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plasma liver enzyme tests are broadly applied to predict biochemical liver functions and to diagnose liver diseases .
alanine transaminase ( alt ) and aspartate transaminase ( ast ) are closely associated with hepatocyte damage and steatosis , including non - alcoholic fatty liver disease ( nafld ) .
elevated liver enzyme levels , as prospective risk factors , have been reported to be involved in type 2 diabetes and coronary heart disease [ 2 , 3 ] . however , alkaline phosphatase and gamma - glutamyltranspeptidase are related with biliary , cholestatic , and alcoholic diseases .
overweightedness in childhood or adolescence is persistent in adulthood obesity and is closely associated with adverse health effects , including metabolic diseases .
a recent cohort study in korean adolescents has shown strong associations of liver enzyme levels with a cluster of metabolic syndrome components .
thus , finding genetic markers of liver enzyme levels in childhood would be important for early prediction of liver function and its related disease in adulthood .
the heritabilities of liver enzyme levels range from approximately 35% to 61% [ 7 , 8 ] . to date , two genome - wide association studies ( gwass ) in european ancestry populations have identified genetic variants located in multiple genes associated with levels of alt ( hsd17b13-mapk10 , trib1 , chuk , and samm50 ) , ast ( nbpf3-alpl ) , and both of them ( cpn1 and pnpla3 ) [ 9 , 10 ] .
in addition , a meta - analysis identified pleiotropic effects between nafld and metabolic traits , such as low - density lipoprotein , high - density lipoprotein , triglyceride , fasting glucose , and insulin resistance . despite the identification of novel genes or loci associated with liver enzymes in cohort populations , the common variants in childhood are still largely unknown . here , we present findings from a gwas in childhood subjects to identify new susceptibility loci for liver enzyme levels .
students , aged between 8 and 13 years , were recruited from gwacheon city and kyunggi province from april to june 2010 . this study was performed as a part of the korean children - adolescents study ( kocas ) , which has been monitored yearly since their entry into elementary school at age 7 in gwacheon city or fourth grade at age 10 in seoul and gyeonggi province , korea .
subjects who were enrolled in a specific diet program or were taking any medications known to affect appetite were excluded from the study .
the study protocol was approved by the institutional review board of seoul paik hospital , inje university , and the korea center for disease control and prevention .
a total of 532 samples were genotyped using the illumina humanomni1-quad beadchip ( illumina inc .
samples that revealed the following properties were excluded : genotyping call < 98% , heterozygosity > 30% , sex inconsistency , cryptic first - degree relative , or duplication .
markers with a high missing call rate ( > 5% ) , minor allele frequency < 0.01 , and significant deviation from hardy - weinberg equilibrium ( p < 1 10 ) were also excluded .
the remaining 747,076 single nucleotide polymorphisms ( snps ) were used in subsequent analyses for association .
the association between genotypes and concentration of liver enzymes in plasma was evaluated with linear regression analysis , controlling for age and body mass index ( bmi ) as covariates .
the manhattan plots of -log10 ( p - value ) were made using haploview v. 4.2 .
the quantile - quantile plots were generated using r statistics package to assess the overall significance of the genome - wide associations .
students , aged between 8 and 13 years , were recruited from gwacheon city and kyunggi province from april to june 2010 . this study was performed as a part of the korean children - adolescents study ( kocas ) , which has been monitored yearly since their entry into elementary school at age 7 in gwacheon city or fourth grade at age 10 in seoul and gyeonggi province , korea .
subjects who were enrolled in a specific diet program or were taking any medications known to affect appetite were excluded from the study .
the study protocol was approved by the institutional review board of seoul paik hospital , inje university , and the korea center for disease control and prevention .
a total of 532 samples were genotyped using the illumina humanomni1-quad beadchip ( illumina inc .
samples that revealed the following properties were excluded : genotyping call < 98% , heterozygosity > 30% , sex inconsistency , cryptic first - degree relative , or duplication .
markers with a high missing call rate ( > 5% ) , minor allele frequency < 0.01 , and significant deviation from hardy - weinberg equilibrium ( p < 1 10 ) were also excluded .
the remaining 747,076 single nucleotide polymorphisms ( snps ) were used in subsequent analyses for association .
the association between genotypes and concentration of liver enzymes in plasma was evaluated with linear regression analysis , controlling for age and body mass index ( bmi ) as covariates .
the manhattan plots of -log10 ( p - value ) were made using haploview v. 4.2 .
the quantile - quantile plots were generated using r statistics package to assess the overall significance of the genome - wide associations .
a total of 747,076 snp genotype assays were tested on the dna samples of 484 korean childhood subjects .
alt and ast levels of the subjects according to age and sex are presented in supplementary table 1 .
linear regression analyses were performed to estimate the p - values for the association between genotype distributions and levels of alt and ast .
manhattan plots of association analyses of all snps with respect to alt and ast are presented with the chromosomal positions ( x - axis ) and negative logarithm of p - values ( y - axis ) of each snp ( fig .
quantile - quantile plots comparing the association p - values with those expected for a null distribution are also presented ( fig .
2 ) . the highest - ranked snps showing the lowest p - values ( p < 1.0 10 ) of association with alt and ast levels are listed in table 1 .
although there was no snp satisfying the criteria of genome - wide significance ( p < 1.0 10 ) , we identified three loci ( rs4949718 , rs80311637 , and rs596406 in st6galnac3 , adamts9 , and celf2 , respectively ) that were suggestively associated with levels of alt and ast .
of these snps , rs4949718 was found to be more strongly associated with alt levels ( p = 1.87 10 ) but less in ast levels ( p = 1.49 10 ) .
the missense rs80311637 ( v1597 m ) and intronic rs596406 snps were more strongly associated with an increased risk of ast than alt .
the levels of alt and ast , known as transaminases , are important indicators for determining hepatocyte injury or hepatocellular diseases .
however , alt is known to be found in the highest level in the liver ; thus , alt levels are more liver - specific indicators .
moreover , it has been reported that alt levels are more significantly associated with a cluster of metabolic syndrome than ast levels in a korean adolescent cohort study . in this study
, we identified three novel genes ( st6galnac3 , adamts9 , and celf2 ) that were multiply associated with elevated levels of alt and ast .
of the three loci , rs4949718 in the intron region of st6galnac3 yielded the lowest p - value for the association with alt , compared to ast levels ( table 1 ) .
in addition , rs80311637 ( adamts9 ) and rs596406 ( celf2 ) were shown to be more strongly associated with ast levels , compared to alt .
these results indicate that the liver - specific feature of alt was not driven as the secondary association for ast . in recent ,
a previous gwas in an amish population identified two genetic variants ( rs17209512 and deletion ) in glutamic - oxaloacetic transaminase 1 ( got1 ) that were associated with serum ast .
however , the rs17209512 snp , which is in incomplete ld with rs76850691 , was not replicated in our study ( table 1 ) .
for the three multiply associated loci , we performed conditional analyses to test whether the association with the other trait was simply secondary to the primary association ( supplementary table 2 ) . in these analyses ,
the association signals on rs4949718 for ast and rs80311637 and rs596406 for alt were abolished by adjustment of the alt and ast , respectively . it is assumed that the multiple effects of these loci on alt and ast resulted from the secondary effects by physiologically correlated features between alt and ast .
