in the chip . with this layout and the potential sources of technical variation
note the goal of our smootharray algorithm will be to remove the technical variation in the log t / c values .
the data used to demonstrate the process consist of 219 head and neck tumor samples obtained from the rpci microarray facility .
process consists of the steps described in table 1 applied sequentially to m , where m denotes the vector of mi spot values for a specific array .
let a denote the vector of ai values , where
( 1)ailog2(tici ) , i=1,2,3, ,37968 .
a is the vector of logarithm products for the two channels in an cgh microarray experiment .
in all subsequent analysis , we will use a typical sample from a dataset designed to examine head and neck tumors as obtained from the rpci microarray facility .
the m vector of values from the scanner represents the input values for the smootharray algorithm .
figure 1 shows the raw dataset ( m values ) in the form of an array image and a genomic plot . note
that since each bac clone is printed twice on the array , the genomic plot is obtained by averaging the two mi values for each bac clone .
the representation of m values and the ranked m values based on their location on the array image is a good way to view the spatial bias present on an array .
the plot shown in figure 1(a ) is commonly referred to as an m - xy plot . by design ,
each sample is hybridized against a sex mismatch hence , as seen in the genomic plot ( figure 1(b ) ) , there is a gain ( increase ) in the ratio on the x chromosome mimicking a single - copy gain .
the data is not centered around 0 in figure 1(b ) indicating a bias in the log t / c values .
the first step in the smootharray algorithm employs a loess smoother on the mi values using the ai values as the explanatory variable .
in short , a loess smoother fits a polynomial surface determined by the set of explanatory variables .
we consider this a global operation since the entire set of probes from an array is used in the loess fitting function , regardless of position on the array or genome .
fitting is by ( weighted ) least squares using the loess function in the r package stats .
the result from this operation is a set of fitted values , gl(mi ) , according to the ai values , where we denote the loess function as gl ( ) . the fitted values , gl(mi ) , represent the intensity bias present at spot i due to the probe intensity ai . by subtracting the fitted values gl(mi ) we account for this technological bias .
hence , we carry forth the m vector to the next step , where m is a vector consisting of
( 2)mi=migl(mi ) , i=1,2,3, ,37968 .
m
i represents the signal for spot i after removing the technological signal due to the product of the intensities from the two channels .
the goal in the next step is to remove the spatial bias present in the acgh technology .
the next step in the algorithm removes the technological noise due to the spatial location in the array .
it is reasonable to expect nearby spots to be correlated with each other due to the reagents process and the hybridization process in microarray technology .
the goal in this step is to accurately determine the spatial pattern present in the array and thus remove it .
the data depicted in figure 3 is the starting point for the spatial smoothing step .
rather than perform the spatial smoothing on the data depicted on figure 3 , we will modify our approach to ensure we preserve the biological signal .
the biological signal present in acgh data can be captured using the circular binary segmentation ( cbs ) algorithm described in . in short ,
the cbs algorithm can be applied to cluster the m values into segments of estimated equal copy number according to their location on the genome .
after cbs , each probe i is a member of a specific segment where we will denote cbs(mi ) as the log t / c group mean for the segment containing probe i. we apply the cbs function ( denoted by cbs ( ) ) to the values in figure 3 and compute the residuals from the cbs operation as m cbs(m ) .
the spatial two - dimensional kernel density smoother is applied to the residuals from the cbs operation .
note that , by subtracting the cbs group we are , in a sense , removing the genome / biological signal to ensure that our kernel density estimate of the spatial signal has a minimal amount of biological signal and is only modeling the technical variation .
this is one of the key ways in which our method differs from the algorithm described in . in short ,
smooth.2d in the fields library in r . the smoothing parameter ( bandwidth ) is chosen via a cross - validation ( cv ) procedure .
namely , a random subset is removed from the data and the surface is fit . after fitting a surface , the absolute value loss ( l1 loss ) or the sum of squares loss ( l2 loss ) for the random subset is computed .
the value of the smoothing parameter that yields the smallest sum of squares is used as the optimal value in the kernel smoothing algorithm . after estimating the spatial technical variation ( figure 4(a ) ) , we compute the resulting set of log t / c values with the spatial variation removed . in other words for spot
i , we compute
( 3)mi=miks(micbs(mi ) ) ,
where ks represents the kernel smoothing function .
mi represents the log t / c value for spot i after the technical signal due to intensity and the spatial location has been removed .
at this point , we remove the technological signal due to the spotting of the array .
the technical issues of the spotting procedure noted in [ 22 , 34 , 39 ] demonstrate a solution for cgh microarray chips .
similar to determining the kernel smoothed spatial surface , we determine the pin , plate , plate row , plate column and repetition effects for the vector m populated with mi for spot i. similar to the estimation of the spatial effect , we will estimate the spotting process effects on the dataset where we preserve the biological signal by employing the cbs algorithm .
we apply the cbs function to the m data and then compute the residuals by subtracting the cbs segment mean .
we compute a median of the residuals for each pin , plate , plate row , plate column , and repetition value and use that as our estimate for the spotting process effect .
we obtain the final set of log t / c values representing the remaining biological signal by subtracting the effect of the pin , plate , plate row , plate column and repetition effect from m , that is , the vector of log t / c values after accounting for intensity and spatial effects . to assess our smootharray procedure , we compare it with five other normalization methods .
specifically , we examine raw , grid loess , background subtraction , global median normalization , and quantile normalization . in the raw normalization method
, we use the raw log ratios without performing any normalization . in a grid loess procedure , within each pin grid , a local polynomial regression fit is performed on the mi values using the ai values as the explanatory variables .
the normalized values from this procedure are obtained by taking the difference between the raw values ( mi ) and the fitted loess values .
the loess fitting is done using the loess function in the r package stats .
the background subtraction method is employed by subtracting the estimated background intensity from the estimated foreground intensity of each spot before taking the logarithm ratios .
the global median normalization procedure is performed by estimating the median log ratio on each array and then subtracting this value from the log ratios on the array . with a global median normalization procedure , the empirical ( sample )
the quantile normalization procedure is enacted according to the procedure outlined in . in this procedure
the goal is to impose the same empirical distribution of log ratios to each array .
a mean array is created by taking the sample mean across the sorted values of the log ratios in each array .
then the distribution of the log ratios in the other arrays in the experiment is matched to the empirical distribution of log ratios in this hypothetical mean array . in the results section
as a tool to quantify the results at each step of smootharray in terms of reduction of noise we employed the median absolute deviation ( mad ) on the x chromosome .
the signal was estimated by the mad on the x chromosome since the x chromosome , by design , was always altered by the virtue of the sex - mismatched controls .
further , we do not expect there to be any disease - specific imbalances to occur on the x chromosome .
the mad as calculated on the x chromosome acts as a good measure of the performance of smootharray and other preprocessing algorithms since the genomic log t / c data is ultimately used to call regions of genomic imbalance , and this measure is based on a subset of the genomic data . also note that none of the smootharray steps require knowledge of the genomic location for a given spot .
we are assured that the improvement ( reduction ) in this measurement must be the result of the removal of technical variation and not biological variation . for the chosen array in our experiment ,
figure 2 shows the loess fit as a function of the intensity ( a ) .
the intensity bias in this case is depicted by a convex curve ( red line ) that shows that probes with a small intensity are more likely to have a large log t / c value .
figure 3 shows the resulting m vector as function of location on the array ( figure 3(a ) ) and genomic location ( figure 3(b ) ) . for the genomic plot ( figure 3(b ) )
we averaged the two probes for each bac clone in order to obtain a value for the loci .
the mad for the x chromosome after this step is 0.102 , indicating a slight increase from the starting mad on the x chromosome . the results from studying the spatial effects for this data are provided in figures 4 and 5 .
figure 4(a ) is the spatial effect image as estimated from a kernel density smoother when using l2 loss as opposed to l1 loss .
figure 4(b ) shows the ranked cross - validation ( cv ) values as estimated using an absolute error or l1 loss ( 1 line ) and using a squared error or l2 loss ( 2 line ) . note that , the smoothing parameter ( bandwidth ) for our kernel density smoother changes slightly when using l2 loss versus l1 loss .
the m data is shown via array location in figure 5(a ) and genomic profile in figure 5(b ) .
contrasting figure 5(a ) with the data in figure 1(a ) , it is clear that the technical spatial variation has been removed .
in fact , the mad for the x chromosome after removing the spatial effect is 0.075 , indicating a reduction from the mad prior to this step .
figures 6 , 7 , 8 and 9 show the side by - side box plots of each of the spotting process effects before and after their removal .
the final dataset after the smootharray process is displayed in an array location image ( figure 10(a ) ) and genomic profile ( figure 10(b ) ) . from a closer analysis in figures 6(a)9(a ) , there is not an obvious outlier in terms of the spotting process effects after accounting for the intensity and spatial bias .
the mad for the x chromosome after removing the spotting process effect is 0.069 , which represents a reduction when compared to the mad score of 0.075 prior to accounting for this effect .
this 20 percent reduction of noise indicates that subsequent acgh calls of gains and losses should be improved due to the smootharray process .
figure 10(c ) demonstrates this reduction in noise as a function of location on the chromosome .
the raw genomic profile is represented in grey points , while the values after smootharray are represented in red . as a global measure of the normalization methods
, we also examined the mad for the x chromosome for each of the 219 samples in the experiment designed to examine biomarkers in head and neck tumors .
the median mad prior to the smootharray process ( raw normalization ) is 0.1465 , while the median mad across the 219 samples after
table 2 displays the median mad across the 219 samples for each of the competing normalization methods while figure 11 shows the distribution of the mad for each method . from table 2 and figure 11
we also examined each of the normalization methods by using a nonparametric one - sample t test to examine the significance of the log ratios on the x chromosome .
for each probe , we obtain a p value measuring the significance from a mean of 0 for each log ratio .
since each sample is hybridized against a sex - mismatched control , we expect each log ratio on the x chromosome to have a significantly small p value . for this analysis , we employed the wilcoxon rank sum test ( a nonparametric test ) for each log ratio .
the cutoff of.05 was chosen since , on a univariate level , this would indicate a single copy gain or loss . using this metric , we see that the smootharray normalization compares favorably to the grid loess , and global median normalization methods . as a further comparison of each normalization scheme ,
the use of m - xy plots allows the users a visual metric to compare the different normalization methods .
figure 12 allows the user to determine if any spatial abnormalities are present on the array . from examining figure 12 ,
through a series of sequential steps we have developed an algorithm called smootharray which normalizes the logarithmic ratios from a cgh- based microarray platform .
secondly the signal due to the spatial location on the microarray is accounted for and removed .
each of these sources of signal is a well - documented problem in acgh literature . throughout the algorithm our philosophy was to employ parsimonious and straightforward approaches to correct for the technical effects at each step .
we note the similarity of our smootharray process with the preprocessing defined in .
specifically , both methods remove noise due to intensity effects , spatial effects , and plate effects .
namely , we employ the cbs algorithm throughout our smootharray algorithm to ensure that we preserve the biological signal , in other words , to ensure that we only remove the technical variation present in the dataset .
although minor , we use a loess rather than the lowess method as in to remove the intensity bias .
we also employ a kernel density smoother with bandwidth chosen via cross - validation to account for the spatial bias rather than a 11 11 window median smoother employed in .
further , we remove the effects due to the pin arrayer procedure and additionally the plate row , plate column , and repetition effect . by comparing these results on the x chromosome
we show the results of our smootharray algorithm . also , our smootharray algorithm has the flexibility to explore the cross - validation steps using an absolute ( l1 ) loss function or squared error ( l2 ) loss function .
note that , currently we have a background option in acghplus which allows the user to subtract a weighted version of the background .
future work will explore employing a step that takes into account the background in preprocessing cgh bac arrays .
smootharray via two ways ( 1 ) by using the blank spots at the end of each grid ( see section 2.1 ) or ( 2 ) by using images obtained from the genepix scanner where segmentation algorithms are applied to determine a background signal for each spot . by examining the spot process effect we can employ violin plots to examine the quality control for each array - design variable pin , plate , plate row , plate column , repetition .
note that the repetition effect acts as a surrogate measure for a potential time effect .
that is , the time elapsed in spotting the probes on the glass slide is represented by the repetition variable , since the second spot for each bac clone is not spotted until all other bac clones have been spotted once .
violin plots have numerous references in current statistical literature as a way of combining the information available from local density estimates with the basic summary statistics inherent in standard box plots . combining the box plot and
the density trace on a single plot , comparing the distributions of several variables via violin plots , is a great tool for cgh microarrays . for the rpci acgh lab
firstly , the spot can be flagged for having a low signal - to - noise value .
the signal - to - noise value is determined by taking the mean value of the pixels in the signal and dividing them by the standard deviation of the background .
if this value is too low ( < 2.5 ) then the spot is of poor quality .
secondly , the spot can be manually flagged by the user to determine poor quality , or thirdly , the spot can be flagged of poor quality because of a dim signal in one of the channels .
dimness is determined by having a mean signal value under a prescribed cutoff in one of the channels . with this set of flags used for poor spot quality , for a given set of samples ( experiment )
, we can compute the rate for each spot being flagged with a qc problem .
then we can examine the distribution of this percentage via violin plots across each spotting process variable .
figure 13(a ) shows the distribution of the spot percentages across each pin , where the spot percentage is the number of times the given spot passed quality control divided by the number of samples in the experiment ( 219 samples ) .
similarly , figure 13(b ) shows the distribution of the percentage of bac clones from each plate that passed the quality control . by studying figure 13
, it is shown that pins 1 and 3 may be suspect in terms of quality control , while several plates have a larger frequency of quality control problems .
specifically , plate 28 consists of bac clones that are consistently flagged for quality control problems . by examining figures 13(c ) , 13(d ) , and 13(e ) for this experiment , there does not appear to be any obvious problems affecting the plate row , plate column , or repetition number for the bac clones .
the concept of violin plots for quality control can further be extended to other commonly reported spot variables such as background mean , background standard deviation , and other potential outlier flags . for future work
, we plan to examine another quality control measure relevant to the bac clones . due to the nature of the bac clones and the updates to the human genome
, it is possible that the bac clones could be mismapped from their position on the genome .
mismapped bac clones can manifest themselves as appearing as an outlier when viewed via their genomic profile . using a mixture model approach
, we plan to subset the number of bac clones under consideration based on estimating the probability for a given bac clone to be mismapped .
this approach shows great promise based on our early attempts at modeling mismapped bac clones .
a key component in preprocessing acgh data lies in understanding the subsequent analysis steps . with our preprocessed data ,
the next step in the acgh bac analysis pipeline involves characterizing the genome in terms of detecting regions of chromosomal copy number variations ( gains and losses ) .
the softwares and algorithms designed for this analysis include cghcall and other breakpoint detection methods , for example , [ 4446 ] .
a slightly different approach allows the researcher to analyze each chromosomal arm rather than examining within each arm for chromosomal breakpoints .