in addition , when comparing with bmi - unadjusted data , the association signals in alt were relatively weaker than those of the bmi - adjusted data . however , there was no significant difference in p - values before and after adjusting for bmi in ast ( data not shown ) .
st6galnac3 encodes a type of sialyltransferase that plays a role in the transfer of sialic acids from cytidine 5 ' monophospho - n - acetylneuraminic acid ( cmp - neuac ) to the carbohydrate termini of glycoproteins and glycolipids .
previous gwass have revealed the associations of st6galnac3 snps with human electroencephalogram and nicotine dependence [ 14 , 15 ] ; however , there has been no report on an association with liver enzyme levels .
although the association with liver enzymes is still unknown , st6galnac3 has been shown to be associated with subscapular skin - fold thickness , a type of obesity - related trait , in a korean adult population .
obesity affects elevated liver enzyme levels by inducing inflammatory responses in the liver , accompanied by increases in c - reactive protein .
the involvements between obesity and liver enzyme levels have been found in cohort studies [ 18 - 20 ] .
therefore , the association of st6galnac3 with obesity may influence liver enzyme levels indirectly . in addition , st6galnac3 protein interacts with 2-heremans - schmid glycoprotein ( ahsg ) , which is also known as fetuin - a , by enzyme - substrate specificity .
ahsg is abundantly distributed in serum protein and is produced mainly in the liver , and elevated levels of this protein are associated with the risk of nafld . considering that liver enzyme levels are closely involved indicators in nafld [ 22 , 23 ]
, the interaction with ahsg might induce an association between st6galnac3 and liver enzyme levels .
adamts9 is a metalloproteinase that plays a role in cleavage of proteoglycans and is expressed in skeletal muscle and pancreas .
this gene has also shown an association with insulin resistance and impaired beta - cell function in the pancreas . taken together
, the association of adamts9 with liver enzymes might be due to its susceptibility to diabetes and insulin resistance .
celf2 ( also known as cugbp2 ) induces the skipping of exon 11 of insulin receptor ( ir )
. the mrna expression level of exon - skipped ir is significantly lower in liver than in other organs in mouse models .
it has been reported that a decrease in the number of irs on target cells can cause insulin resistance .
therefore , it is assumed that celf2 affects liver enzyme levels by lowering expression of ir in liver that induces insulin resistance . in summary ,
our data demonstrate new multiple associations with alt and ast levels in a korean childhood cohort .
further replication and functional studies remain to be done to clarify the genetic factors of liver dysfunction .
supplementary data including two tables and one figure can be found with this article online at http://www.genominfo.org/src/sm/gni-11-149-s001.pdf . | liver enzyme elevations , as an indicator of liver function , are widely associated with metabolic diseases .
genome - wide population - based association studies have identified a genetic susceptibility to liver enzyme elevations and their related traits ; however , the genetic architecture in childhood remains largely unknown .
we performed a genome - wide association study to identify new genetic loci for liver enzyme levels in a korean childhood cohort ( n = 484 ) .
we observed three novel loci ( rs4949718 , rs80311637 , and rs596406 ) that were multiply associated with elevated levels of alanine transaminase and aspartate transaminase .
although there are some limitations , including genetic power , additional replication and functional characterization will support the clarity on the genetic contribution that the st6galnac3 , adamts9 , and celf2 genes have in childhood liver function . |
the adoption of the latin cortex to describe the outer gray matter covering of the brain paints this structure as a thin bark or rind enveloping the remainder of the brain.1 the cerebral cortex , however , is not a homogeneous , two - dimensional mantle .
rather , the cortex is a multilayered sheet with rich laminar interconnections.26 accordingly , there is a notable spatial anisotropy in the radial ( laminar ) arrangement of cortical cell bodies and their projections . what is the consequence of this anatomical organization for neural activity patterns measured in the cortical microcircuit ?
for example , might the intrinsically generated , spontaneous activity be highly coordinated within a cortical column but relatively independent between cortical columns ? addressing this type of question requires simultaneous measurements at known spatial intervals in the cortex .
recent advances in neurophysiological techniques have exploited high - density multicontact recordings of extracellular electrical activity in vivo79 to facilitate comparison of neuronal signals collected simultaneously .
this approach allows one to assess the spatial characteristics of neural coordination between known positions within the three - dimensional cortical extent . in this review ,
we highlight key findings from our group that shed light on neuronal activity patterns within the three - dimensional volume of the cerebral cortex .
we focus on primate visual cortex as a model sensory system with well described anatomy that has been the subject of a large body of neurophysiological studies . to contextualize current work , we first provide an overview of the cortex s anatomical features as well as methods to assess spatial coordination of neural processes .
we then briefly summarize neurophysiological data that demonstrate the fundamental anisotropy of neural coherence in the tangential versus radial dimensions of the cortex .
as early as the 19th century , neuroanatomists began to parcel the mammalian cortical sheet across the tangential dimension into distinct areas that differ in their cytoarchitecture.10 these areas were subsequently shown to exhibit distinct patterns of connectivity and neuronal function .
some of the neocortical areas defined in these ways are conserved among primates and to some extent among other mammalian orders in their topological position and pattern of interareal projections.1113 within each cortical area , certain axonal projections do not enter the white matter , but instead remain in the neuropil .
these horizontal connections , which are laterally extending branches of pyramidal cell axons,14,15 are marked by a high degree of functional specificity and often target neurons or columns with broadly similar response properties.16 the extent of tangential cortical distance covered by these horizontal connections varies considerably between areas and increases at higher stages in the cortical hierarchy.17 whereas axons in the primary visual cortex ( v1 ) may reach 2 mm,18 those in the inferior temporal cortical area ( te ) have been shown to project up to 9 mm.19 horizontal connections tend to be reciprocal,20 commonly remain within their cortical layer,21,22 and are largely excitatory both in origin as well as in their postsynaptic targets.23,24 functionally speaking , horizontal connections are mostly modulatory in nature,25 and likely contribute to the high activity coherence measured over short distances in the cortex ( see measuring the reach of activity coherence in the cortex ) .
early neuroanatomists also recognized the importance of the radial dimension to the composition of the cortex , with sheets of cells arranged in segregated layers that form early in development.26,27 four to six cytoarchitectonic layers can be visualized with a variety of histological staining techniques ( figure 1a ) .
the exact delineation of the respective layer boundaries depends somewhat on the type of histological stain used , the cortical areas examined , and the animal species.28 the resulting variability in layer count has prompted the adoption of slightly different labeling schemes over the years,29,30 but broad consensus follows neuroanatomist korbinian brodmann s original plan of dividing the neocortex into six major laminae.10 these individual layers are further grouped into three visibly distinct laminar domains .
layer 4 and its various sublayers are commonly denoted as granular , since the high density of spiny cell bodies in this domain appears grainy in nissl - type histological stains .
superficial layers 13 thus occupy a supragranular position , whereas deeper layers 5 and 6 are infragranular.31 in contrast with the pronounced morphological differences between cortical layers , functional responses across layers are similar , and have given rise to the concept of a cortical column.3236 the cortical column is conceptualized as a repeated motif of radial units , each supported by a stereotypical pattern of intrinsic connections,37 the functional homogeneity of which is often considered a universal and critical feature of the cerebral cortex.38 in the primary visual cortex , for example , the same basic orientation and eye preferences can be observed across layers of a given column.39 moreover , studies point to a basic model of sequential excitation across particular laminae within a column following the arrival of sensory information from the thalamus.40,41 this model suggests that thalamic input to cortical layer 4 is relayed to the supragranular layers , where it is integrated with various types of modulatory signals,2,4244 and then relayed to the infragranular layers .
infragranular neurons send local projections to granular and supragranular targets in the same cortical column , as well as long - range projections to cortical and subcortical structures inside and outside of the telencephalon.45 while there are many ways in which information can be integrated across columns , and the generality of functional columns in the cortex remains a topic of debate,4658 this idealized microcircuit model emphasizing intracolumnar processing has played an important role in understanding the complexity of cortical projection patterns and their bearing on measured sensory signals.59,60 to summarize , the anatomical organization of the cortex varies considerably across both the radial and tangential dimensions . within a given area ,
horizontal connections account for much of the tangential connectivity , whereas the interlaminar projections account for the radial connectivity .
the local field potential ( lfp ) represents a collective measure of neural activity , including subthreshold synaptic processes that are synchronized in space and time.6166 its inherent heterogeneity limits the precision of the lfp for probing details of microcircuit function.67,68 nonetheless , empirical studies have shown that both sensory - evoked lfp responses69 and high frequency gamma range ( > 30 hz ) lfp power70,71 are often well correlated with local spiking activity in the cortex .
thus , it is possible to use the lfp , and in particular its upper frequency range , to study neural coordination at different spatial scales .
however , a conspicuous feature of the lfp signal is its 1/f spectral distribution , where f denotes frequency and is the exponent of a power law.72,73 this means that the high frequency gamma range is at least one order of magnitude smaller in amplitude than the lower frequency fluctuations , which dominate the raw signal fluctuations . experimental approaches to measure the spatiotemporal coordination of neural signals involve simultaneous electrophysiological measurement from multiple electrodes ( figure 1b ) .
having obtained such simultaneous signals , most attempts to evaluate the interaction between sites amount to a measure of temporal correlation of two or more neural signals . to this end
, neurophysiologists employ a wide range of correlational measures that will not be reviewed here . here
we describe one common measure , the mean squared coherence , which measures signal correlation as a function of frequency . whether or not frequency is a natural domain across which to divide neural signals is a matter of debate , although this practice is very common .
what is clear is that doing so allows one to isolate aspects of the lfp signal that correlate with particular behavioral or neural events , such as the close relationship between the local spiking and the low amplitude gamma range mentioned above .
it is important to mention that the precision of lfp coherence as a measure of neural coordination is limited by electric volume conduction .
in contrast with spiking activity , where the signal source can be identified as one or more nearby neurons , the lfp reflects an ambiguous combination of superimposed local and distant neural events.65,67 in the following section , we use mean squared coherence as a measure to assess the coherence across the different cortical dimensions . of particular interest
is how the pronounced anatomical anisotropy of the cortex described in the previous section influences the spatial coordination of neural activity across these different dimensions .
given the columnar cortical architecture outlined above , one might predict that lfp coherence would also follow a columnar pattern .
interestingly , the data suggest nearly the opposite : the spatial stretch of lfp coordination is perpendicular to the cortical columns . the lfp is spatially coherent along zones extending tangentially , but is sharply discontinuous within a column . between electrodes
spaced tangentially along the cortical surface , lfp coherence falls off continuously and in a frequency - dependent fashion .
one study of the macaque visual cortex examined the coherence of ongoing activity across distances spanning from a few hundred microns to over a centimeter as monkeys rested idly in a dark room.74 the main findings of that study were that lfp coherence falls off monotonically , and that the falloff is steeper for high frequency than for low frequency lfp components ( figures 2a and 3a ) .
a subsequent study showed that this falloff reflects the cortical distance rather than the absolute distance between the electrode contacts , since lfp coherence between electrodes placed on either side of a sulcus showed a drop reflecting the span of the infolded cortex.75 this general pattern of coherence is qualitatively similar during rest and when monkeys perform a simple behavioral task ( ie , when they are behaviorally engaged ) and is comparable in amplitude and slope between areas v1 , v2 , and v4 .
the results are consistent with other findings that show steep ( < 5 mm ) falloff in high frequency lfp and spiking76 and more gradual ( > 5 mm ) falloff in low frequency lfp coherence as a function of lateral cortical distance.67,69,74,77 in the radial dimension , coherence has recently been assessed between contacts occupying different laminar falloff in lfp coherence along the cortical surface , the pattern of laminar coherence is discontinuous and strongly compartmentalized , particularly for frequency ranges below the gamma range ( figures 2b and 3b).78,79 probing the spatial pattern of coherence from different seeds reveals two prominent laminar zones of coherent lfp activity separated by the deep granular layer , with one zone dominating within the superficial layers ( 13 ) and another zone in the deep layers ( 5 and 6 ) . while the lfp coherence within these zones was high , that between them was nearly zero across a wide frequency range ( see figure 3b ) .
this spatial structure was present in the ongoing activity both during rest and when the animals were engaged in a behavioral task .
taken together , the studies outlined above suggest that the spatial extent of ongoing lfp coherence spreads broadly in the tangential direction but is restricted in the radial direction .
the resulting spatial pattern can be conceptualized as two laminar sheets of coherent activity ( figure 4 ) , which constitutes a marked deviation from the radial anisotropy predicted by the columnar architecture described above .
the findings reviewed here point to an intricate three - dimensional structure of neural coherence that reflects a laminar compartmentalization emerging when the brain is not explicitly stimulated ( with the animals being either at rest or behaviorally engaged ) .
neither known interlaminar connections nor innervation from the thalamus or other cortical areas can fully explain the strictly separate domains of intracortical coherence37,8083 we observed in the upper and lower cortical layers .
nonetheless , it is interesting to speculate that in the absence of specific sensory input ( ie , during periods of rest or between sensory events ) , cortical activity may switch from a columnar mode , where the sensory world is parsed by a large number of columnar units dedicated to extracting features of the world , to a laminar mode , where functional differences in the tangential direction become less important , and activity segregates by layer
one possibility is that it reflects a division between intracortical versus thalamocortical interactions , with one contributing most strongly to the upper layer fluctuations and the other to the lower layer fluctuations .
while clearly a speculation at this point , this explanation would account for the apparently separate laminar sheets of ongoing cortical activity that are largely uncoordinated across their boundary in the granular layer . at first sight
, our observation of radially anisotropic ongoing activity seems at odds with the columnar structure of stimulus - evoked responses because of the apparent compartmentalization that has been reported in certain studies .
for example , neurophysiological recordings with multiple laminar electrodes inserted simultaneously into rat auditory cortex found that the spatial pattern of spiking activity within the supragranular layers was highly localized , sparse , and strongly specific to a small subset of sensory stimuli .