this approach allows the researcher to characterize a chromosome in terms of overall imbalance ( with confidence ) rather than focus on specific regions of gains and losses .
our smootharray method clearly shows improvement in reducing the noise for a dataset of 219 samples designed to study head and neck tumors .
further , when using several quality control metrics , our method performs favorably to five other competing normalization methods described in materials and methods . for future work , we plan to extend our comparisons to quantify the amount of improvement over other competing pre - processing methods such as those in [ 2327 ] .
experiments and comparisons such as those employed in [ 32 , 48 , 49 ] can be used to assess the performance and determine the best analysis routes for identifying genomic imbalances in bac acgh datasets .
this future study would also compare the subsequent algorithms that assess gains and losses across the genome .
this novel method compares favorably against several other normalization measures when evaluated using several quality control metrics . for this study
, we focused on data obtained from the rpci microarray facility on a study of 200 head and neck tumor samples . for this experiment ,
our algorithm reduced the noise by approximately 23 percent . by removing the technological noise due to the intensity effect , spatial effect , and spotting process , the resulting data has reduced noise and is suitable for subsequent analyses to determine chromosomal regions of gains and losses
. the smootharray method also offers the user the option to examine several quality control figures which allows the researcher to pinpoint problems that may arise in the spotting process . | the main focus in pin - tip ( or print - tip ) microarray analysis is determining which probes , genes , or oligonucleotides are differentially expressed . specifically in array
comparative genomic hybridization ( acgh ) experiments , researchers search for chromosomal imbalances in the genome . to model
this data , scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise . in this paper
we propose smootharray ,
a new method to preprocess comparative genomic hybridization ( cgh ) bacterial artificial chromosome ( bac ) arrays and we show the effects on a cancer dataset . as part of our r software package
acghplus , this freely available algorithm removes the variation due to the intensity effects , pin / print - tip , the spatial location on the microarray chip , and the relative location from the well plate .
removal of this variation improves the downstream analysis and subsequent inferences made on the data .
further , we present measures to evaluate the quality of the dataset according to the arrayer pins , 384-well plates , plate rows , and plate columns .
we compare our method against competing methods using several metrics to measure the biological signal . with this novel normalization algorithm and quality control measures
, the user can improve their inferences on datasets and pinpoint problems that may arise in their bac acgh technology . |
functional abdominal pain ( fap ) is one of the most common diseases , and large percentages of children suffer from it .
some studies showed a prevalence of 9% - 15% ( 1 ) . according to the rome iii criteria
, the functional abdominal pain must include all of the following criteria : persistent or episodic abdominal pain insufficient criteria for other functional gastrointestinal disorders no evidence of an anatomic , inflammatory , metabolic or malignancy disorders these criteria should be present at least once per week for at least 2 months prior to diagnosis ( 2 ) .
existence of fap in childhood can increase the risk of irritable bowel syndrome ( ibs ) ( 3 ) , predictors of psychiatric disorders and other psychosomatic symptoms in adulthood ( 4 ) .
the prevalence of celiac disease ( cd ] in children with fap was about two times the normal population ( 5 ) .
current recommendations for treatment of fap include : reassuring parents , high - fiber diet , antidepressants and peppermint oil ( 6 ) .
it was indicated that probiotics are more effective than placebo in the treatment of fap ( 7 ) .
they also prevent bacteria from proliferating in the small intestine and are also immune system modulators ( 8) .
most of the studies have shown that the use of these agents is safe , effective and well - tolerated ( 9 ) .
some studies have demonstrated that the number of intestinal lactobacillus and bifidobacterium in children with fap and ibs are decreased ( 12 ) .
therefore , the use of these microorganisms can be effective in the treatment of fap .
the purpose of this study was to determine the efficacy of lactobacillus reuteri ( biogaia ) for treating chronic functional abdominal pain in children .
it was conducted on 80 children aged 4 to 16 years , who visited the gastroenterology clinic of ayatollah mousavi hospital in zanjan from 2012 - 2013 ( around 14 months ) .
all 4 - 16 years old children with at least 1 attack of abdominal pain per week during the past 2 months were evaluated for functional abdominal pain ( fap ) based on rome iii criteria ( 2 ) .
all parents were given enough information about the study and consent to participation in the project obtained .
inclusion criterion included : - all children aged 4 to 16 years with functional abdominal pain ( fap ) according to rome iii diagnostic criteria exclusion criteria included : - having an abdominal pain with known organic cause - having a history of abdominal and gastrointestinal surgery - having ftt or weight loss more than 5% of body weight - having any abnormal paraclinical finding including complete blood count , urinalysis , stool examination for occult blood , biochemistry , abdominal ultrasound , liver function tests , serum amylase and lipase - having a history of drug use in the past 3 months including antidepressants or laxatives - having any kind of chronic illness - having a history of abdomen blunt trauma the patients were divided into two groups based on the randomized allocation ( computer registration ) .
the first group ( case group ) was given probiotic from lactobacillus family and reuteri strain ( biogaia brand manufactured by sweden health care company ) 5 drops per day orally equivalent to 108 cfu ( colony - forming units ) for 4 weeks .
the physicians and the patients were unaware of the contents of the medications prescribed ( double blind study ) .
patients were followed up for 4 weeks after cessation of treatment . in follow up period frequency and severity of pain , other symptoms and drug side effects were carefully recorded .
demographic data and pain characteristics ( frequency , location , presence or absence of associated symptoms , need to sedative for pain relief ) were recorded .
pain intensity was assessed according to the wong - baker faces scale system ( figure 1 ) ( 13 ) .
the scale includes six face images showing the effect of the pain , ranging from relax face ( no pain ) in the left side and intense face ( most severe pain ) in the right side .
follow up the researcher assessed intensity pain scores , frequency of pain , and ultimately response to treatment . finally the data were analyzed with spss software and using descriptive statistics , the frequency distribution chart , chi - square test and relative risk was calculated . in all stages of the analysis ,
p - value equal or less than 0.05 ( p - value 0.05 ) was considered statistically significant .
during a 14-month study , 270 children with abdominal pain were referred to the pediatrics gastroenterology clinic . of these , only 80 children fulfilled the criteria of functional abdominal pain ( rome iii ) , which were divided into two groups .
forty children were in the case group ( biogaia recipient group ) half of whom were boys .
other children ( 40 cases ) were in the control group ( placebo recipient group ) , 52.5% of these were males and 47.5% females .
the mean and standard deviation of age in the case and control group were 6.26 2.10 and 6.26 2.61 years , respectively .
there was no statistically significant difference in terms of mean and standard deviation of weight ( table 1 ) ( p = 0.99 ) . at baseline 97.5% of children in case group and 90% of the control group did not have any associated symptoms .
fever and anorexia were seen in 2.5% and 7.5% of children in control group , respectively . in this sense , both groups were consistent ( p = 0.16 ) , 95% of patients in the case group and 97.5% in the control group had periumbilical pain , and the remaining had hypogastric pain .
intensity of abdominal pain in the case group according to scoring system of wong - baker faces scale was about 30% more , 57.7% even more and 12.5% whole . in the control group
, there was no significant difference between the groups ( table 2 ) . at the end of the second month , 40% in case group and 65% in control group were no hurt .
zero score is used for no hurt and 6 for whole . applying this scoring system ,
average score obtained in the control group at baseline was 4.05 0.71 , at the end of first month 2.08 1.56 and at the of second month 2.25 1.46 .
in the case group corresponding scores were 3.83 0.63 , 2.50 1.45 and 2.53 1.43 , respectively . according to these results
, there was significant difference between the groups at baseline and first month after treatment ( p = 0.0001 ) , while there was no significant difference at the end of second and first month in both groups ( in case group p = 0.317 and control group p = 0.227 ) . at first month , 50% of children in case group and 65% in control group had no pain and at second month 47.5% in case group and 52.5% in control group did not report any pain . in this respect
average pain episodes per week in the control group was 1.33 0.47 at baseline , 0.40 0.59 at first month and 0.530.59 at second month . in the case group
it was 1.45 0.55 at baseline , 0.68 0.76 at first month and 0.70 0.75 at second month . here also , there was a significant change in both groups compared to the first month ( p = 0.0001 ) , but the change was not significant in the second month compared with the first month ( in case group p = 0.317 and control group p = 0.059 ) .
this study investigated the effects of lactobacillus reuteri and placebo in reducing severity and frequency of pain in 80 children aged 4 to 16 years with chronic functional abdominal pain .
functional gastrointestinal disorders ( fgid ) are one of the most common childhood disorders , pathophysiology of which is still not well defined ( 14 ) .
some of the studies have shown that the number of lactobacillus is significantly reduced in the intestine of these patients ( 15 , 16 ) .
thus , replacement of these microorganisms was an acceptable hypothesis for treatment of functional abdominal pain . in our study both cases and control groups in terms of age , sex ,
weight , number of pain episodes per week , location of pain and associated symptoms were consistent .
however , there was no significant difference between the groups in the intensity and frequency of pain episodes .
after one month of cessation of drugs , no significant change was observed compared to the first month .
our study revealed that use of probiotics in treating fap had no preference to placebo . in literature reports regarding the effect of lactobacillus reuteri in the treatment of fap
niv et al . as in our study , found that the effect of lactobacillus reuteri was similar to placebo in reducing abdominal pain and no more than that ( 17 ) . unlike our study , claudio romano et al reorted that lactobacillus reuteri reduces the severity of pain in children but , similar to our results does not reduce the frequency of pain ( 18 ) .
the dose of probiotic was chosen based on previous published studies ( 19 ) , its manufacturer s recommendations ( 109 cfu ) and duration of treatment as mentioned in the literature ( 20 ) .
however , the optimal dose and duration of drug therapy in the treatment of fap is not clearly identified yet . in this study
, we treated the samples randomly divided into two groups , so that the groups were matched for age and sex .
other limitation of the study was not having access to patients over 13 years old .
most of them were referred to the adults gastroenterology clinic and could not be included in the study .
so , average age of the patients in our study was considered lower than that in other studies . to achieve appropriate management of fap
our results showed that , although lactobacillus reuteri may be effective in reducing severity and frequency of pain , its effect is not greater than placebo .
again our study proved that the use of placebo in the treatment of functional gastrointestinal disorders can be helpful , which emphasizes the fact that main causes of the disorder are mental and emotional problems , and also environmental stress , whereas the organic causes are less involved .
the major contribution to the treatment of fap is to ensure the child and parents , so stress could be reduced . | background : functional abdominal pain ( fap ) is one of the most common diseases , and large percentages of children suffer from it.objectives:the purpose of the study was to evaluate the effect of lactobacillus reuteri in treatment of children with functional abdominal pain.patients and methods : this study was a randomized double - blind placebo - controlled trial .
children aged 4 to 16 years with chronic functional abdominal pain ( based on rome iii criteria ) were enrolled in the study .
they were randomly divided into two groups , one receiving probiotic and the other placebo .
results : forty children received probiotic and forty others placebo . there were no significant differences in age , weight , sex , location of pain , associated symptoms , frequency and intensity of pain between the groups .
the severity and frequency of abdominal pain in the first month compared to baseline was significantly less and at the end of the second month , there was no significant difference between both groups compared to the end of the first month.conclusions:this study showed that the severity of pain was significantly reduced in both groups
. there was no significant difference in pain scores between them .
the effect of probiotic and placebo can probably be attributed to psychological effect of the drugs . |
the administrative records from a large , private workers compensation insurance company in the united states were used in this study .
these claims come from various different organizations , industries , company sizes , and geographic locations .
all claims with complete data from january 1 , 2002 until december 31 , 2008 were assessed for inclusion . only claims with at least 1 day of compensated lost work time were included .
lost work time included both days of temporary partial disability ( tpd ) and days of temporary total disability ( ttd ) .
claims were followed for 1 year from the date at which lost work time began or until indemnity payments finished if that occurred within a year .
several restrictions were applied to the claims ( initial pool = 427,571 ) . for claimants who had multiple claims within a single calendar year from 2002 to 2008 , only the first claim within the calendar year was included in analyses ( 13,222 excluded ) .
this was done to ensure that we only followed a single claim from a claimant within 1 calendar year .
claimants who received a lump sum payment within 1 year of claim initiation were also excluded ( 5701 excluded ) . in several instances ,
claimants first received compensation for lost work time more than 1 year after claim initiation .
the sample was restricted to only include claims wherein lost work time first occurred within 1 year of claim initiation ( 6038 excluded ) . finally , some claimants experienced multiple episodes of lost work for a single claim resulting from the same condition .
this occurred in cases wherein an individual went back to work after an injury / illness for some time but then had to go back out of work again as a result of the same condition .
for the current study , claimants were considered to have multiple episodes of lost work time when they returned to work for at least 14 days before having to go back off again .
cases wherein the individual returned to work for less than 14 days before going back out were considered a single episode .
age and tenure at the time of claim initiation were the main predictor variables for the analyses .
the outcome variable was the length of disability , calculated based on the date that a claimant first received tpd or ttd until the date at which tpd or ttd ended .
tpd or ttd were considered to have ended when no paid disability days were taken for at least 14 calendar days consecutively .
when the length of disability exceeded 1 year , which occurred in 28,733 claims , the value for the length of disability was censored at 365 days . to address issues with normality ,
several variables that might confound the relationship between age , tenure , and length of disability were included in the analyses .
these variables included gender , annual income , litigation status , and year of injury / illness .
the first 15 were in increments of $ 10,000 ( eg , $ 0 to $ 9999 ) .
litigation status was coded 1 if the insurer assigned an attorney to the claim and 0 if not .
a series of dichotomous variables was used to represent the year of injury / illness with 2002 as the reference year . the crossed effect between industry and the diagnosis chapter
industry was categorized into 10 groups including the following : ( 1 ) agriculture , forestry & fishing , ( 2 ) mining , ( 3 ) construction , ( 4 ) manufacturing , ( 5 ) transportation , communications , electric , gas , & sanitary services , ( 6 ) wholesale trade , ( 7 ) retail trade , ( 8) finance , insurance , & real estate , ( 9 ) services , and ( 10 ) public administration .
the most commonly used international classification of diseases , ninth revised edition ( icd-9 ) diagnosis appearing in the claimant 's medical file for the first 15 days of medical bills was used as the primary diagnosis for the claim .
the crossed - effect approach was selected instead of treating diagnosis chapter and industry separately because it is likely that certain diagnoses are more prevalent in some industries than others .
in addition , the impact of specific diagnoses on the length of disability is likely to be dependent on the industry in which an individual works .
for example , the length of disability might be shorter following a leg fracture in the finance , insurance , & real estate industry than in the construction industry . by using a crossed - effect approach we are better able to take this into account .
age and tenure at the time of claim initiation were the main predictor variables for the analyses .
the outcome variable was the length of disability , calculated based on the date that a claimant first received tpd or ttd until the date at which tpd or ttd ended .