in contrast , spiking within the infragranular layers was widespread , constant , and promiscuous with respect to sensory stimulation.84 furthermore , analysis of spontaneous lfp recorded in several visual areas found a peak of low frequency ( 10 hz ) coherence between lfp and multiunit spiking activity in the infragranular layers that was absent in the supragranular layers.85 in contrast , the recently described repeating patterns of clustered cortical synchronization termed neuronal avalanches
seem to be confined to the superficial layers of primate sensory cortex.86 stimulus - evoked lfp power87 and multiunit spiking activity88 of macaque v1 have been demonstrated to exhibit pronounced anisotropies between upper and lower cortical layers . moreover , reversible inactivation ( cooling ) of the supragranular layers of cat primary visual cortex had little influence , if any , on the response properties of infragranular neurons.89,90
lastly , some authors have reported stark differences in orientation tuning between supragranular and infragranular neurons within the same cortical column of macaque visual cortex,9193 which they interpreted as evidence for a functional dichotomy between deep and superficial layers.94 these results , taken together with the coherence differences in lfp discussed above , hint at a strict functional division between the main laminar compartments of visual cortex .
the laminar pattern of lfp activity described here thus may provide a unique window on the spatiotemporal structure of neuronal population activity that warrants further investigation into the relationship between structure and function within the cortical microcircuitry . | the mammalian neocortex features distinct anatomical variation in its tangential and radial extents .
this review consolidates previously published findings from our group in order to compare and contrast the spatial profile of neural activity coherence across these distinct cortical dimensions .
we focus on studies of ongoing local field potential ( lfp ) data obtained simultaneously from multiple sites in the primary visual cortex in two types of experiments in which electrode contacts were spaced either along the cortical surface or at different laminar positions .
these studies demonstrate that across both dimensions the coherence of ongoing lfp fluctuations diminishes as a function of interelectrode distance , although the nature and spatial scale of this falloff is very different . along the cortical surface ,
the overall lfp coherence declines gradually and continuously away from a given position .
in contrast , across the cortical layers , lfp coherence is discontinuous and compartmentalized as a function of depth .
specifically , regions of high lfp coherence fall into discrete superficial and deep laminar zones , with an abrupt discontinuity between the granular and infragranular layers .
this spatial pattern of ongoing lfp coherence is similar when animals are at rest and when they are engaged in a behavioral task .
these results point to the existence of partially segregated laminar zones of cortical processing that extend tangentially within the laminar compartments and are thus oriented orthogonal to the cortical columns .
we interpret these electrophysiological observations in light of the known anatomical organization of the cortical microcircuit . |
heterologous synthesis of selected ntts and mutant proteins in e.
coli the heterologous synthesis of pamntt1 and of two
atp / adp transporters from the rickettsia - related bacteria
caedibacter caryophilus ( ccntt ) and holospora
obtusa ( hontt ) was performed on the basis of existing pet16b
constructs ( 13 ,
22 ) , which were transformed
into blr cells ( merck ) .
heterologous expression of the full - length cdna
encoding the plastidial atp / adp transporter from arabidopis thaliana
( atntt1 ) led to very low amounts of recombinant transporters in blr
cells ( data not shown ) .
however , the truncation of the n - terminal extension
( the putative leader peptide of 87 amino acid residues ) and application of
rosetta2 ( de3 ) plyss expression cells resulted in an increased
synthesis of atntt1 .
the truncated atntt1 sequence was
amplified from the expression construct
( 34 ) by pcr using pfu
polymerase and the following oligonucleotides : atntt1shortndei_sense ,
5-ggccgcggctcatatggacggagctg-3 , and the standard primer
t7-term .
the pcr construct contained one ndei restriction site inserted by the
sense primer and the xhoi restriction site of the plasmid .
the amplificate was
cloned into an appropriately ( ndei / xhoi ) restricted pet16b vector . for generation of k446 mutant proteins , the pamntt1 encoding
sequence ( inserted in pet16b )
was modified by site - directed mutagenesis using
the quikchange site - directed mutagenesis kit ( stratagene ) .
the triplet
encoding lysine 446 ( aaa ) was substituted by the triplets cgc , cag , and gaa ,
respectively , by pcr . to eliminate possible pcr - caused mistakes in the
amplified expression vector , we excised the mutated sequences by using bamhi
( 13 ) and inserted them into
the correspondingly prepared original pet16b vector .
the correctness of all
constructs was proven by complete sequencing ( nanobiocenter , tu
kaiserslautern , germany ) .
briefly , transformed
bacterial cells were grown at 37 c in tb medium under selective
conditions . at an a600 of 0.5 , heterologous
expression
four
hours post - induction the cells were harvested by centrifugation ( 5,000
g , 10 min , 20 c ) , resuspended in buffer medium ( 1 mm
edta , 15% glycerol , 10 mm tris , ph 7.0 ) , and immediately frozen in
liquid n2 . purification and reconstitution of heterologously expressed
ntts enrichment of e. coli membranes and purification and
reconstitution of heterologously expressed ntts were conducted as described by
trentmann et al .
the recombinant , histidine - tagged proteins were purified
by immobilized metal affinity chromatography with nickel - sepharose 6 fast flow
( ge healthcare ) according to the manufacturer 's instructions .
buffers used for
washing and elution contained 0.5% n - dodecyl--maltoside . to
rule out pi incorporation during reconstitution , we omitted
pi not only in the elution buffer but also in the last washing
step .
additionally , the remaining liquid at the immobilized metal affinity
chromatography material and in the column tip was removed thoroughly prior to
elution .
50 l of the eluate were reconstituted into liposomes loaded with
the given interior substrates ( 10 mm atp or adp or 5 mm
pi ) .
protein concentrations of the purified ntts were quantified in
a bio - photometer ( eppendorf , hamburg , germany ) according to the method
described by bradford ( 35 ) .
purity of the recombinant ntts was analyzed by sds - page ( 3% stacking and 12%
separating gel ) as described by laemmli
( 36 ) .
import of radioactively labeled substrates into
proteoliposomes uptake studies were performed as previously
described ( 32 ) . for transport
measurements
, proteoliposomes were incubated at 30 c in presence of the
given concentrations of -p - labeled nucleotides or
radioactive pi [ p ] ( perkinelmer life sciences ) .
optionally , import was conducted in the presence of the given concentrations
of nonlabeled nucleotides or pi .
removal of substrates by anion
exchange chromatography ( dowex 1 8 cl , 200400 mesh ; sigma ) was
used to terminate import .
liposomes were eluted , and internal radioactivity
was quantified in a scintillation counter ( canberra - packard , rsselsheim ,
germany ) .
pi influences nucleotide exchange the observation
that pamntt1 mediated adenine nucleotide exchange is electroneutral
( 32 ) was surprising .
this
carrier obviously transports a counterion that compensates for the charge
difference of the hetero - exchange .
the catalytic activity of bacterial and
plastidial atp / adp transporters in contrast to bacterial proton - driven ntts is
not affected by the proton gradient across the membrane
( 1215 ,
1923 ) .
, we analyzed the influence of diverse
salt compositions on pamntt1-mediated nucleotide transport in the
liposomal system .
we identified that the comparably low rates of adp import
into atp loaded vesicles ( adpim / atpex ) were increased by
500 m external pi ( 3.7 times ) and also by its
structural analogue , arsenate ( 2.6 times ) ( supplemental table s1 ) .
higher
concentrations ( 2 mm ) of latter compounds further increased the
stimulatory effects .
the addition of other components had lower stimulatory or
even inhibitory influences on the corresponding transport ( supplemental table
s1 ) .
figure 1.influence of the presence of pi in the liposomal lumen on
pamntt1 mediated adenine nucleotide hetero - exchange .
atpim / adpex transport and
adpim / atpex transport was analyzed in absence ( light
gray bars , set to 100% ) and in presence of internal pi
( dark gray bars , calculated according to the transport in absence of
pi ) .
for this , pamntt1 was reconstituted into liposomes
loaded with 10 mm adp or atp or into vesicles containing 5
mm pi plus 10 mm adp or 5 mm
pi plus 10 mm atp .
import of 50 m
-p - labeled atp or adp was allowed for 5 min and stopped by
anion exchange chromatography .
the data represent net values calculated by
subtraction of uptake into vesicles lacking counter - exchange nucleotides and
are the means of three independent experiments .
the presence of pi and adenine nucleotides at
the liposomal interior and exterior is graphically displayed .
influence of the presence of pi in the liposomal lumen on
pamntt1 mediated adenine nucleotide hetero - exchange .
atpim / adpex transport and
adpim / atpex transport was analyzed in absence ( light
gray bars , set to 100% ) and in presence of internal pi
( dark gray bars , calculated according to the transport in absence of
pi ) .
for this , pamntt1 was reconstituted into liposomes
loaded with 10 mm adp or atp or into vesicles containing 5
mm pi plus 10 mm adp or 5 mm
pi plus 10 mm atp .
import of 50 m
-p - labeled atp or adp was allowed for 5 min and stopped by
anion exchange chromatography .
the data represent net values calculated by
subtraction of uptake into vesicles lacking counter - exchange nucleotides and
are the means of three independent experiments .
the presence of pi and adenine nucleotides at
the liposomal interior and exterior is graphically displayed . in a previous study we demonstrated that only those reconstituted
pamntt1 proteins , which were inserted in the native orientation
( right side out ) , displayed catalytic activity
( 32 ) . to mimic the presence of
pi in the bacterium , we tested whether pi application at
the liposomal interior also influences adenine nucleotide hetero - exchange .
when compared with the control ( 0 mm luminal pi ) , the
addition of internal pi stimulated atp import in exchange with adp
( atpim / adpex ) 4-fold , whereas
adpim / atpex transport was not highly affected by
internal pi application ( fig .
1 ) .
accordingly , not only external but also internal pi
supports atp / adp exchange .
however , stimulation of the hetero - exchange
exclusively occurred when adp and pi were present at the same side
of the liposome ( adp plus pi ) ( supplemental table s1 and fig .
1 ) .
it had been suggested that pi plays a regulatory role in atp / adp
exchange across the rickettsial membrane
( 37 ) .
this based on the
observation that high pi concentrations marginally enhanced atp but
highly stimulated adp import into isolated bacteria cells .
furthermore , only
in the presence of pi , adp was able to compete with atp for import ,
leading the authors to the assumption that pi might increase ( at
least ) the affinity of the rickettsial atp / adp transporter for adp
( 37 ) . to investigate the role
of pi in ntt - mediated adenylate exchange in more detail and
separated from the complex cellular metabolism
, we analyzed the effect of
rising external pi concentrations on adenine nucleotide import into
pamntt1-proteoliposomes loaded with interior pi plus the
given counter - exchange nucleotides .
figure 2.influence of rising exterior pi concentrations on adenine
nucleotide hetero - exchange mediated by pamntt1 .
pamntt1
was reconstituted into liposomes loaded with 5 mm pi
plus 10 mm of the given nucleotides .
the effects of rising external
pi concentrations on atp import into adp plus pi - loaded
proteoliposomes ( light gray bars ) and on adp import into atp plus
pi - loaded vesicles ( dark gray bars ) were analyzed .
import
of 50 m -p - labeled atp or adp was stopped
after 5 min .
the applied external pi concentrations ( in
mm ) are displayed at the x axis .
km
and vmax values ( in parentheses ) were calculated
for adenine nucleotide hetero - exchanges in the absence ( 0 mm ) and
in the presence of 5 mm external pi , respectively , and
are given above the bars of the corresponding exchanges .
the
data represent net values calculated by subtraction of uptake into vesicles
lacking counter - exchange nucleotides and are the means of three independent
experiments .
standard errors of the kinetic
parameters were below 12% and of adpim / atpex transport
in absence of external pi ( 0 mm ) were at 20% . in
the graphic depicting the presence of pi and nucleotides at the
liposomal interior and exterior ,
influence of rising exterior pi concentrations on adenine
nucleotide hetero - exchange mediated by pamntt1 .
pamntt1
was reconstituted into liposomes loaded with 5 mm pi
plus 10 mm of the given nucleotides .
the effects of rising external
pi concentrations on atp import into adp plus pi - loaded
proteoliposomes ( light gray bars ) and on adp import into atp plus
pi - loaded vesicles ( dark gray bars ) were analyzed .
import
of 50 m -p - labeled atp or adp was stopped
after 5 min .
the applied external pi concentrations ( in
mm ) are displayed at the x axis .
km
and vmax values ( in parentheses ) were calculated
for adenine nucleotide hetero - exchanges in the absence ( 0 mm ) and
in the presence of 5 mm external pi , respectively , and
are given above the bars of the corresponding exchanges .
the
data represent net values calculated by subtraction of uptake into vesicles
lacking counter - exchange nucleotides and are the means of three independent
experiments .
standard errors of the kinetic
parameters were below 12% and of adpim / atpex transport
in absence of external pi ( 0 mm ) were at 20% . in
the graphic depicting the presence of pi and nucleotides at the
liposomal interior and exterior ,
changes in external pi availability had low impact on atp
homo - exchange parameters and only slightly affected adp homo - exchange ( 4-fold
increased vmax at 5 mm external pi )
( data not shown ) .
in contrast to the homo - exchanges , the hetero - exchanges were
substantially influenced by pi addition
( fig .
as observed in the
effector analysis ( supplemental table s1 ) adpim / atpex
hetero - exchange was significantly stimulated by exterior pi .
the
highest rise in stimulation was obtained between 50 m and 2
mm pi ( fig .
2 , dark gray bars ) .
increasing the external pi
concentration above 2 mm caused a further but comparably slight
stimulation of the adp import rates and suggests the beginning of a
pi saturation phase .
determination of the kinetic parameters
revealed that the stimulatory influence of external pi on
adpim / atpex exchange can be traced back to an enhanced
affinity ( from 0 to 5 mm : 14-fold lower km ) and
an increased vmax ( from 0 to 5 mm : 3-fold
higher vmax ) of pamntt1 for adp .
concentrations of more than 2 mm pi in the transport
medium led to a substantial reduction of the atpim / adpex
transport rates .
the vmax of this exchange was high in the
presence as well as in the complete absence of external pi , and
thus the lowered import rates were mainly caused by a decreased affinity of
pamntt1 for atp import ( from 0 to 5 mm : 27-fold lower
km ) ( fig .