tpd or ttd were considered to have ended when no paid disability days were taken for at least 14 calendar days consecutively . when the length of disability exceeded 1 year , which occurred in 28,733 claims , the value for the length of disability was censored at 365 days . to address issues with normality
several variables that might confound the relationship between age , tenure , and length of disability were included in the analyses .
these variables included gender , annual income , litigation status , and year of injury / illness .
the first 15 were in increments of $ 10,000 ( eg , $ 0 to $ 9999 ) .
litigation status was coded 1 if the insurer assigned an attorney to the claim and 0 if not .
a series of dichotomous variables was used to represent the year of injury / illness with 2002 as the reference year .
the crossed effect between industry and the diagnosis chapter was estimated using random effects in the analyses .
industry was categorized into 10 groups including the following : ( 1 ) agriculture , forestry & fishing , ( 2 ) mining , ( 3 ) construction , ( 4 ) manufacturing , ( 5 ) transportation , communications , electric , gas , & sanitary services , ( 6 ) wholesale trade , ( 7 ) retail trade , ( 8) finance , insurance , & real estate , ( 9 ) services , and ( 10 ) public administration .
the most commonly used international classification of diseases , ninth revised edition ( icd-9 ) diagnosis appearing in the claimant 's medical file for the first 15 days of medical bills was used as the primary diagnosis for the claim .
the crossed - effect approach was selected instead of treating diagnosis chapter and industry separately because it is likely that certain diagnoses are more prevalent in some industries than others .
in addition , the impact of specific diagnoses on the length of disability is likely to be dependent on the industry in which an individual works .
for example , the length of disability might be shorter following a leg fracture in the finance , insurance , & real estate industry than in the construction industry . by using a crossed - effect approach we are better able to take this into account .
to analyze the relationship between age , tenure , and the length of disability , random - effects models were utilized instead of ordinary least squared regression models , due to the non - independence of observations across the major diagnosis chapters and industry groupings . using a crossed - effect approach , random effects for each diagnosis chapter by industry grouping
only pairings with at least 20 claims per pairing were included in the analyses . in the first stage of the analyses ,
possible nonlinearity in the relationship between age and the length of disability was examined . the linear term for age
was added to the first model . in the second model , the squared term for age was added . in the second stage of the analyses ,
the linear and nonlinear age and tenure terms reaching statistical significance were then included in a combined model with both age and tenure . in the final stage of the analyses ,
analyses were estimated using stata 13 ( stata corporation , college station , tx ) .
the analyses presented have a large sample size and thus a p value of less than 0.001 was considered statistically significant .
in addition , we present the predicted lengths of disability for the different statistically significant relationships addressed . we did not use a specific cutoff for determining the meaningfulness ( clinical significance ) of a difference in the predicted lengths of disability , as this number likely varies dependent on the stakeholder group ( ie , employers , policy makers , or clinicians ) .
of all claims ( 361,754 ) , 31% were for women , half ( 50% ) had an annual income greater than $ 30,000 , and approximately a quarter ( 27% ) were involved in litigation .
the number of claims by year declined from 58,793 claims in 2002 to 47,097 claims in 2008 .
the largest industries represented in the claims were manufacturing ( 23% ) , transportation , communications , electric , gas , & sanitary services ( 23% ) , and services ( 22% ) .
the most common diagnosis chapters were injury and poisoning ( 53% ) and diseases of the musculoskeletal system ( 29% ) .
length of disability ranged from 3 days to 365 days , with an average of 88 days .
the age of claimants in the sample ranged from 18 to 80 years , with an average of 41 years , and the length of tenure ranged from 0 to 30 years , with an average of 6 years . in our sample ,
the correlation between age and tenure was 0.38 ( p < 0.001 ) .
for the first stage of the analyses , the relationship between age and the length of disability was estimated .
, we found that the relationship was slightly nonlinear ( figure 1 ) , with both the age and age - squared terms reaching statistical significance .
the relationship changes very little when adjusted for tenure ( model 5 ) than when not adjusted for tenure ( model 2 ) .
when adjusted for tenure , the predicted length of disability varies by approximately 18 days across ages 18 to 80 , ranging from 32.3 days at age 18 to 50.9 days at age 80 . regarding the adjustment of the model for tenure , we compared the age coefficients from model 2 , with the age coefficients from model 5 using a wald test .
we found that the age coefficients were statistically similar across the models ( = 3.71 , p > 0.05 ) .
plot of the relationship between age and the length of disability when adjusted and unadjusted for tenure .
this figure is based on predicted values for the length of disability at different ages . in the second stage of the analyses ,
, we found a linear relationship with the length of disability , with the tenure - squared term ( model 4 ) not reaching statistical significance .
the tenure relationship was estimated unadjusted for age ( model 3 ) and adjusted for age ( model 5 ) .
we found the relationship to be quite different when the model was adjusted for age .
a wald test comparing the tenure coefficients for the adjusted and unadjusted models revealed a significant difference ( = 49.82 , p < 0.001 ) .
the adjusted and unadjusted relationships are plotted in figure 2 . when the tenure - length of disability relationship was not adjusted for age
, there was a positive relationship with the predicted length of disability increasing as tenure increased .
the predicted length of disability ranged from a low of 42.1 days at 1 year of tenure to a high of 43.7 days at 20 years of tenure .
in contrast , when the relationship was adjusted for age , the relationship became negative with the predicted length of disability decreasing as tenure increased . in this model ,
the predicted length of disability ranged from a high of 43.8 days at 1 year of tenure to a low of 40.3 days at 20 years of tenure .
plot of the relationship between tenure and the length of disability when adjusted and unadjusted for age .
this figure is based on predicted values for the length of disability at different lengths of tenure .
in addition to examining the main effect relationships of age and tenure with the length of disability separately , we also investigated differences between the relationships in their relative strength . using a wald test
, we compared the absolute values of the coefficients for age and tenure in the combined model ( model 5 ) .
the results revealed that the age - length of disability relationship was considerably stronger than the tenure - length of disability relationship ( = 526.82 , p < 0.001 )
. in the final stage of analyses , the interaction between age and tenure with the length of disability was estimated .
a significant interaction was found between age and tenure , which is plotted in figure 3 following the procedure of aiken and west .
the age - length of disability relationship was plotted for various different lengths of tenure so that variation in the relationship across tenure levels might be observed .
this figure is based on predicted values for the length of disability at different ages and lengths of tenure . as seen in figure 3 , at low levels of tenure ( less than 5 years ) ,
the relationship between age and the length of disability is a slightly inverted u - shaped relationship wherein the length of disability increases with age until around age 70 at which point the length of disability then begins decreasing slightly up to age 80 .
in contrast , at higher levels of tenure ( more than 10 years ) , the length of disability continuously increases with age .
the positive relationship between age and the length of disability is greater as tenure increases .
prior to age 70 , as tenure increased , the predicted length of disability decreased ; however , after age 70 , as tenure increased , the predicted length of disability began to increase .
in general , the highest predicted length of disability was for the oldest workers with high tenure , whereas the shortest predicted length of disability was for younger workers with the lowest tenure .
the values of tenure might be thought to be conditional on age as certain values of tenure are not possible at younger ages ( eg , 20 years of tenure at 30 years of age ) .
a series of analyses were therefore conducted with this conditional relationship in mind by attempting to estimate models wherein the full sample of claimants has the potential to have any of the lengths of tenure regardless of their age . in the first model
, we examined the interaction between age and tenure with tenure dichotomized into less than 2 years of tenure compared with 2 or more years of tenure .
the results of this model were largely similar to the model with tenure measured continuously ( supplemental figure 1 ) . in the second model
, we restricted the sample to claimants age 30 or older and we also restricted the sample to claimants with less than 10 years of tenure .
as seen in supplemental figure 2 , a similar finding emerged as in the full model . due to the large number of claims ( n = 28,733 ) with a length of disability greater than 365 days , sensitivity analyses assessed whether the study findings varied when those claims were not included in the model .
all analyses were replicated on the 333,021 claims with a length of disability of less than a year .
the results were found to be consistent within this subsample . as with the full sample , the age - length of disability relationship was found to be nonlinear , whereas the tenure - length of disability relationship was found to be linear .
the findings for age did not change when the model was adjusted for tenure . for tenure ,
the relationship with the length of disability did reverse when adjusted for age . also , the age - length of disability relationship was found to be stronger than the tenure - length of disability relationship .
finally , the interaction pattern between age and tenure was similar in the subsample model .
this study examined a model of age , tenure , and the length of disability in a large sample of workers compensation claims from the united states with the goal of better understanding the complexity among these factors . for age and the length of disability ,
in general there was a positive relationship with the length of disability increasing as age increased and this relationship varied very little based on whether or not it was adjusted for tenure . over a 2.5 week difference in the predicted length of disability
specifically , the relationship began to flatten around age 70 and the length of disability increased very little with age from 70 to 80 years .
whereas few studies have explored possible nonlinearity in the age - length of disability relationship , most studies have found a generally positive relationship between the two . in the one study that did assess potential nonlinearity ,
the relationship was still linear , but in this previous study , the age range only extended to 65 . in the present study , the observed nonlinearity was primarily for workers over age 65 , which might explain the divergence in our results .
it is possible that the plateauing of the age - length of disability relationship after age 65 reflects the healthy worker effect , in which as age increases , only
. the lack of variability in the length of disability at older ages could be the result of only healthier workers still being in paid employment and therefore eligible for workers compensation or it could result from the oldest workers feeling the need to go back to work sooner to keep their jobs secure .
although the positive relationship between age and the length of disability has been shown in previous studies , there has been little empirical work testing the reasons for this relationship .
there are , however , several possible explanations , such as the increasing likelihood of having comorbidities with age that might complicate the recovery process and physical changes that occur with age that might increase recovery time .
there might also be aspects of individuals working conditions that might influence the length of disability .
employers might be more likely to offer accommodations to younger workers or to give more encouragement to younger workers to rtw quickly after injury .
interestingly , the relationship differed greatly when it was adjusted for age compared with unadjusted for age .
when unadjusted for age , there was a positive relationship such that as tenure increased , the length of disability also increased .
although the reverse was true when adjusted for age with the length of disability decreasing as tenure increased .
this finding suggests that when the model did not include age , tenure was partially acting as a proxy for age .
our finding of a negative relationship when adjusted for age is in line with previous research .
we found approximately a 3-day decrease in the predicted length of disability going from 1 year to 20 years of tenure .
it is important to note that although the relationship was statistically significant , even after the adjustment for age , the predicted length of disability only varied by a relatively small number of days over the range of tenure . from a clinical standpoint
, a 3-day difference might not be very meaningful , in which case tenure would not be considered a primary factor in predicting the length of disability ; however , for employers and insurers who might be considering the length of disability for thousands of workers , even a 1 day difference in the length of disability can be important . in terms of the relative strength of the relationship with the length of disability for age and tenure , although both age and tenure remained statistically significant , age was found to be a stronger predictor of the length of disability than tenure .
one possible explanation for this finding is that the mechanism behind the relationship between age and the length of disability , which might primarily reflect delayed recovery as a result of physiological changes associated with aging , has a greater impact on duration of disability than the mechanism behind the relationship between tenure and the length of disability .
although open to conjecture , the tenure - length of disability relationship likely involves a desire to rtw quickly because of a stronger organizational attachment ; however , despite wanting to rtw quickly , older workers might need to remain out of work longer due to the additional time required for them to recover .
this would result in the worker 's age having a greater impact on the length of disability than the worker 's length of tenure .
we also found an interaction between age and tenure with the length of disability . at the youngest ages
, we found very little variability in the length of disability across the tenure groups .
this is logical as there is a far smaller range for the lengths of tenure for individuals who recently entered the workforce .
in contrast , for midlife workers aged 45 to 50 , there was approximately a 4-day difference in the predicted length of disability across the tenure groups .
the predicted length of disability was the shortest for midlife workers with high tenure compared with low tenure .
this finding might reflect that in comparison to lower tenure midlife workers , workers in midlife that have been with their employers longer are more motivated to rtw quickly and know about the availability of resources at their organization , such as workplace accommodations , which facilitate a faster rtw . for workers in the typical retirement ages of 65 to 70 ,
the predicted length of disability was found to vary very little across the tenure groups , but by age 80 , the predicted length of disability varied by approximately a week , with lower tenure workers having a shorter predicted length of disability than high - tenure workers .
it is possible that older workers with high tenure feel secure in their positions , and thus might not feel the need to rush back to work after a work - related injury / illness .
in contrast , older workers with low tenure might hurry to rtw for fear of losing their jobs and being unable to find other employment due to their age .
this might have contributed to differences in the length of disability across lengths of tenure in the oldest workers .
the interaction between age and tenure found in our study is somewhat different from previous studies . in one study , for workers over the age of 53 , the number of days off of work following a work - related accident was higher for low - tenure workers than high - tenure workers . in this previous study ,
it is possible that this might contribute to the divergence of the findings as in our study , there was a turning point in the interaction between ages 50 and 80 . at age 50 ,
our findings were consistent with this previous study with the predicted length of disability being highest for low - tenure workers , but by age 80 the reverse was true . in another study , for homecare workers where the maximum age of participants was 64 years , the length of disability was found to be greater for the oldest workers with high tenure than those with low tenure , whereas in residential care workers , the oldest workers with medium tenure had the longest length of disability , followed by the high - tenure and then the low - tenure workers .
this previous study focused on certified sickness absence as opposed to work - related injury / illness specifically that could account for the differences as certified sickness absence involves a large number of chronic medical conditions .
first , the stronger relationship between age and the length of disability than between tenure and the length of disability has implications for research focused on the prognostic factors for rtw . by quantifying the magnitude of the relationships in a single model
, we were able to show that age might be a more important factor to focus on than tenure .
if tenure were to be included in models , the models should also include age , as the relationship between tenure and the length of disability actually reversed when adjusted for age .
further , the age by length of disability relationship varied across tenure levels . from a clinical perspective , our results point to the need to gain a better understanding of the factors leading to the relationship between age and the length of disability .
age - related changes in recovery time after any type of condition are likely to play a major role in this relationship , but to the extent that other factors might also influence this relationship might have the potential to inform interventions and treatment plans .
for example , older workers might have a greater length of disability because suitable accommodations in the workplace are not made to facilitate returning to work .
regarding the interaction between age and tenure , as the workforce continues to age and the nature of careers also evolves , it is important to consider the interplay between these two factors on the length of disability .
up until the typical retirement age , for workers over the age of 50 , the length of disability was higher for workers with lower tenure .
workers in midlife and those approaching retirement age who are new to their organizations might need additional support in the rtw process
. these workers might be less familiar with the resources available to them and might not have strong organizational and supervisor relationships to rely on . from an intervention perspective , it might be important to identify what types of programs and policies would be most helpful in these workers rtw process .
although our study had strengths including the use of a large number of workers compensation claims covering a wide range of employees of various ages and lengths of tenure in many different states and industries , there were important limitations to note .
the large sample size afforded us the ability to examine the age and tenure interaction , but it also resulted in relationships being statistically significant despite there being relatively small differences in the predicted lengths of disability . when interpreting the results , it is important to consider what differences in the length of the disability are most meaningful to a given stakeholder group . due to our reliance on already existing administrative data , we were unable to include in our analyses variables such as comorbidities , relationships with coworkers , and availability of job accommodations .
being able to have done so would likely have helped us to better understand the complexity of the age , tenure , and length of disability relationships .
although it is a study strength that the age ranged from 18 to 80 years , individuals working after the traditional retirement years likely represent select groups of workers who delayed retirement , either due to financial necessity or personal preference . as
such , the length of disability for the oldest workers might be influenced by factors beyond those at younger ages .
in addition , in comparison with workers of other ages , there were relatively few workers over the age of 70 ( n = 2492 ) , with workers aged 70 to 80 representing less than 1% of the total sample . with this in mind
the length of disability was calculated based on the beginning and end of indemnity payments . in doing so
, we assume that the end of indemnity payments coincides with rtw ; however , in the administrative data used in this study , there is no way to verify if this was indeed the case .
it is possible that in certain cases , the end of indemnity payments actually signified an individual taking permanent disability or retirement .
a final limitation was that the primary methodology used in this study did not fully account for the conditional relationship between age and tenure . at the youngest ages ,
we did supplemental analyses including only distributions of age and tenure that would be possible showing that our findings held under these conditions ; however , the results from the main analyses should be interpreted with this limitation in mind .
the results of the current study pointed to a stronger relationship between age and the length of disability than for tenure and the length of disability . in terms of risk factors for longer work - disability durations , age seems like a more important factor than tenure ; however , there was variability in the age - length of disability relationship based on the length of tenure suggesting that tenure might still be an important aspect of the work - disability process . from a case management perspective , older workers might be a group in need of extra support as their rtw process seems to be delayed relative to younger workers .
for some older workers , retirement is not an option due to financial necessity or needing to continue to have access to health insurance benefits beyond those provided by the government .
such workers might feel unable to leave their jobs after a workplace injury / illness , and experience extra difficulty getting back to work . with the continued aging of the workforce and the changing nature of career paths , furthering our understanding of how and why age and tenure influence
the current study was a first step in examining the interplay between age and tenure with the length of disability , but future research is necessary to uncover the mechanisms behind these relationships . | objective : the aim of the study was to examine the relationships among age , tenure , and the length of disability following a work - related injury / illness.methods : this study utilized 361,754 administrative workers compensation claims .
the relationships between age , tenure , and disability duration was estimated with random - effects models.results:the age - disability duration relationship was stronger than the tenure - disability duration relationship . an interaction was observed between age and tenure . at younger ages , disability duration varied little based on tenure . in midlife , disability duration was greater for workers with lower tenure than for workers with higher tenure . at the oldest ages , disability duration increased as tenure increased.conclusions:findings indicate that age is a more important factor in disability duration than tenure ; however , the relationship between age and disability duration varies based on tenure , suggesting that both age and tenure are important influences in the work - disability process . |
a recent surge in interest in molecule - based electronics has highlighted the need for a better understanding of conduction mechanism in biomaterials .
plant cuticle membranes have been found to contain high proportions of insoluble and acid resistant cutin biopolyester .
current - voltage characteristics of nandi flame seed cuticle biomaterial have shown electrical switching and memory properties .
conducting biomaterials have been demonstrated to be potentially useful in making eco - friendly electronic devices and probes for sensors fabrication [ 47 ] , solid electrolyte systems , barrier materials , and controlled release polymers , and in tissue engineering because they can easily be used as a platform that supports electrical stimulation of cell - tissue constructs .
generally charge conduction in semiconducting polymers is thought to take place by hopping of charge carriers in an energetically disordered landscape .
vrh conduction mechanism originally proposed by mott for amorphous semiconductors assuming a phonon - assisted process has been employed in previous studies [ 1315 ] to explain charge transport in conducting synthetic polymers and their composites at low temperatures . hopping
charge transport has also been noted in double helix dna and in ion - doped biopolymers [ 8 , 17 ] . while considerable time has been devoted to investigate conduction mechanism in synthetic polymers [ 1823 ] , only little attention has been paid towards electrical conductivity of pristine biopolymers .
consequently , there are notably few studies that can reliably explain conductance observations made with biopolymers .
davis suggested that electron and resonance tunneling is responsible for detectable charge transport in biomolecules sandwiched between metal electrodes . from measurements that probed changes in oxidized guanine damage
yield with response to base perturbations , armitage et al . noted that charge transfer through base - base of dna molecules takes place via the - bond overlap .
tao et al . reported electron and bridge - assisted super - exchange charge transfer between donor and acceptor groups in peptide systems .
similar studies on proteins have also shown that electron transfer can occur across hydrogen bonds and that the rate of such transfer is greatly increased when the electron motions are strongly coupled with those of the protons .
radha and rossen studied energy transport in biopolymers and suggested , based on the experimental results , that a soliton in biopolymers is an energy packet ( similar to the conformon which is the packet of conformational strain on mitochondria ) associated with a conformational strain localized in region much shorter than the length of a macromolecule .
it was noted that as the soliton ( localized curvature ) moves on the polymer , it could trap an electron and drag it along .
this mechanism may be important in understanding charge transport in biological molecules , where curvatures abound . in this paper , we report dc conductivity measurements and charge transport mechanisms in nandi flame seed cuticle ( a pristine biopolymer ) in the temperature range 350450 k. fourier transform infrared ( ft - ir ) spectra analysis of pristine nandi flame seed cuticle in comparison with ft - ir spectra of cellulose and surface probe analysis using atomic force spectroscopy ( afm ) are presented before electrical conductivity .
discussion of charge transport mechanism is presented based on the fact that at least one mechanism can predominate within a specific temperature range .
the following charge transfer mechanisms : electric field dependent schottky and poole - frenkel emission , space charge limited current ( sclc ) ( from which some electrical parameters of the sample are obtained ) , tunneling current , and vrh are discussed in that order .
samples of thin seed cuticles ( figure 1 ) were obtained from nandi flame trees , also known as african tulip , which are used as ornamental trees in kenya .
samples were well cleaned with acetone and dried at a temperature of 310 k for 12 hours inside a lindberg blue tube furnace of model tf55035c and then stored in polythene bags containing silica gel desiccators .
ft - ir studies were done in order to compare the ir spectrum of the cuticle to that of cellulose .
samples for ft - ir measurement were prepared by grinding the sample ( 0.12.0 percent by weight ) with potassium bromide ( kbr ) and compressing the whole into a wafer .
thickness of the samples was determined by an interferometric method and found to be about 4 m .
electrode coating on the film of pristine cuticles was done using quick drying and highly conducting flash - dry silver paint obtained from spi supplies , pa , usa .
a mask ( made of stiff and thin transparent polythene paper ) of circular aperture of 0.56 cm diameter was used while coating , to ensure uniformity in size of coated surface .
circular aluminum foil of the same diameter was placed on freshly coated surface such that the sample was sandwiched between two aluminum electrodes .
these metal - cuticle - metal sandwiches were left to dry at room temperature for a period of 24 hours to ensure that there was good ohmic contacts between aluminum electrode and the sample cuticle .
flash - dry silver paint was used to connect thermally insulated thin wires onto the aluminum electrodes .
a cuticle sandwiched between aluminum electrodes was placed inside the lindberg / blue tube furnace and temperature varied at steps of 5 k between 350 k and 500 k at constant electric fields while measuring current - voltage ( i - v ) using keithley 2400 series source meter . surface structural characterization of the pristine cuticles was done using afm ( a digital instrument multimode / nanoscope iii scanning probe microscope was used ) .
taking images at the fundamental resonance frequency of si cantilevers ( 300 khz ) while operating the instrument in the tapping mode , the height and phase images were obtained simultaneously .
the ft - ir spectra of the cuticle and cellulose are shown in figure 2 .
the ir bands ( given with accuracy of 2 cm ) and band assignments are listed in table 1 .
vibrations in the 1800 to 1200 cm range mostly arise from side chains or side groups ( such as ester and oh ) while vibration in the 1200 to 900 cm are related to backbones of polysaccharides such as cellulose , hemicelluloses , and pectin .
the strong ir bands of the cuticle at 1278 cm assigned to ( ch ) , and 1106 cm assigned to (co ) , (cc ) are overlapped with strong cellulose bands at 1277 cm and 1107 cm , respectively .
generally , the cuticle ir bands in the range 1050 to 1400 cm are strongly correlated with the bands of cellulose .
however , ir absorption peaks of the cuticle in the carbonyl side group region ( 17501600 cm ) and in the region related to backbone of polysaccharides ( 1025950 cm ) do not correspond to cellulose bands .
figures 3(a ) and 3(b ) are afm scans showing surface structural characteristics of the cuticle sample .
the interstitial regions between the ridges represented by dark area are cavities on the membrane having an approximate width of 0.5 nm .
this results are similar to afm studies on the surface of cellulose which showed that surface structural characteristics of native cellulose consists of planes of cellulose molecules which are regularly interspaced at about 0.53 nm . forward and reverse bias current density - electric field dependence at high fields ( 1010 v / cm ) are displayed in figure 4 .
the current levels in the reverse bias are higher than forward bias , showing relatively linear relations of ln j versus e. this behavior may be interpreted either in terms of schottky effect which is a field lowering of interfacial barrier at the blocking electrode or by poole - frenkel effect which is due to thermal excitation of trapped charges via field assisted lowering of trap depth .
expressions for these processes are shown in ( 1 ) and ( 2 ) :
( 1)js = jsoexp [ ( se1/2)/kt ]
for the schottky effect and
( 2)jpf = jpfoexp [ ( pfe1/2)/kt ]
for the poole - frenkel effect .
j
so and jpfo are preexponential factors , s is the schottky coefficient , pf is the poole - frenkel coefficient , and e is the electric field .
theoretical values of schottky and poole - frenkel coefficient are related by ( 3 ) :
( 3)s = e340=pf2 .
with the assumption that the value of for our samples is 3.0
, the reported value for ethyl cellulose , the theoretical values of s and pf were obtained from ( 3 ) and found to be 3.51 10 j v m and 7.01 10 j v m , respectively .
experimental values of obtained from slopes ( linear regression parameters ) of linear fit plots of ln j versus e ( figure 4 ) at different temperatures are listed in table 2 .
the large discrepancy in experimental values of listed in table 2 and theoretical values of s and pf leads to a conclusion that current transport mechanism in our samples governing the high field at a temperature range of 320370 k can not be explained in terms of shottky or poole - frenkel emission .
figures 5(a ) and 5(b ) show j - v characteristics over a temperature range of 320350 k for the forward bias and reverse bias , respectively
. figure 5(a ) shows that forward bias characteristics has two regions : low voltage region below a threshold voltage ( vth ) where current density follows ohms law relations ( ohmic region ) , and higher voltages region , above vth at which , there is a pronounced power - law behavior given by j v where n 1.8 0.3 ( sclc region ) .
vth has negative temperature coefficient as noticed in figure 5(a ) where it decreases as temperature increase a case that is identical to tunneling of charge carriers through the entire metal - insulator - metal junction for a structure with multiple tunnel barriers in the coulomb blockade regime . at a poling temperature of 400 k
existence of vth elucidates electrical switching of the material from high impedance region where the current densities are very low , to low impedance region which allows large currents to flow .
figure 5(b ) shows that our samples remained in the low impedance region even as the voltage was decreased ( reverse bias ) leading to high levels of leakage current in the reverse bias than in the forward bias ( see figure 4 ) .
this can also be explained in terms of built up of image charges at high field of the reverse bias which lowers potential barrier at the metal - polymer junction hence allowing higher current to flow in the reverse bias .
this phenomenon of electrical switching with memory has also been observed in some synthetic polymers [ 3336 ] and has been used as active cells in modern nonvolatile memory chips .
polymers which exhibit electrical switching and memory effect can also be applied in electrical switching circuit and in gas sensing .
electrical switching is explained in terms of formation of semiquinones and quinoid radicals and in terms of sclc . as shown in figure 5(a ) , the low current densities at low voltages is due to charge capture in traps ( intrinsic charges ) present in the cuticle .
increase in bias voltage results in an increase in injected charge , thereby filling the limited traps .
this injection is provided by tunneling of the electrons ( holes ) into the conduction ( valence ) bands of the polymer .
injection of charges from metal electrode causes a reduction in the number of traps which , leads to a rapid increase in effective carrier mobility and therefore a rapid power - law increase in current . at sufficiently high injection levels ,
all the traps are filled reaching the trap filled limit ( tfl ) and consequently the current becomes sclc .
the total trap concentration nt in the cuticle sample is defined in ( 4 ) :
( 4)nt=20ed2vtfl ,
where e is the electronic charge relative permittivity of the material 0 is the permittivity of free space , d is thickness of sample ( 4 m ) and , vtfl is the upper limit of the voltage at which sufficient charge has been injected into the insulator to fill the traps .
equilibrium carrier concentration n0 can be obtained from the expression ( 5 ) :
( 5)n0=980ed2vtr ,
where vtr is the voltage at which the transition from ohmic to sclc behavior takes place . using experimental values of vtfl = 15 v and vtr = 6 v obtained from j - v curve at 320 k ( figure 5(a ) ) , the values of nt and n0 were calculated from ( 4 ) and ( 5 ) .
current density at low voltage ( the ohmic conduction region ) is given by ( 6 ) :
( 6)j = p0evd ,
where p0 is the concentration of thermally activated holes expressed as shown in ( 7 ) :
( 7)p0=nvexp ( edkt ) .
current density then becomes
( 8)j = nvevdexp ( ed / kt ) ,
where nv is effective density of states , e is electron charge , is charge mobility , v is the applied voltage , d is the sample thickness , ed is the ionization energy of donor charges , k is the boltzmann 's constant , and t is the temperature . by plotting ln j versus 1000/t it is possible to determine and ed .
variation of ln j with 1000/t , at a voltage of 0.9 v ( ohmic region ) is shown in figure 6 . assuming that nv = 10 cm which is within the range of the effective density of states for thin films of other organic semiconductors , the value of p o , , and ed are determined from ( 7 ) and ( 8) .
these electrical parameters , p o , , and ed being obtained from analysis of temperature dependence of ohmic region and nt , n0 obtained from analysis of sclc regime are listed in table 3 . the low charge mobility and
the electrical parameters shown in table 3 are comparable to same parameters calculated for other organic semiconductors [ 31 , 38 ] .
thermal activation plots ( log versus 10/t ) for different polarizing fields were plotted to analyze the effect of temperature on conductivity of the samples ( figure 7(a ) ) .
the observed dependence can be described by the arrhenius equation ( 9 ) :
( 9)=0exp ( eakt ) ,
where is conductivity , o the preexponential factor , and ea the activation energy .
conductivity was obtained from the relations in ( 10 ) :
( 10)=ivla ,
where i is measured current , v is measured voltage , l is the thickness of the samples ( 4.0 10 cm ) , and a is the electrode active area .