2 ) .
it has to be mentioned that the transport rates of optimal
atpim / adpex exchange ( 0 mm external and 5
mm internal pi , 1000 nmol mg protein 5
min ) were much higher than that of pi stimulated adp
import into atp plus pi - loaded vesicles ( 5 mm external
and 5 mm internal pi , 350 nmol mg
protein 5 min )
( fig .
it
might be assumed that pi in the vesicle lumen hindered optimal atp
export .
however , depletion of luminal pi did not result in a
further relevant stimulation of adpim / atpex exchange
( data not shown ; see also adpim / atpex in
fig .
the opposed effects of
external pi on adpim / atpex versus
atpim / adpex exchange could be explained by ( i ) a
cotransport of adp and pi and by ( ii ) a possible competition of
pi with atp during hetero - exchange .
pi is substrate of pamntt1 but only in conjunction with
adp to analyze whether pamntt1 is able to transport
pi , we performed import studies using radioactively labeled
pi ( 50 m p ) .
pi import was
conducted with proteoliposomes loaded with interior pi ,
pi plus adp , or pi plus atp . in absence of exterior
nucleotides
no pi uptake into the vesicles was measurable when only
pi , or pi plus atp were internally present
( fig .
3a , white
circles and black diamonds ) . to our surprise pi was
imported into liposomes loaded with pi plus adp
( fig .
3a , gray
squares ) . in a similar experiment we tested the effect of externally
added adp ( fig .
application of external adp did not markedly affect
the rates of pi import into pi plus adp loaded liposomes
( fig .
3 , compare a and
b , gray squares ) but stimulated pi
import into liposomes containing solely pi , or pi plus
atp ( fig .
3 , compare a and
b , white circles and black diamonds ) .
external atp led to no or to comparably low pi import ( compare the
import rates of fig .
the
latter effect further argues for a possible competition of pi and
atp for import .
our results show that pi indeed is a substrate of
pamntt1 but only when adp is present at least at one side of the
liposomal membrane .
the observation that pi transport occurs also
in the absence of adenine nucleotide counter - exchange
( fig .
3 ) suggests that
pamntt1 catalyzes , in addition to a possible pi plus adp
cotransport in exchange with atp , an adp - dependent pi
homo - exchange . to provide further evidence for a pi homo - exchange
,
we analyzed pi import into adp - loaded liposomes lacking luminal
pi .
the measured pi import rates were 10-fold lower
than the corresponding rates obtained with liposomes containing adp plus
pi ( data not shown ) .
this is a further argument supporting the
postulated pi homo - exchange . to analyze whether pi
homo - exchange is accompanied by a simultaneous unidirectional translocation of
adp , we additionally investigated [ -p]adp import into
proteoliposomes solely loaded with pi .
because in the absence of
counter - exchange nucleotides pi but not adp import occurred ( data
not shown ) , we concluded that adp - dependent pi homo - exchange is not
associated with a unidirectional adp transport .
pamntt1 catalyzes pi homo - exchange and cotransport with
adp in a further study we analyzed adp and pi transport
in more detail to address two important questions .
first , is pamntt1
capable of transporting pi as cosubstrate of adp during
hetero - exchange , and second , what is the stoichiometrical ratio of the
pi plus adp cotransport . figure 3.analysis of pi import mediated by reconstituted
pamntt1 .
time - dependent import of 50 m radioactive
pi into pamntt1-proteoliposomes loaded with 5
mm pi or 5 mm pi plus the given
adenine nucleotides ( 10 mm ) .
pi import was allowed for
the given time spans and stopped by anion exchange chromatography .
pi uptake studies were performed in the absence of external
nucleotides ( a ) , in the presence of 50 m nonlabeled
external adp ( b ) , or in the presence of 50 m
nonlabeled external atp ( c ) to investigate a possible influence of
external adenine nucleotides on pi transport .
pi uptake
into proteoliposomes loaded with 5 mm pi ( open circle ) ,
5 mm pi plus 10 mm adp ( gray square ) , or 5
mm pi plus 10 mm atp ( black diamond ) .
time - dependent import of 50 m radioactive
pi into pamntt1-proteoliposomes loaded with 5
mm pi or 5 mm pi plus the given
adenine nucleotides ( 10 mm ) .
pi import was allowed for
the given time spans and stopped by anion exchange chromatography .
pi uptake studies were performed in the absence of external
nucleotides ( a ) , in the presence of 50 m nonlabeled
external adp ( b ) , or in the presence of 50 m
nonlabeled external atp ( c ) to investigate a possible influence of
external adenine nucleotides on pi transport .
pi uptake
into proteoliposomes loaded with 5 mm pi ( open circle ) ,
5 mm pi plus 10 mm adp ( gray square ) , or 5
mm pi plus 10 mm atp ( black diamond ) .
atp import in strict stoichiometrical exchange with one adp plus one
pi ( h2po4 , with one negative
charge ) would be electroneutral . in this context
it is important to mention
that under the applied conditions an important part of pi carries
one negative charge .
unfortunately , determination of the adp plus pi export
parameters of the reconstituted pamntt1 was impossible .
we were not
able to measure the slight decrease of the high internal label .
however , we demonstrated above that exterior pi
stimulates adpim / atpex transport ( supplemental table s1
and fig .
2 , black bars ) and that similarly interior pi
stimulates atpim / adpex transport
( fig .
, it seems
justified to assume that pi acts as cosubstrate of adp during
hetero - exchange independent of the transport direction
( adpim+piim / atpex or
atpim / adpex+piex ) .
accordingly , we
investigated the coimport of pi and adp . for this , import measurements were performed with radioactively labeled adp
in the presence of nonlabeled pi as well as with labeled
pi in the presence of nonlabeled adp .
one set of experiments was
done with liposomes containing atp plus pi , and one was done with
vesicles loaded with solely atp ( to disconnect the adp - dependent pi
homo - exchange from coimport with adp ) . for determination of the adp to
pi coimport stoichiometry , we calculated pi import in
relation to adp import ( adp import was set as 1 ) .
the presence of pi in the liposomal lumen led to a high
pi to adp import ratio [ 4.03 ( 0.45 ) pi to 1
adp ] ( supplemental fig .
s1 ) . therefore , pi homo - exchange
substantially exceeds coimport with adp . however , depletion of luminal
pi led to a significantly reduced pi homo - exchange and
allowed the calculation of a nearly one - to - one stoichiometry for pi
and adp coimport ( 1.21 ( 0.16 ) pi to 1 adp ) in exchange
with interior atp ( supplemental fig .
it has to be mentioned that
pi contaminations in the liposomal lumen or a marginal
unidirectional pi transport might have caused a slight
overestimation of the exact pi - to - adp ratio .
determination of the kinetic properties and identification of the
phosphate binding center of pamntt1as demonstrated above , already
low external atp concentrations inhibited pi import
( fig .
3 , compare b and
c ) , whereas high exterior pi concentrations
were required to substantially reduce atpim / adpex
transport ( fig .
these
characteristics suggest that the two anions , atp and pi , compete
for uptake and that atp is the preferred import substrate .
on the other hand ,
pi exhibits positive effects on adp transport ( import and export )
rather than competitive interference ( figs .