activation energy , ea was calculated from the average curve ( figure 7(b ) ) of log versus 10/t at different temperature regions .
the increase in the electrical conductivity and the decrease in the activation energy for the films sample could be attributed to the amorphous - crystalline transformation .
a polycrystalline film material contains a large number of microcrystallites with grain boundaries between them . at the grain boundary of each of the crystallites
these trap centers trap charge carriers at the grain boundaries , and hence a space charge can be built up locally .
increase of temperature , increases probability of thermal ionization of the trapping centers thus causing a shift in the quasi - fermi level and the possibility of new trap states in the mobility gap [ 31 , 40 ] .
electrical conduction mechanism at the temperature region where activation energy is low , ( ea 0.77 ev ) and where conductivity is almost constant ( figure 7(b ) ) is by tunneling of charge carriers across the grain boundaries .
this is supported by almost linear variation of versus t as shown in figure 8 .
the log versus 10/t variation at 350 < t < 440 k region is linear ( figure 7(b ) ) . investigation of conduction mechanism in this temperature range was made on the basis that tunneling or hopping mechanisms can predominate .
temperature - dependence data in figure 7(b ) were reused to plot versus t , and ln ( t ) versus t. from this analysis , we noted that tunneling conduction mechanism in the range of 350440 k was inadequate due to nonlinear variation of versus t. thermionic emission was also ruled out because this mechanism does not take place when the applied voltage ( v ) is greater than 0.8 v , and at low activation energy .
figure 9 shows linear variation of ( t ) versus t thus indicating that vrh conduction process can predominate in this temperature range 350450 k . a good fit of conductivity - temperature data to the expression in ( 11 ) :
( 11)t1/2=0exp [ (t0t)1/4 ]
is necessary for applicability of vrh model . the preexponential factor 0 and the degree of disorder ( mott temperature ) t0
n(ef ) , and the inverse - fall - off length of the wave function of a localized state near the fermi - level by the following relations :
( 12)0=3e3ph(8k)1/2[n(ef)]1/2 ,
( 13)t0=18.113kn(ef ) .
to check the validity of the vrh model , ln ( t ) versus t variation was plotted ( figure 9 ) . t0 , and n(ef ) were determined from ( 13 ) using the slope in figure 9 , and assuming that 3 which is approximately equal to inverse length of unit cell ( monomer ) of native cellulose [ 4244 ] .
other mott parameters , the distance r and average energy w were determined at t = 400 k by using the following relations in ( 14 ) and ( 15 ) :
( 14)r=[98ktn(ef)]1/4 ,
( 15)w=34r3n(ef ) .
the obtained mott parameters are listed in table 4 which shows that the product r and the average energy , w , satisfy mott 's requirements ( r > 1 , w > kt ) for variable range hopping at this temperature range .
degree of localization of the carriers in the trap states indicated by r > 1 , shows that the charges are highly localized .
table 5 shows the variations of the mott parameters with temperature in our samples .
it is evident from this table that r > 1 and w > kt , which agrees with mott 's condition for variable range .
it can also be noted from table 5 that when the temperature decreases , the average energy w decreases and the average distance r increases , supporting the fact that when the phonon energy is insufficient ( low temperature ) , carriers will tend to hop larger distances in order to locate in sites which are energetically closer than their nearest neighbours .
dc conductivity measurements were performed at a temperature range of 320 k450 k. conductivity increased with temperature .
charge mobility analyzed from j - v curves was found to be 4.06 10 ( mvs ) .
analysis of current density - electric field dependence showed that both schottky and poole - frenkel emission can not explain conduction mechanism in the high fields where sclc was seen to be present .
plots of dc conductivity versus temperature were used to investigate mott 's vrh conduction model at a temperature range of 320440 k. this model was originally developed for amorphous silicon by mott and davis . when applied to conducting polymers , it assumes that electron transport originates from localized or fixed states within the polymer chain .
the charge transfer between the chains takes place by phononassisted hopping between two localized states .
analysis of semilogarithmic plots of ln(jt ) versus t gave the following mott parameters : the degree of disorder to was in the order of 10 , the density of states n(ef ) 10 ( evcm ) while the distance r 10 cm and energy ( w ) were in the range of 0.72 ev0.98 ev . the product r and the average energy , w , satisfy mott 's requirements ( r > 1 , w > kt ) for variable range hopping in this temperature range .
the study has shown that the cuticle is a promising alternative to synthetic polymers used in electrical switching applications .
due to environmental concerns posed by nonbiodegradable synthetic polymers , exploring this material would provide information on how to use it in addressing environmental problems and as a precursor in the design of novel materials with electrical functionality such as the modern nonvolatile memory chips , biosensors , and biological transistors . | metal - sample - metal sandwich configuration has been used to investigate dc conductivity in 4 m thick nandi flame [ spathodea campanulata p. beauv . ]
seed cuticles .
j - v characteristics showed ohmic conduction at low fields and space charge limited current at high fields .
charge mobility in ohmic region was 4.06 105 ( m2v1s1 ) .
temperature - dependent conductivity measurements have been carried out in the temperature range 320 k
< t > 450 k. activation energy within a temperature of 320 k440 k was about 0.86 ev .
variable range hopping ( vrh ) is the main current transport mechanism at the range of 330440 k. the vrh mechanism was analyzed based on mott theory and the mott parameters : density of localized states near the fermi - level n(ef ) 9.04 1019 ( ev1cm3 ) and hopping distance r 1.44 107 cm , while the hopping energy ( w ) was in the range of 0.72 ev0.98 ev . |
the study was a randomized controlled trial and included a pre - test , post - test and delayed post - test with a control group .
the independent variable was mindfulness training and the dependent variable was the quality of life of the warfare veterans with ptsd .
all participants of the study were the warfare victims of iran - iraq war diagnosed with ptsd . from among the hospitals affiliated to the foundation of martyrs and veterans affairs of isfahan , shahid rajaee hospital was randomly selected . hidden and confidential assignment to put people in groups of forklift , after being determined by participants
, one of the psychologists did not have knowledge of the research process , the hospital of the spinal cord was called veterans . he is based on the number of people counted in the crash and two group numbers and put the individual couples .
the sample size for group therapy in clinical trials of psychology and counseling of 5 to 12 people is defined ( methods of research in psychology and educational sciences , delaware , 1390 ) . as well as the knowledge and methods of the mindfulness founder kabat zinn , segal and tizdel of the researchers called this the realm of mind - awareness group therapy with the setup on the volume of 8 to 15 people
, it is worth noting the participants until after the intervention and control groups is determined ( after the post test ) had no more information about the research process .
having administered psychiatric interviews with the aim of diagnosing ptsd according to dsm - iv - tr criteria by the hospital s psychiatrist , 32 individuals were randomly selected and assigned into two equal groups . besides , the effect of gender was controlled by including only males in the study .
the participants had to fall within the age range of 35 - 60 and have secondary education .
moreover , it was checked whether they suffered any active psychotic disease , showing symptoms of bipolar disorder , borderline personality disorder , anti - social and active suicidal tendencies and drug abuse during the last three months prior to the study ( 36 , 37 ) .
two participants dropped out in each group as the study continued ( due to reasons like being discharged from the hospital or stopping participation in the study ) .
the experimental group received mindfulness - based stress reduction ( mbsr ) training ( 39 ) . given the flexibility of mindfulness - based treatments , mindfulness training was designed with regards to the structure and features of the quality of life of the warfare victims with ptsd . on the whole
the sessions are described as follows , during the session , a pre - test was administered .
cordial relationship with the participants was established and the intended concepts were explained . also , the need for mindfulness training was justified and the issue of relaxation was introduced .
the focus was on training relaxation for 14 muscle groups including forearm , calf , shin , thighs , abdomen , chest , shoulders , neck , lips , eyes , jaws , temples and forehead .
the participants received relaxation training for the following six muscle groups : hands and arms , feet and thighs , abdomen and chest , neck and shoulders , jaws and forehead and lips and eyes .
the inhale and exhale technique with calmness and no thinking and the breath watching technique were trained .
also , as their homework , the participants were asked to practice mindfulness - based breathing for 20 minutes before going to bed . in this session ,
the participants were trained how to pay attention to body movements while breathing , to focus on body muscles and their movements , and to trace physical feelings , hearing , taste , etc . they were required to do mindfulness - based eating at home as their homework ( i.e. , paying attention to the taste and the appearance of foods ) .
the participants were taught how to scan mind , positive and negative thoughts and their desirability and undesirability , to allow positive and negative thoughts into and out their minds without passing any judgment and to pay deep attention to them . as homework
the training sessions four , five and six were repeated for 20 to 30 minutes .
the following tools were used to collect data.demographic information sheet : the individual and demographic information of the participants were collected through the demographic information sheet .
the items included in the sheet were age , gender , profession , education , marital status , disease history and. 1 .
clinical interviews : in order to diagnose ptsd ( 12 ) and investigate other criteria for selecting the participants ( 19 , 25 ) clinical interviews were conducted.2 .
clinical interviews were administered by the hospital s psychiatrist according to the fourth revised version of diagnostic and statistical manual for mental disorders ( dsm - iv - tr ) ( 12).3 .
world health organization quality of life questionnaire ( whoqol-26 ) : the 26-item questionnaire on the quality of life proposed by the world health organization was used .
the items were scored based on five - option likert scale . questions no . 3 , 4 , and 26
the questionnaire tests four categories ; namely , hygiene and physical well - being , psychiatric well - being , social life and living environments and conditions .
the categories are used as a comprehensive scale and generally , include overall quality of life and general well - being levels . in a study conducted abroad ,
the validity of the questionnaire was found to be 90% for patients with chronic diseases , and 86% for normal people .
the validity of the questionnaire in iran has been estimated to be 89% ( 40 ) .
demographic information sheet : the individual and demographic information of the participants were collected through the demographic information sheet .
the items included in the sheet were age , gender , profession , education , marital status , disease history and .
clinical interviews : in order to diagnose ptsd ( 12 ) and investigate other criteria for selecting the participants ( 19 , 25 ) clinical interviews were conducted .
clinical interviews were administered by the hospital s psychiatrist according to the fourth revised version of diagnostic and statistical manual for mental disorders ( dsm - iv - tr ) ( 12 ) .
world health organization quality of life questionnaire ( whoqol-26 ) : the 26-item questionnaire on the quality of life proposed by the world health organization was used .
the questionnaire tests four categories ; namely , hygiene and physical well - being , psychiatric well - being , social life and living environments and conditions .
the categories are used as a comprehensive scale and generally , include overall quality of life and general well - being levels . in a study conducted abroad
, the validity of the questionnaire was found to be 90% for patients with chronic diseases , and 86% for normal people .
the validity of the questionnaire in iran has been estimated to be 89% ( 40 ) .
having explained the aims of the study , the participants approval for their participation was obtained . in order to take research ethics into the account ,
both groups took the pre - test and then , the experimental group received eight 90-minute mt sessions . at the end of the treatment , both experimental and control groups took the post - test .
also , the control group underwent drug therapy until the delayed - post test was administered . during that time
therefore , to observe research ethics , eight mt sessions were held for them too .
data were analyzed using the repeated measure anova function of spss version 17 . according to descriptive statistics , from among the participants assigned to the experimental group , 42.9 per cent % had secondary education , 42.9 per cent % had a high school diploma , 14.2and 14.2 per cent % had a bachelor s degree .
for the control group , the numbers were 57.1 , 35.8 , and 7.1 , respectively , for secondary education , diplomas , and bachelor degrees . in terms of marital status , 92.9 per
cent % of the participants in the experimental group was married and 7.1 per cent % was were divorced .
for the control group , 71.1 per cent% of the participants were married , 7.1 per cent % was single , and 21.4 per cents % were remarried .
the employment statistics of the experimental group indicated 57.1 per cent % employed , 35.7 per cent % unemployed , and 7.1 per cent % retired . the same statistics for the control group were 64.3 per cent% , 28.6 per cent% , and 7.1 per cent % respectively for the employed , unemployed , and retired , respectively .
the mean and standard deviation for the experimental and control groups were , respectively , 84/350 and 26/457/49 , respectively . with regards to the type of paralysis ,
92.9 per cent % of the participants in the experimental group was nero - psycho and the rest ( 7.1 percent% ) were suffering from a mixture of paralyses ( nero - psycho , physical , and chemical ) .
64.3 ; 64.3 percent % of the participants in the control group was were suffering from nero - psycho paralysis and the rest were suffering from a mixture of paralyses ( table 1 ) . table 2 shows demonstrates the mean and standard deviation of the investigated variables for both the experimental and control groups in the pre - test , post - test , and delayed post - test .
as the table 2 presents , demonstrates , there was not a statistically significant difference for the quality of life and its relevant dimensions based on box and levin tests .
the results of the repeated measures anova showed a statistically significant difference for the quality of life and its relevant dimensions between the means of pre - test , post - test , and delayed post - test for the experimental and control groups .
moreover , the effect size was found to be 0.689 ( eta squared = 0.698 ) which shows the effectiveness of the treatment .
the interactive effect was significant for all the variables , too ( table 3 . ) .
therefore , given the mean scores , it can be said that there was a decrease in the post - test and delayed post - test scores for the experimental group in comparison with that of the control group .
the experimental group received mindfulness - based stress reduction ( mbsr ) training ( 39 ) . given the flexibility of mindfulness - based treatments , mindfulness training was designed with regards to the structure and features of the quality of life of the warfare victims with ptsd . on the whole , eight 90- minute mindfulness training sessions were held .
the sessions are described as follows , during the session , a pre - test was administered .
cordial relationship with the participants was established and the intended concepts were explained . also , the need for mindfulness training was justified and the issue of relaxation was introduced .
the focus was on training relaxation for 14 muscle groups including forearm , calf , shin , thighs , abdomen , chest , shoulders , neck , lips , eyes , jaws , temples and forehead .
the participants received relaxation training for the following six muscle groups : hands and arms , feet and thighs , abdomen and chest , neck and shoulders , jaws and forehead and lips and eyes .
the inhale and exhale technique with calmness and no thinking and the breath watching technique were trained .
also , as their homework , the participants were asked to practice mindfulness - based breathing for 20 minutes before going to bed . in this session ,
the participants were trained how to pay attention to body movements while breathing , to focus on body muscles and their movements , and to trace physical feelings , hearing , taste , etc .
they were required to do mindfulness - based eating at home as their homework ( i.e. , paying attention to the taste and the appearance of foods ) .
the participants were taught how to scan mind , positive and negative thoughts and their desirability and undesirability , to allow positive and negative thoughts into and out their minds without passing any judgment and to pay deep attention to them . as homework
the training sessions four , five and six were repeated for 20 to 30 minutes .
during the session , a pre - test was administered . cordial relationship with the participants
was established and the intended concepts were explained . also , the need for mindfulness training was justified and the issue of relaxation was introduced .