1 and
2 ) .
accordingly , pi
and adp simultaneously fit into the binding center , whereas pi
probably interacts with the ntt domain , which is otherwise occupied by the
-phosphate of atp . in a previous study we showed that mutations of a lysine residue at
position 527 in the plastidial atp / adp transporter from a. thaliana
( atntt1 ) reduced atp transport to a higher extent than adp transport
( 34 ) .
remarkably , this lysine
residue is conserved in all plastidial and bacterial atp / adp transporters
( supplemental fig .
s2 ) . to test whether this amino acid residue is not only
important for atp but also for pi transport
, we generated different
pamntt1 mutant proteins and analyzed their biochemical properties in
the liposomal system .
this was mandatory because e. coli possesses
endogenous pi transporters that obscure ntt mediated pi
translocation .
we substituted the positively charged lysine by a positively
charged arginine ( k446r ) , by the neutral glutamine ( k446q ) , or by the
negatively charged glutamate ( k446e ) to investigate a correlation between the
charge of the amino acid residue 446 and the kinetic properties
( km and vmax values ) of
pamntt1 .
the biochemical parameters of unmodified pamntt1 for atp and adp
transport were in the same range as previously described
( 32 ) .
furthermore , the
reconstituted wild type protein exhibited moderate affinities
( km in the range of 140 m ) and high
vmax values ( 916 mol mg
protein h ) for adp - dependent pi transport
( table 1 ) .
application of
internal atp slightly and of external atp highly reduced the affinity of
pamntt1 for pi uptake .
table 1comparison of the kinetic constants of reconstituted pamntt1
and k446 mutant proteins kinetic parameters of nucleotide or pi
exchanges ( exterior substrates / interior substrates ) mediated by reconstituted
pamntt1 and the three mutant proteins ( pamntt1-k446r ,
pamntt1-k446q , and pamntt1-k446e ) were determined by
application of rising concentrations of the respective labeled import
substrates ( 51,500 m ) .
the used proteoliposomes
contained 5 mm internal pi and the given nucleotides ( 10
mm ) .
kinetic parameters of pi import were analyzed in
the absence or presence of 50 m nonlabeled external nucleotides
( added external nucleotides in squared brackets ) .
km
values are given in m , and vmax values ( in
parentheses ) are in mol mg of protein
h , respectively .
transport was allowed for time spans in
the linear phase of the corresponding import at 50 m .
standard errors of the low
affinity and low velocity imports were below 20% and for the remaining imports
below 14%.exchanges
pamntt1
pamntt1 mutant proteins
k446rk446qk446e atp / atp + pi 95 ( 27.6 )
123 ( 0.5 )
187 ( 6.5 )
620 ( 2.3 )
atp / adp + pi 8.6 ( 31.3 )
66 ( 1.0 )
274 ( 7.2 )
552 ( 2.4 )
adp / atp + pi 1401 ( 5.5 )
179 ( 0.3 )
68 ( 4.5 )
29 ( 1.6 )
adp / adp + pi 120 ( 7.1 )
96 ( 0.7 )
44 ( 4.0 )
159 ( 15.4 )
pi / adp + pi 125 ( 13.6 )
271 ( 2.1 )
pi[+adp]/pi 154 ( 10.0 )
152 ( 0.5 )
pi[+adp]/adp + pi 139 ( 15.9 )
220 ( 1.9 )
544 ( 0.4 )
pi[+adp]/atp + pi 295 ( 9.0 )
275 ( 0.7 )
550 ( 0.4 )
pi[+atp]/adp + pi
506 ( 13.3 )
598 ( 2.0 )
comparison of the kinetic constants of reconstituted pamntt1
and k446 mutant proteins kinetic parameters of nucleotide or pi
exchanges ( exterior substrates / interior substrates ) mediated by reconstituted
pamntt1 and the three mutant proteins ( pamntt1-k446r ,
pamntt1-k446q , and pamntt1-k446e ) were determined by
application of rising concentrations of the respective labeled import
substrates ( 51,500 m ) .
the used proteoliposomes
contained 5 mm internal pi and the given nucleotides ( 10
mm ) .
kinetic parameters of pi import were analyzed in
the absence or presence of 50 m nonlabeled external nucleotides
( added external nucleotides in squared brackets ) .
( in
parentheses ) are in mol mg of protein
h , respectively .
transport was allowed for time spans in
the linear phase of the corresponding import at 50 m .
standard errors of the low
affinity and low velocity imports were below 20% and for the remaining imports
below 14% .
the conserved exchange of lysine 446 ( k446r ) led to a remarkable reduction
of the vmax , in particular of the atp homo - exchange
( table 1 ) .
furthermore , the
affinity for atp import in exchange with interior adp plus pi was
reduced , and that for adp import into atp plus pi - loaded vesicles
was enhanced .
nevertheless , the basic properties of the mutant protein
pamntt1-k446r , such as the affinities for ( adp - dependent )
pi import or atp and adp import during homo - exchange , as well as
the preference of the atpim / adpex transport over
adpim / atpex exchange , still resemble that of the
unmodified pamntt1 . in comparison with the wild type protein ,
the mutant protein with the
neutral substitution ( pamntt1-k446q ) exhibited reduced affinities
( and vmax values ) for atp import , whereas the affinities
for adp import , in particular in exchange with interior atp , were
significantly enhanced ( table
1 ) .
these changed biochemical characteristics led to a general
adjustment of the import rates , the atp homo - exchange equals
atpim / adpex transport , and also the high difference
between adp homo - exchange and adpim / atpex transport of
the wild type protein was reduced in pamntt1-k446q
( table 1 and supplemental fig .
s3 ) . because pi transport of pamntt1-k446q was nearly
completely diminished ( supplemental fig .
solely in the presence of
external adp and internal pi plus adenine nucleotides , very low
affinities and vmax values for pi import were
calculable ( table 1 ) .
interestingly , the replacement of the positively charged lysine by the
negatively charged glutamate completely blocked adp - dependent pi
uptake ( supplemental fig .
s3 and table 1 ) . in comparison with the unmodified
pamntt1 and the mutant pamntt1-k446q , the mutation in
pamntt1-k446e led to a further reduction of the
km and vmax values of the atp import
( table 1 ) .
furthermore , the
affinity for adp import into atp - loaded proteoliposomes was enhanced , whereas
the vmax of this exchange was reduced .
the moderate
affinity for adp import into adp plus pi - loaded vesicles , however ,
was accompanied by the highest vmax .
generally , the
observed differences in the biochemical characteristics between the wild type
protein pamntt1 and the mutant pamntt1-k446q are more
pronounced in the mutant pamntt1-k446e .
our analyses of the mutant
proteins revealed that a positively charged amino acid residue at position 446
is required for proper pi import , for the preference of
atpim / adpex transport , and for the discrimination of
adpim / atpex exchange .
pi is substrate of several nonmitochondrial atp / adp
transporters the fact that pamntt1 accepts pi
as third substrate might resolve inconsistencies in the phosphate metabolism
of p. amoebophila . to analyze whether also phylogenetically more
distantly related atp / adp transporters from other organisms possess the
capacity to transport pi , we investigated two carriers from the
rickettsial species c. caryophilus ( ccntt ) and h.
obtusa ( hontt ) and one plastidial transporter from the higher
plant a. thaliana ( atntt1 ) .
these representative rickettsial
and plastidial ntts were heterologously expressed in e. coli ,
purified ( supplemental fig .
s4 ) , and reconstituted into liposomes . all
recombinant carriers mediated atp and adp transport and therefore were
functional in the liposomal system ( supplemental fig .
generally ,
hontt exhibited highest net uptake rates that suggest a high activity
of this reconstituted protein ( fig .
4 and supplemental fig .
import studies with radioactive
pi showed that atntt1 and also the two selected
rickettsial ntts were able to import pi . in
fig .