the focus was on training relaxation for 14 muscle groups including forearm , calf , shin , thighs , abdomen , chest , shoulders , neck , lips , eyes , jaws , temples and forehead .
the participants received relaxation training for the following six muscle groups : hands and arms , feet and thighs , abdomen and chest , neck and shoulders , jaws and forehead and lips and eyes .
the inhale and exhale technique with calmness and no thinking and the breath watching technique were trained .
also , as their homework , the participants were asked to practice mindfulness - based breathing for 20 minutes before going to bed .
the participants were trained how to pay attention to body movements while breathing , to focus on body muscles and their movements , and to trace physical feelings , hearing , taste , etc . they were required to do mindfulness - based eating at home as their homework ( i.e. , paying attention to the taste and the appearance of foods ) .
the participants were taught how to scan mind , positive and negative thoughts and their desirability and undesirability , to allow positive and negative thoughts into and out their minds without passing any judgment and to pay deep attention to them . as homework
the training sessions four , five and six were repeated for 20 to 30 minutes .
the following tools were used to collect data.demographic information sheet : the individual and demographic information of the participants were collected through the demographic information sheet .
the items included in the sheet were age , gender , profession , education , marital status , disease history and. 1 .
clinical interviews : in order to diagnose ptsd ( 12 ) and investigate other criteria for selecting the participants ( 19 , 25 ) clinical interviews were conducted.2 .
clinical interviews were administered by the hospital s psychiatrist according to the fourth revised version of diagnostic and statistical manual for mental disorders ( dsm - iv - tr ) ( 12).3 .
world health organization quality of life questionnaire ( whoqol-26 ) : the 26-item questionnaire on the quality of life proposed by the world health organization was used .
the questionnaire tests four categories ; namely , hygiene and physical well - being , psychiatric well - being , social life and living environments and conditions .
the categories are used as a comprehensive scale and generally , include overall quality of life and general well - being levels . in a study conducted abroad ,
the validity of the questionnaire was found to be 90% for patients with chronic diseases , and 86% for normal people .
the validity of the questionnaire in iran has been estimated to be 89% ( 40 ) .
demographic information sheet : the individual and demographic information of the participants were collected through the demographic information sheet .
the items included in the sheet were age , gender , profession , education , marital status , disease history and .
clinical interviews : in order to diagnose ptsd ( 12 ) and investigate other criteria for selecting the participants ( 19 , 25 ) clinical interviews were conducted .
clinical interviews were administered by the hospital s psychiatrist according to the fourth revised version of diagnostic and statistical manual for mental disorders ( dsm - iv - tr ) ( 12 ) .
world health organization quality of life questionnaire ( whoqol-26 ) : the 26-item questionnaire on the quality of life proposed by the world health organization was used .
the questionnaire tests four categories ; namely , hygiene and physical well - being , psychiatric well - being , social life and living environments and conditions .
the categories are used as a comprehensive scale and generally , include overall quality of life and general well - being levels . in a study conducted abroad ,
the validity of the questionnaire was found to be 90% for patients with chronic diseases , and 86% for normal people .
the validity of the questionnaire in iran has been estimated to be 89% ( 40 ) .
having explained the aims of the study , the participants approval for their participation was obtained . in order to take research ethics into the account ,
both groups took the pre - test and then , the experimental group received eight 90-minute mt sessions . at the end of the treatment ,
two months later , the groups took the delayed post - test . also , the control group underwent drug therapy until the delayed - post test was administered . during that time
therefore , to observe research ethics , eight mt sessions were held for them too .
data were analyzed using the repeated measure anova function of spss version 17 . according to descriptive statistics , from among the participants assigned to the experimental group , 42.9 per cent % had secondary education , 42.9 per cent % had a high school diploma , 14.2and 14.2 per cent % had a bachelor s degree .
for the control group , the numbers were 57.1 , 35.8 , and 7.1 , respectively , for secondary education , diplomas , and bachelor degrees . in terms of marital status ,
92.9 per cent % of the participants in the experimental group was married and 7.1 per cent % was were divorced . for the control group , 71.1 per cent% of the participants were married , 7.1 per cent % was single , and 21.4 per cents % were remarried .
the employment statistics of the experimental group indicated 57.1 per cent % employed , 35.7 per cent % unemployed , and 7.1 per cent % retired . the same statistics for the control group were 64.3 per cent% , 28.6 per cent% , and 7.1 per cent % respectively for the employed , unemployed , and retired , respectively .
the mean and standard deviation for the experimental and control groups were , respectively , 84/350 and 26/457/49 , respectively . with regards to the type of paralysis
, 92.9 per cent % of the participants in the experimental group was nero - psycho and the rest ( 7.1 percent% ) were suffering from a mixture of paralyses ( nero - psycho , physical , and chemical ) .
64.3 ; 64.3 percent % of the participants in the control group was were suffering from nero - psycho paralysis and the rest were suffering from a mixture of paralyses ( table 1 ) .
table 2 shows demonstrates the mean and standard deviation of the investigated variables for both the experimental and control groups in the pre - test , post - test , and delayed post - test .
as the table 2 presents , demonstrates , there was not a statistically significant difference for the quality of life and its relevant dimensions based on box and levin tests .
the results of the repeated measures anova showed a statistically significant difference for the quality of life and its relevant dimensions between the means of pre - test , post - test , and delayed post - test for the experimental and control groups .
moreover , the effect size was found to be 0.689 ( eta squared = 0.698 ) which shows the effectiveness of the treatment .
the interactive effect was significant for all the variables , too ( table 3 . ) .
therefore , given the mean scores , it can be said that there was a decrease in the post - test and delayed post - test scores for the experimental group in comparison with that of the control group .
the results of the bonferroni test showed a decrease in the scores of the quality of life and its relevant dimensions in both the post - test and delayed post - test for the experimental group .
also , for the control group , there was a statistically significant difference between the mean scores of the post - test .
however , it was much less in comparison with that of the experimental group ( table 3 ) .
the present study aimed at investigating how mindfulness training would influence the quality of life of warfare veterans with ptsd .
the results of the repeated measures anova indicated a decrease in the scores of the quality of life and its relevant dimensions for the experimental group in both post - test and delayed post - test . also , the results of the post hoc showed a drop in the scores of the quality of life and its relevant dimensions in the post - test and delayed post - test in comparison with the pre - test for the experimental group .
the results of the study are similar to those of earlier studies supporting the effectiveness of the mindfulness - based interventions in reducing the symptoms of psychological disorders and medical conditions ( 30 ) , anxiety disorders ( 30 ) , cancers ( 31 ) , depression ( 32 ) on the one hand and improving affection and temperament ( 34 ) and mental health ( 35 ) on the other .
also , since the previous researches have shown the effectiveness of mindfulness training in ameliorating depression , psychological disorders , anxiety and the quality of life of those experiencing stressful and traumatic situations ( 25 , 38 , 37 , 36 ) , it can be claimed that the results of the present study confirm the earlier body of research .
ptsd produces changes in the feelings and behaviors of the sufferers such as an increase in their intimacy , social and sexual activities , feeling of detachment and alienation and affective and excitement coldness ( 19 , 20 ) .
therefore , employing effective therapeutic and rehabilitative strategies to reduce the resultant problems can improve and promote the quality of life of the warfare veterans with pstd .
findings of the study indicated a significant relationship between the effectiveness of mindfulness techniques and other dimensions of life such as mental health which is similar to the findings of earlier studies .
some studies have reported the effectiveness of mindfulness training in improving mental well - being and health ( 20 , 22 , 24 ) . also , the results of this study showed that mindfulness techniques can boost physical health .
from this perspective , the findings confirm those of earlier studies ( 21 , 23 ) .
the same positive effects are supported by the results of the study in terms of social relationships which have already been reported by other studies ( 16 , 20 , 25 ) . as shown before , positive and statistically significant changes in the quality of life of the warfare veterans with pstd
this can be accounted for by the idea that since mindfulness trainings modify judgeless feelings , it may bring about more physical and psychological consciousness , and may help a judgeless acceptance of excitement and physical phenomena ( 41 ) , and it can also play an important role in improving the quality of life of warfare veterans .
this conclusion is defendable when seen in the light of the findings of the previous studies which show the potentials of mindfulness trainings for modifying negative thoughts and behaviors that lead to positive health - related changes ( 42 ) .
moreover , attending mindfulness workshops causes individuals to change their thoughts in a way that helps them to conceive thoughts as just mental and transient phenomena rather than unchangeable realities ( 43 ) . to put it simply ,
it can be noted that since mindfulness training raises the individuals consciousness of psychological and physical feelings , it results in a realistic acceptance of life events and confidence , deep sympathy and love toward others ( 44 ) .
post and veid believe that the effectiveness of mindfulness - based treatments is due to the intervention of spiritual and religious beliefs . in general , the researchers have a positive orientation towards the effectiveness of those psychotherapy - oriented treatments in which the focus is on religion , mindfulness and praying , or religious trainings ( 36 ) . reducing stress by means of mindfulness techniques significantly increases psychotherapy - based well - being and mindfulness and deceases stress and physical signs . the time spent in practice sessions is connected to an improvement in well - being and mindfulness level .
therefore , mindfulness can be considered as a mediator which ameliorates psychological performance and reduces stress ( 28 ) .
it has been proved that mindfulness training is effective in decreasing depression and anxiety of ptsd sufferers and improving their quality of life ( 16 , 27 , 28 , 29 ) .
also , there are reports of the efficacy of the method in heightening affection , temperament and mental health of chemical warfare victims ( 25 , 26 ) . in general , mindfulness - based interventions have been effective in decreasing temperament and anxiety problems , bipolar and borderline personality disorders , chronic pains and cancers ( 37 ) . directly and indirectly
, mindfulness training improves health within a biological - psychological - social framework ( 45 ) .
since mindfulness training helps individuals to understand their feelings , direct their attention , accept experiences as they are and prevent passing judgments on their thoughts , it is likely that the method can reduce the symptoms of those suffering from ptsd . indirectly
, mindfulness techniques such as body monitoring can lead to a better understanding of signals and a decrease in unrealistic judgments through making the sufferers more capable of recognizing their feelings ( 25 ) .
although individuals have their own idiosyncrasies , a mindfulness training team paves the way for them to understand and learn more about their feelings , thoughts , excitements and the people around them .
consequently , this makes them capable of leading a more efficient life when they are out of the groups ( 28 ) .
this increases their sensitivity and mistrust and keeps them fearful ; this state , in turn , causes individuals to obsessively and relentlessly think of past and future events ( 36 ) . in such cases , practicing conscious breathing as the locus of attention and reference can help such people to get back to their normal condition . getting back to oneself
is experienced once individuals ponder over their past or future and operate according to their thoughts and feeling patterns ( 33 ) .
pstd sufferers have problems with controlling their annoyance , and this negatively influences their marital relationship and affection and reduces family performance and leads to low quality of life .
mindfulness practices improve mental ability in a way that getting back to the normal state even in stressful situations becomes possible ( 33 ) .
in fact , conscious attention to the present time teaches individuals new ways of understanding and responding to all intrinsic feelings and thus they develop the ability to identify feelings , experiences and thoughts ( 27 ) . paying conscious attention to the present time is composed of two concepts : self - management attention and acceptance which is accompanied by a tendency towards experiencing ( 27 ) .
the concept of acceptance consists of three stages : observing psychological events , freeing oneself from a tendency to a change in form or the frequency of the event , and finally distinguishing between reality and psychological experiences .
in other words , acceptance as a main component of mindfulness - based treatments helps individuals look at psychological events as understandable and transient states instead of considering them as disastrous , intolerable and unavoidable ( 33 ) .
the members of the experimental group reported an increase in their daily performance and activity during the workshop and the period following that .
this can be due to a decrease in the number of avoiding experiences and experience - seeking motivation in them .
it is supposed that mindfulness - practices encourage participants to keep a positive attitude towards curiosity and openness to experiences ( 38 ) .
in addition , the practices develop accuracy and clarity , enhance understanding and insight towards incompatible , destructive and automatic behaviors , and make it possible for individuals to respond or stop the behavior instead of reacting to it ( 30 , 27 , 25 ) .
the new research trend exploring the pathological roots of psychological disorders based on evaluating mindfulness shows the inadequacy of this ability in a number of patients with psychiatric problems .
also , available literature suggests that mindfulness - based treatments play a role in reducing and ameliorating the symptoms of anxiety and depression . by considering mindfulness as a social cognition ability of the brain
, researchers believe that employing strategies for assessing and training social cognition to treat patients suffering from psychiatric disorders is a necessity ( 46 ) .
it can be claimed that since mindfulness training draws the attention and consciousness of individuals to physical and psychological feelings , it can help them to identify their abilities to cope with stress , to become professionally influential and to interact and cooperate with other members of the society ( 34 ) .
this is due to the fact that one of the main dimensions of mindfulness - based treatments is that individuals learn to accept their negative emotions and thoughts without judging them and experience mental events in a positive way ( 42 ) .
another point which should be borne in mind with regards to mindfulness - based treatments is the mental re - presentation of real life objects which are out of the control of human beings .
hence , mindfulness and its training can play a significant role in improving the quality of life of those warfare veterans with ptsd and can modify the social behaviors stemming from their nero - psycho problems .
mindfulness training is one of the therapeutic methods working through psychotherapy and stress reduction . in this method ,
individuals are trained to re - present objects in their mind which are out of immediate control .
actually , the method is a combination of body relaxing and mindfulness ( 27 ) .
rayon and dosy ( as cited in 35 ) showed that mindfulness help individuals to modify negative behavioral patterns and automatic thoughts , resulting in adaptive positive health - related behaviors .
also , it should be noted that one of the positive dimensions of mindfulness is the increase in the amount of individuals attention to thoughts , excitements and tendencies , causing adaptive and positive psychiatric behaviors to coordinate .
additionally , it can improve the abilities of individuals in a way that directs them to individual and social activities and raises their interest in them ( 42 ) .
therefore , providing mindfulness training for the ptsd warfare veterans , who suffer from nero - psycho , professional and inter - individual problems due to the experiencing damaging and traumatic situations , can have instructive effects . like all research projects , this study had limitations , some of which were the small number of the participants , the short interval between the post - test and delayed post - test and gender ( females were not included ) .
larger samples , longer intervals between post - tests and delayed post - tests and inclusion of females with ptsd can make the findings of the present study more generalizable and reliable .
attending war zones and facing stressful events cause physical problems and psychological disorders and may reduce the quality of life of the people experiencing such events .
the findings of the study suggest that mindfulness training can improve the quality of life of such people . | objectivethose veterans suffering from post traumatic stress disorder ( ptsd ) experience a low quality of life .
this study investigated how the quality of life of warfare victims with ptsd was influenced by mindfulness training ( mt)methodthis study followed a randomized controlled trial and included a pre - test , post - test and delayed post- and was conducted in 2012 at shahid rajaee hospital in isfahan , iran .
the participants were 28 randomly selected male warfare victims with ptsd who were assigned into control and experimental groups .
data were collected using world health organization quality of life questionnaire-26 ( whoqol-26 ) .
repeated measures anova was run to analyze the data.resultsthe findings of this study indicated a statistically significant decrease in the mean score of the experimental group in both post - test and delayed post - test . also , in comparison with the control group , there was a statistically significant increase in the mean scores of the physical and psychiatric health , social relationship and social setting and condition of the experimental group in the post - test and delayed post - test ( p < 0.01).conclusionthe findings of this study lend support to the effectiveness of mt in improving the quality of life of those veterans with ptsd and have significant implications for drawing our attention to mindfulness - based treatments as a way to enhance the quality of life of warfare victims suffering from ptsd . |
an 80-year - old female visited our clinic due to involuntary movement on her oromandibular area and both extremities .
she was diagnosed with hypertension and chronic small vessel disease about 10 years before and treated with clopidogrel 75 mg and amlodipine besylate 5 mg daily .
other family members including parents , siblings , 2 sons and 3 daughters were healthy and do not have any history of movement disorder .
about 2 years before , she first experienced abnormal movement and discomfort on her oromandibular area .
about 1 year later , abnormal involuntary movement that characterized by continuous restlessness was started on both extremities and frequent falling to forward was noted when she was walking .
the restless movements worsened with stress and emotion , and were progressive , resulting in speech problems and gait disturbance .
caregivers also complained about memory decline and insomnia . on examination , she showed dysarthria and continuous omd with generalized choreitic restlessness on her both extremities .
choreitic movement was spread to the trunk and affected sitting or standing posture . when she was ordered to maintain a sustained posture or to close her eyes , she could not maintain even longer than several seconds , which presenting motor impersistence .
cognitive function was also impaired , so it was very difficult to concentrate on or conduct doctor s request .
she scored 21 points on the korean version of mini - mental state examination . on detailed neuropsychological study ( seoul neuropsychological screening battery ) ,
her cognitive function was impaired especially in comprehension , calculation , praxis , language and visuospatial memory and frontal lobe function . on laboratory evaluation including complete blood cell counts , routine chemistry , thyroid function , coagulation profile , tumor markers , peripheral blood smear , serum ceruloplasimin and 24 hour urine cupper were within normal range .
anti - nuclear antibody ( ana ) was weak positive , but ana titer has no clinical significance .
brain magnetic resonance imaging ( mri ) shows no specific findings except diffuse brain atrophy ( figure 1 ) .
positron emission tomography using [ f]-fluoro - deoxyglucose shows severe hypometabolism in both basal ganglia ( figure 2 ) .
genetic testing for hd revealed 40 cag repeats on one huntington allele and 12 repeats on the other .
here we report a patient with late - onset hd who presented omd as an initial symptom .
the typical age of onset for adult - onset hd is between the ages of 30 and 50,2 but the disorder can manifest at any time between infancy and senescence .
the age of symptom onset is associated with cag repeats length.3 there is negative correlation between the age of onset and the repeat length . in late - onset hd ,
the expansion size of cag repeats is relatively low . in previous study , which reviewed thirty- four patients with late - onset hd ( onset range 6079 years ) ,
another study noted that in persons beyond the age of 60 , the effect of the cag repeat length on age of onset seemed to diminish .
8 according to the study about the prevalence of late - onset hd , the expanded cag repeat sequence was found with a narrow range of 3638 repeats.6 it is also known that in lower cag repeat reduced penetrance is present.7 most of the patient were the first in their family to have a diagnosis of hd and , this case was also the first in her family.5 it is considered that the lack of family history of hd is related to the small cag repeat size occurring in other family members.9,10 early signs of adult - onset hd are general restlessness , hygienic neglect , sleep disturbances , behavioral changes , anxiety , and depression.1 motor signs follow and include involuntary movement that can be suppressed by the patient but not for long.1 in patients with late - onset hd , however , motor symptoms are most common first signs and presented with mild form.5 a study of late - onset hd also reported predominantly motor disturbance at onset with relatively mild disability and a favorable outlook for both independent living and for life expectancy . 6 among the initial motor symptoms ,
there are typical facial movements with the characteristic raising of the eyebrows and the special facial expression of an astonished look.1 in this case , however , initial manifestation was omd and progressed slowly to generalized chorea .
omd is abnormal , involuntary , aimless , repetitive movements affecting the tongue , lips , and jaw .
the issue of the occurrence of spontaneous ( or unmedicated ) omd in normal aging remains blurred , since complete drug history and other dyskinesigenic conditions ( e.g. , orodental and cognitive status ) are often incompletely documented.14 other neurological disorders associated with omd include chronic hepatic encephalopathy , infectious or paraneoplastic encephalitis , and subcortical infarcts .
rarely , omd occurs in a variety of brain conditions , such as mental retardation , rett syndrome , and neurodegenerative conditions like hd .
so , it is not easy to diagnose hd when old patients present omd as initial symptom .
typical finding in hd that contributes to overactivity is motor impersistence , the inability to maintain a voluntary muscle contraction at a constant level.11 motor impersistence is independent of chorea and is linearly progressive , making it a possible surrogate marker of disease severity.12 it is a classic physical sign in hd that differentiates hd from other disorders , such as tardive dyskinesia.13 in this case , motor impersistence on tongue protrusion and on eye closing was noticed .
so it is considerable to suspect late - onset hd when old patient presenting omd and motor impersistence , even without other cognitive or psychiatric symptoms .
we describe late onset hd patient with a 40 cag repeats in the huntingtin gene and omd as an initial manifestation .
we suggest that hd should be considered in omd patient with motor impersistence irrespective of the family history . | huntington s disease ( hd ) is a neurodegenerative disorder characterized by a triad of choreoathetosis , dementia and dominant inheritance .
the cause of hd is an expansion of cag trinucleotide repeats in the hd gene .
typical age at onset of symptoms is in the 40s , but the disorder can manifest at any time .
late - onset ( 60 years ) hd is clinically different from other adult or juvenile onset hd and characterized by mild motor problem as the initial symptoms , shorter disease duration , frequent lack of family history , and relatively low cag repeats expansion . we report a case of an 80-year - old female with oromandibular dyskinesia as an initial manifestation of hd and 40 cag repeats . |
metabolic syndrome ( mets ) , a cluster of medical disorders including central obesity , glucose intolerance , dyslipidemia , hypertension and hyperglycemia , is known to increase the risk of developing cardiovascular diseases ( cvd ) and type 2 diabetes ( t2d ) ( 1 ) .
the alarming prevalence of mets worldwide varies based on populations , lifestyle status and socioeconomic factors .
evidence reveals that about 24 and 42% of iranian males and females have mets , respectively ( 2 ) .
previous studies have shown the role of genetics and socio - behavioral factors on educational levels , smoking , physical inactivity and inappropriate dietary habits in the incidence of mets ( 3 ) .
genetic studies on mets , including classical twin studies ( 4 ) , family - based studies using a modeling technique ( 5 ) and genome - wide linkage scans ( 6 ) coincide with a great challenge , due to the complex traits and association with these socio - behavioral factors .
these studies found the occurrence of mets to be positively associated with low educational levels ( 7 ) , low levels of physical activity ( pa ) ( 8) , the western diet ( 9 ) and also inversely associated with dairy consumption .
furthermore , the negative effect of some nutritional habits like frequent coffee consumption , the absence of vegetables in daily meals and the desire for high - calorie dishes on the occurrence of mets has been documented ( 10 ) . since genetic and most environmental factors shape the context of families ,
for instance , first - degree relatives of persons with diabetes are more prone to have the syndrome ( 11 ) ; likewise , persons with the syndrome are more likely to have members with similar cardio - metabolic risk factors ( 12 ) .
while several studies have assessed genetic and environmental factors related to the development of mets , there is a paucity of population - based family studies to elucidate these contributions , particularly in an iranian population .
limited investigations in this field have been conducted in east and south asia but there is no survey available from the middle east region concerning familial aggregation and the impact of socio - behavioral factors on mets .
this study is the first attempt to explore the possibility of a relationship between familial aggregation and the development of mets in tehranian families .
this case - control / family - based study was conducted on all 3,958 families of the tehran lipid and glucose study ( tlgs ) .
the design of the tlgs included two major components : a cross - sectional study to investigate the prevalence of cardiovascular disease and its risk factors , and a prospective 20-year follow - up with approximately 3.5 year intervals .
details of the tlgs design and its preliminary results have been described elsewhere ( 1 ) .
all data for the tlgs was collected through face - to - face interviews administered by trained research staff . for the present study ,
1,776 families that have at least one member with mets in the fourth phase ( 2009 - 2012 ) of the tlgs were recruited .
participants provided written informed consent and the study was approved by the ethics committee of the research institute for endocrine sciences , shahid beheshti university of medical sciences , tehran , iran ( ethics code 750ec , 2012 ) .
the authors are committed to protecting and ensuring the privacy and confidentiality of the personal health information of their participants .
of 12,823 subjects participating in the fourth phase of the tlgs , those aged < 19 years ( n = 1,758 ) , family members other than first and second degree ( n = 30 ) , and those with missing data ( n = 175 ) were excluded , with 10,860 subjects remaining .
metabolic syndrome , diagnosed by the joint interim statement ( jis ) criteria , led to 4,422 and 6,438 subjects with and without mets , respectively . using random sampling , 635 case - probands and 1,142 control - probands
were selected from subjects with and without mets , respectively . finally , family members of probands were recruited , yielding 635 families of case probands ( 1,175 subjects with mets and 1,423 subjects without mets ) and 1,142 families of the control probands ( 1,350 subjects with mets and 3,187 subjects without mets ; figure 1 ) .
data on mets , its components , and the socio - behavioral and reproductive characteristics of participants were obtained from the tlgs data bank .
all variables including body mass index ( bmi ) , waist circumference ( wc ) and systolic and diastolic blood pressure ( sbp and dbp ) were measured according to standardized protocols ( 13 ) .
furthermore , blood samples were taken after 12 - 14 hours of overnight fasting for biochemical analysis of triglycerides ( tgs ) , high - density lipoprotein cholesterol ( hdl - c ) , and fasting blood sugar ( fbs ) .
all assays , including those of fbs , tg and hdl - c levels were performed on the day of sampling .
fbs levels were measured using the glucose oxidase method ( glucose kit ; pars azmun , tehran , iran ) .
tg levels of the samples were determined using the enzymatic colorimetric method ( tg kit ; pars azmun , tehran , iran ) .
hdl - c levels of the samples were determined using the precipitation and enzymatic colorimetric method ( hdl - c kit ; pars azmun , tehran , iran ) .
coefficients of variation ( cv ) for hdl - c and tg measurements were < 5% .
levels of education were categorized as primary ( < 6 years ) , secondary ( 6 - 12 years ) , and higher ( 12 years ) .
in addition , based on the occupation status , participants were placed in three groups of unemployed , employed , and unemployed but having income .
the tlgs questionnaire included information on three major sections regarding lifestyle habits : smoking , pa and regular diet .
information about pa was collected using the modifiable activity questionnaire ( maq ) ( 14 ) to calculate the metabolic equivalent ( met ) based on min / wk .
high reliability ( 98% ) and moderate validity ( 47% ) were reported for the persian version of maq ( 14 , 15 ) .
physical activity levels were categorized as low ( met < 600 min / wk ) , moderate ( met 600 - 1499 min / wk ) , and high ( met 1500 min / wk ) ( 16 ) .
dietary data were collected using a valid and reliable 147-item semi - quantitative food frequency questionnaire ( ffq ) ( 17 ) , which asked the participants to provide their regular intakes over last year on a daily , weekly or monthly basis .
the daily intakes were calculated and the portion sizes of foods converted to grams using household measures .
each food and beverage was analyzed for energy and nutrient intake using the united states department of agriculture ( usda ) food composition table ( fct ) . in the current study ,
dietary factors including energy , leafy vegetables , red vegetables , orange vegetables , starchy vegetables , coffee , soft drinks , tea , fruits , dairy products and synthetic fruit juice were measured and categorized into quartiles of intake .
the first and the last quartiles of dietary factors have been considered as the least and the most consumption , respectively .
reproductive variables considered were age at menarche , number of abortions and number of children .
metabolic syndrome was defined based on the jis criteria ( 18 ) as having three or more of the following components : tgs 150 mg / dl or drug treatment , hdl - c < 40 mg / dl in men and < 50 mg / dl in women or drug treatment , sbp 130 mmhg and/or dbp 85 mmhg or drug treatment , fbs 100 mg / dl or drug treatment , and wc 89 cm for men and 91 cm for women ( 19 ) .
normal distribution of continuous data was assessed by the kolmogorov - smirnov test and non - normal variables were transformed to logarithms .
the liang and pulver method was used as a statistical model ( 20 ) , enabling the assessment of familial aggregation , environmental risk factors and relevant interactions allowing for correlation .
the generalized estimation equation ( gee ) method was used to estimate the regression coefcients for familial aggregation and environmental factors using spss software ( version 20 ; spss , chicago , il , usa ) .
adjusted odds ratios ( or ) and their respective 95% cis for environmental risk factors were also reported .
this case - control / family - based study was conducted on all 3,958 families of the tehran lipid and glucose study ( tlgs ) .
the design of the tlgs included two major components : a cross - sectional study to investigate the prevalence of cardiovascular disease and its risk factors , and a prospective 20-year follow - up with approximately 3.5 year intervals .
details of the tlgs design and its preliminary results have been described elsewhere ( 1 ) .
all data for the tlgs was collected through face - to - face interviews administered by trained research staff . for the present study ,
1,776 families that have at least one member with mets in the fourth phase ( 2009 - 2012 ) of the tlgs were recruited .
participants provided written informed consent and the study was approved by the ethics committee of the research institute for endocrine sciences , shahid beheshti university of medical sciences , tehran , iran ( ethics code 750ec , 2012 ) .
the authors are committed to protecting and ensuring the privacy and confidentiality of the personal health information of their participants .
this was a case - controlled , family - based study . of 12,823 subjects participating in the fourth phase of the tlgs , those aged < 19 years ( n = 1,758 ) , family members other than first and second degree ( n = 30 ) , and those with missing data ( n = 175 ) were excluded , with 10,860 subjects remaining
. metabolic syndrome , diagnosed by the joint interim statement ( jis ) criteria , led to 4,422 and 6,438 subjects with and without mets , respectively . using random sampling ,
635 case - probands and 1,142 control - probands were selected from subjects with and without mets , respectively . finally , family members of probands were recruited , yielding 635 families of case probands ( 1,175 subjects with mets and 1,423 subjects without mets ) and 1,142 families of the control probands ( 1,350 subjects with mets and 3,187 subjects without mets ; figure 1 ) .
data on mets , its components , and the socio - behavioral and reproductive characteristics of participants were obtained from the tlgs data bank .
all variables including body mass index ( bmi ) , waist circumference ( wc ) and systolic and diastolic blood pressure ( sbp and dbp ) were measured according to standardized protocols ( 13 ) .
furthermore , blood samples were taken after 12 - 14 hours of overnight fasting for biochemical analysis of triglycerides ( tgs ) , high - density lipoprotein cholesterol ( hdl - c ) , and fasting blood sugar ( fbs ) .
all assays , including those of fbs , tg and hdl - c levels were performed on the day of sampling .
fbs levels were measured using the glucose oxidase method ( glucose kit ; pars azmun , tehran , iran ) .
tg levels of the samples were determined using the enzymatic colorimetric method ( tg kit ; pars azmun , tehran , iran ) .
hdl - c levels of the samples were determined using the precipitation and enzymatic colorimetric method ( hdl - c kit ; pars azmun , tehran , iran ) .
coefficients of variation ( cv ) for hdl - c and tg measurements were < 5% .
levels of education were categorized as primary ( < 6 years ) , secondary ( 6 - 12 years ) , and higher ( 12 years ) .
in addition , based on the occupation status , participants were placed in three groups of unemployed , employed , and unemployed but having income .
the tlgs questionnaire included information on three major sections regarding lifestyle habits : smoking , pa and regular diet .
information about pa was collected using the modifiable activity questionnaire ( maq ) ( 14 ) to calculate the metabolic equivalent ( met ) based on min / wk .
high reliability ( 98% ) and moderate validity ( 47% ) were reported for the persian version of maq ( 14 , 15 ) .
physical activity levels were categorized as low ( met < 600 min / wk ) , moderate ( met 600 - 1499 min / wk ) , and high ( met 1500 min / wk ) ( 16 ) .
dietary data were collected using a valid and reliable 147-item semi - quantitative food frequency questionnaire ( ffq ) ( 17 ) , which asked the participants to provide their regular intakes over last year on a daily , weekly or monthly basis .
the daily intakes were calculated and the portion sizes of foods converted to grams using household measures . each food and beverage was analyzed for energy and nutrient intake using the united states department of agriculture ( usda ) food composition table ( fct ) . in the current study ,
dietary factors including energy , leafy vegetables , red vegetables , orange vegetables , starchy vegetables , coffee , soft drinks , tea , fruits , dairy products and synthetic fruit juice were measured and categorized into quartiles of intake .
the first and the last quartiles of dietary factors have been considered as the least and the most consumption , respectively .
reproductive variables considered were age at menarche , number of abortions and number of children .
metabolic syndrome was defined based on the jis criteria ( 18 ) as having three or more of the following components : tgs 150 mg / dl or drug treatment , hdl - c < 40 mg / dl in men and < 50 mg / dl in women or drug treatment , sbp 130 mmhg and/or dbp 85 mmhg or drug treatment , fbs 100 mg / dl or drug treatment , and wc 89 cm for men and 91 cm for women ( 19 ) .
normal distribution of continuous data was assessed by the kolmogorov - smirnov test and non - normal variables were transformed to logarithms .
the liang and pulver method was used as a statistical model ( 20 ) , enabling the assessment of familial aggregation , environmental risk factors and relevant interactions allowing for correlation .
the generalized estimation equation ( gee ) method was used to estimate the regression coefcients for familial aggregation and environmental factors using spss software ( version 20 ; spss , chicago , il , usa ) . adjusted odds ratios ( or ) and their respective 95% cis for environmental risk factors were also reported .
the mean for cardio - metabolic risk factors was higher in cases than controls except for hdl , which was lower in cases ( p < 0.001 ) ( data not shown ) .
a comparison of socio - demographic , behavioral , reproductive characteristics and dietary factors among the 7,135 participants between case and control groups is shown in table 1 .
the frequency of males in the case and control groups was 46.1 and 59.2 % , respectively .
subjects in the case group were older ( 52.0 14.7 vs. 36.6 14.4 years ) , with more females ( 53.9% ) , married ( 83.2% ) , current smokers ( 13.2% ) , unemployed but having income ( 19.8% ) , less high educated ( 16.8 vs. 32.0% ) , higher mean of abortion ( 0.56 0.96 vs. 0.22 0.59 ) and children ( 3.76 2.51 vs. 1.34 1.74 ) ( p < 0.001 ) . mean intake of dietary factors including leafy , red and orange vegetables and tea were higher in cases , while intakes of coffee and synthetic fruit juice were higher in controls ( p < 0.05 ) .
the difference of pa levels was nearly significant between the case and control groups ( p = 0.067 ) ( table 1 ) .
as shown in table 2 , the odds ratio for having mets among family members for case versus control probands was 2.19 ( 95% ci : 1.68 - 2.84 ) , which , after adjusting for potential confounders including age , sex , educational level , marital status , occupation , age at menarche , and energy , soft drink and starchy vegetable intake , increased to 2.31 ( 95% ci : 1.81 - 2.94 ; p < 0.05 ) . or : adjusted odds ratio for each variable by including factors ( age and gender of case and control proband and kcal ) and age and gender of family members from each case or control proband in the model . or , adjusted for familial aggregation and signicant factors . in the final model , after excluding non - significant variables , the odds ratios ( 95%ci ) were 1.62 ( 1.02 - 2.59 ) , 2.35 ( 1.40 - 3.96 ) , 1.51 ( 1.00 - 2.27 ) , 0.90 ( 0.83 - 0.97 ) , 0.70 ( 0.50 - 0.98 ) , and 1.72 ( 1.18 - 2.49 ) , for lower education ( r : higher education ) , married status ( r : single ) , unemployment with having income ( r : employed ) , age at menarche , starchy vegetables ( r : the fourth quartile ) and higher intakes of soft drinks ( r : the fourth quartile ) , respectively ( table 2 ) . compared to control probands , the risk of high wc , high bp , hyperglycemia , high tg , and low hdl increased significantly to 1.28 ( 1.10 - 1.48 ) , 1.28 ( 1.09 - 1.49 ) , 1.49 ( 1.28 - 1.75 ) , 1.72 ( 1.50 - 1.98 ) , and 1.49 ( 1.31 - 1.69 ) , respectively in cases .
familial aggregation inherited from the father was significantly observed in all mets components ( from or = 1.63 for hyperglycemia to or = 2.69 for high wc ) except for low hdl , after controlling for potential confounders .
familial aggregation resulting in offspring was significantly found for high wc ( or = 1.58 ) and hyperglycemia ( or = 1.64 ) .
familial aggregation attributed from mothers and spouses were significantly predictive for high tg ( or = 0.29 and or = 0.57 , respectively ) in both and high wc ( or = 0.33 ) just for mothers ( table 3 ) .
other independent variables controlled in the model included age , sex , level of education , marital status , occupation , starchy vegetables and soft drinks for central obesity , high blood pressure , hyperglycemia , triglycerides and hdl .
the present study reveals the familial aggregation of mets , even after adjusting for potential socio - demographic and behavioral confounders .
compared to the control group , case - proband families had approximately a > two - fold risk of having mets . in cases , among socio - demographic and reproductive factors , level of education , marital status , occupation , age at menarche , and some dietary factors were significantly associated with an elevated risk of mets compared to the control group .
the risk of all mets components significantly increased from 1.28-fold for both high wc and bp to 1.72-fold for high tg among the case - proband group .
considering spouses and siblings , higher risk for mets components among families whose fathers and offspring had mets components implies the pivotal role of genetic inheritance in the incidence of this syndrome and its components .
however , lack of the same association among families whose mothers had mets components could emphasize the probable role of the other socio - behavioral factors .
our results are consistent with a previous tlgs study , which showed that children of parents with mets had a > four - fold risk of having this syndrome ( 21 ) . in addition , more findings from the tlgs show a high recurrence risk ratio ( five - fold ) of mets among siblings and a higher significantly higher risk of mets aggregation among probands families affected , even after adjustment for age , gender and smoking ( 22 ) . the recurrence risk of sibling and familial aggregation of mets and its components has been previously studied ( 23 ) . a strong tendency to familial aggregation of mets
has been reported among taiwanese people , even after controlling for potential confounders ( 10 ) .
another study among the chinese population revealed familial similarity regarding body fat , lipid profiles , fbs , insulin levels and blood pressure ( 24 ) .
similar results from the framingham heart and offspring cohort have also shown family pair correlations for some of cardio - metabolic risk factors , specifically for bmi and lipid levels ( 25 ) . although these studies discuss genetic influences on the mets and its components , the spousal correlations observed for this syndrome , as a whole or some of its components , in some of participants imply the role of environmental factors in shaping this familial aggregation ( 26 , 27 ) . in the current study ,
compared to higher - educated subjects , familial aggregation of mets was significantly higher in less - educated case probands . despite considering education as a socio - demographic indicator in most previous studies ,
only a few discussed educational level as a determinant factor for incidence of mets and its components ( 10 , 28 , 29 ) . in line with our findings , another study showed that the familial aggregation of mets was more likely among those with lower educational levels ( 10 ) .
in addition , there is more evidence showing that the incidence risk of mets decreases with an increase in the years of education among norwegian men and women ( 29 ) .
however , ngo et al . emphasized the role of area - level of education in shaping the distribution and development of mets rather than individual - level of education ( 30 ) .
in addition to educational level , in the current study , marital status and occupation was significantly associated with familial aggregation of mets .
growing literature has documented the relationship between socioeconomic position and a wide range of health outcomes , including cardio - metabolic diseases ( 30 ) .
for example , associations between state - level income inequality and cardio - metabolic risk factors such as bmi , hypertension and sedentarism have been found to be stronger in low - income individuals ( 31 ) . based on our findings , lower age at menarche
was associated with higher risk of mets among families , whereas , in contrast , another study showed no significant association between age at menarche and the familial aggregation of mets ( 10 ) .
our study findings are in agreement with those of the bogalusa heart study conducted among 1,479 adult women , which reported that early menarche is characterized by a higher prevalence of clustered risk variables as mets components ( 32 ) .
this effect could be a combination of lifestyle risk factors and/or biological changes associated with age at menarche . in the current study , higher consumption of soft drinks was associated with greater risk of mets .
similarly , among middle - aged adults of the framingham heart study , a significant association was reported between a higher prevalence of mets and consumption of more than one serving of soft drink per day .
in a prospective analysis , a 44% increase of incidence of mets was seen among adults who consumed at least one serving of soft drink per day and daily consumption of soft drinks was also associated with a higher incidence of each mets component ( 33 ) .
. showed that among participants of the multi - ethnic study of atherosclerosis , sugar - sweetened beverages were directly related to a 36% higher risk of incidence of mets along with a greater risk of high fbs and enlarged wc , but not with other mets components ( 34 ) .
it may be explained that higher consumption of added synthetic sugar such as fructose corn syrup can lead to weight gain , increased insulin resistance , lower hdl - c , and increased tgs ( 35 - 37 ) .
the increased sweetness of diet foods may lead to conditioning for a greater preference of sweetened items .
the caramel content of both regular and dietary drinks may be a potential source of advanced glycation end products , promoting insulin resistance and inflammation ( 38 , 39 ) . in the current study ,
the proband parents , offspring , siblings , spouses and other second - degree relatives were studied and the impact of each family member was assessed separately .
the higher risk found in parents compared with the offspring , siblings and spouses suggest that genetic inheritance plays a key role , although the contribution of lifestyle habits conferred by parents can not ruled out .
when considering individual components of mets after adjusting for environmental factors , paternal influence was shown to play major role in the incidence of abdominal obesity , hypertension , hyperglycemia and high tgs .
the strength of the current study is its representation of tehranians , because results were based on a large general population of tehran .
furthermore , data on lifestyle , reproductive history and multiple cardiovascular risk factors were available and measured according to a standardized protocol .
first of all , this study was conducted in an urban community , which could be different from rural areas in some aspects .
in addition , missing data on behavioral factors such as pa and smoking could affect the results associated with these variables .
however , to the authors knowledge , this is the first report to use a genetic epidemiology approach to dissect the genetic , socio - behavior components and odds ratio of mets in an iranian population . in conclusion
, the risk of all mets components were significantly raised from 1.28 fold for both high wc and bp to 1.72 fold for high tg among case probands .
considering spouses and siblings , the higher risk for mets components among families whose fathers and offspring had mets components implies a pivotal role of genetic inheritance in incidence of the syndrome and its components .
however , lack of the same association among families in which the mothers had mets components could emphasize the probable role of the other socio - behavioral factors .
the strength of the current study is its representation of tehranians , because results were based on a large general population of tehran .
furthermore , data on lifestyle , reproductive history and multiple cardiovascular risk factors were available and measured according to a standardized protocol . nevertheless , some limitations should be mentioned .
first of all , this study was conducted in an urban community , which could be different from rural areas in some aspects .
in addition , missing data on behavioral factors such as pa and smoking could affect the results associated with these variables .
however , to the authors knowledge , this is the first report to use a genetic epidemiology approach to dissect the genetic , socio - behavior components and odds ratio of mets in an iranian population . in conclusion
, the risk of all mets components were significantly raised from 1.28 fold for both high wc and bp to 1.72 fold for high tg among case probands .
considering spouses and siblings , the higher risk for mets components among families whose fathers and offspring had mets components implies a pivotal role of genetic inheritance in incidence of the syndrome and its components .
however , lack of the same association among families in which the mothers had mets components could emphasize the probable role of the other socio - behavioral factors . | backgroundsince genetic and most environmental factors shape the context of families , some studies have been initiated to investigate the role of familial relationships in metabolic syndrome ( mets).objectivesto estimate the familial aggregation of mets and its components by identifying both case and control probands among tehranian adults with different socio - behavioral and reproductive characteristics.patients and methodsthis case - controlled / family - based study was conducted on 1,777 families ( 635 case probands ) who participated in the tehran lipid and glucose study ( tlgs ) .
socio - demographic and reproductive information including levels of education , marital status , occupation status , age at menarche , number of abortions , number of children , and lifestyle habits such as smoking , physical activity and regular diet were obtained from the tlgs data bank .
metabolic syndrome was defined according to the joint interim statement ( jis ) criteria . to estimate the regression co - efficient for familial aggregation and environmental factors ,
the generalized estimation equation method was used.resultsthe risk of having mets among family members for case versus control probands was 2.19 ( 95% ci : 1.68 - 2.84 ) , which , after adjusting for potential confounders including age , sex , educational level , marital status , occupation , age at menarche and energy , soft drink and starchy vegetable intake , increased to 2.31 ( 95% ci : 1.81 - 2.94 ; p < 0.05 ) .
compared to control probands , the risk of having mets components increased significantly from or = 1.28 for both high waist circumference ( wc ) and blood pressure ( bp ) to or = 1.72 for high triglycerides in cases .
familial aggregation inherited from the father was significantly observed in all mets components , from adjusted or = 1.63 for hyperglycemia to adjusted or = 2.69 for high wc , except for low hdl , after controlling for potential confounders.conclusionsconsidering spouses and siblings , there was a higher risk for mets components among families whose fathers and offspring had mets components , implying the pivotal role of genetic inheritance in the incidence of the syndrome and its components . |
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