4 the time - dependent
pi uptake in presence of exterior adp is presented .
pi
import into vesicles loaded with adp plus pi exceeded pi
import into proteoliposomes loaded with atp plus pi or with solely
pi . the rates of atntt1- and ccntt - mediated
pi homo - exchange were lower than the rates of pi ( plus
adp ) import into vesicles loaded with atp plus pi , whereas
hontt exhibited higher rates for pi homo - exchange than for
pi import in presence of exterior adp and interior atp plus
pi . apart from slight differences in the substrate preference
pattern , our results clearly demonstrate that atp / adp exchanging ntts from
distantly related organisms are capable for an adp - dependent pi
transport , which resembles that of pamntt1 ( figs .
3 and
4 ) .
figure 4.pi import mediated by phylogenetically distantly related
ntt - type atp / adp transporters .
heterologously expressed and purified
atp / adp transporters from a. thaliana ( atntt1 ) , c.
caryophilus ( ccntt ) , and h. obtusa ( hontt )
were reconstituted into liposomes loaded with 5 mm pi or
with 5 mm pi plus the given nucleotides ( 10
mm ) .
time - dependent import of 50 m radioactive
pi in presence of 50 m nonlabeled adp mediated by
atntt1 ( a ) , ccntt ( b ) , and hontt
( c ) .
import into proteoliposomes loaded with pi ( open
circle ) , pi plus adp ( gray square ) , or pi plus atp
( black diamond ) .
the given values are net values ( calculated by subtraction of
pi import in absence of external and internal nucleotides ) .
the
data are the means of at least three independent experiments , and the standard
errors are displayed .
pi import mediated by phylogenetically distantly related
ntt - type atp / adp transporters .
heterologously expressed and purified
atp / adp transporters from a. thaliana ( atntt1 ) , c.
caryophilus ( ccntt ) , and h. obtusa ( hontt )
were reconstituted into liposomes loaded with 5 mm pi or
with 5 mm pi plus the given nucleotides ( 10
mm ) .
time - dependent import of 50 m radioactive
pi in presence of 50 m nonlabeled adp mediated by
atntt1 ( a ) , ccntt ( b ) , and hontt
( c ) .
import into proteoliposomes loaded with pi ( open
circle ) , pi plus adp ( gray square ) , or pi plus atp
( black diamond ) .
the given values are net values ( calculated by subtraction of
pi import in absence of external and internal nucleotides ) .
the
data are the means of at least three independent experiments , and the standard
errors are displayed .
metabolically impaired energy parasites depend on atp import from the host
cell , and also plant plastids rely on energy supply from the surrounding
cytosol when photosynthetic activity is reduced or missing .
atp / adp exchange
would lead to substantial pi accumulation in the organelle or cell
if no interacting export mechanism for phosphate exists . in this study we
identified that pi transport is a previously not identified
intrinsic feature of nonmitochondrial atp / adp transporters ( figs .
3 and
4 ) .
pi is
transported simultaneously with adp , and this cotransport is facilitated in a
one pi to one adp stoichiometrical exchange with atp ( supplemental
fig .
the cotransport of one h2po4 ( with
one negative charge ) would be in complete agreement with the recently
documented electroneutrality of pamntt1-mediated atp / adp exchange
( 32 ) . during hetero - exchange
,
pi could act as a counterion compensating for the generation of a
charge difference across the membrane
. a stimulatory influence of high pi concentrations ( in the
millimolar range ) on adp import into isolated rickettsia prowazekii
cells had been observed more than three decades ago
( 38 ) .
although a regulatory
effect of pi on the transport characteristics was discussed , the
exact role of pi in atp / adp exchange was not known
( 37 ) .
interestingly , the
simplest explanation , a possible cotransport of pi and adp , was
never suggested for ntts .
detailed analyses of pamntt1 ( figs .
2 and
3 and supplemental fig .
4 , b and
c ) suggest that the regulatory principle of pi
is tightly associated to its function as a cosubstrate of adp .
the addition of
pi indeed increased the vmax and the affinity
of pamntt1 for adp transport into atp loaded proteoliposomes .
it is
imaginable that a simultaneous entry of adp and pi might mimic the
presence of a triphosphorylated adenine nucleotide ( atp ) , which is the
preferred import substrate .
the capacity of the phylogenetically different ntts to transport adp plus
pi in exchange with atp ( i ) is in line with the electroneutrality
of nucleotide exchange ( 32 ) ,
( ii ) prevents harmful pi accumulation in intracellular living
bacteria and plant plastids , and ( iii ) implies that the phosphate exporter
that was proposed for a long time in these bacteria and in plastids is
represented by the
very recent analyses demonstrated that energy parasitism is not only
restricted to chlamydiales and rickettsiales but also occurs
in further important mammalian pathogens
( 23 ,
24 ) .
ntt - type carriers reside
in the plasma membrane and in the mitochondrial relict ( the so called
mitosome ) of the intracellular protist encephalitozoon cuniculi
( 24 ) . by catalyzing a highly
specific atp / adp exchange ,
these ntts provide energy to the cell or to the
mitosome that lacks atp synthesis .
it is tempting to speculate that also these
eukaryotic ntts catalyze a pi coexport with adp . to our surprise
, ntt - type atp / adp transporters mediate a nucleotide
exchange - independent but adp - induced homo - exchange of pi in
addition to the pi cotransport with adp ( figs .
3 and
4 , white circles ) .
the
pi homo - exchange was induced by external as well as by internal adp
( fig .
we
propose a scenario that could explain the induction of pi
homo - exchange independent of the side of adp application .
adp and pi enter the binding center , for example at the
proteoliposomal lumen , and are transported to the opposed side
( fig .
, adp stays at the binding center , whereas the nonlabeled
pi is displaced by radioactive pi . a subsequent
reversely directed translocation of the two bound substrates and replacement
of radioactive pi by nonlabeled pi at
the interior
causes the observed pi homo - exchange
( fig . 5 ) .
this hypothesized
mechanism presupposes that only one single binding center exists for import
and export that opens from one side of the membrane to the other where
nucleotides and/or pi are displaced by other substrates .
by the help of mutant proteins we identified a correlation between the
charge of the amino acid residue at position 446 and the transport properties
of pamntt1 .
a positive amino acid residue at position 446 allowed the
insertion of atp or pi ( plus adp ) , a neutral amino acid residue or
even more a negative amino acid residue stimulates adp but suppresses
pi or atp entry ( table
1 ) .
substitution of lys by arginine ( k446r ) entailed
substantially lowered import rates for all substrates .
we assume that
structural differences between lysine and arginine rather than an unfavorable
charge might have caused the decreased transport velocity
( table 1 ) .
the biochemical
properties of the mutant proteins suggest that lysine 446 is an essential
component of the atp and pi binding center or translocation
pathway .
we conclude that the positively charged lysine 446 interacts with the
negative charge introduced either by the -phosphate of atp or
alternatively by pi .
figure 5.model explaining the adp - dependent pi homo - exchange mediated
by pamntt1 .
adp ( ado - p - p , p - p - ado ) supports entry of interior
pi ( oval 1 ) , adp plus pi are translocated
( white arrow ) to the exterior .
pi is displaced by
radioactive pi ( pi ) , whereas adp remains at
the binding center ( oval 2 ) .
the substrate complex is translocated
( white arrow ) to the interior and radioactive pi is
displaced by nonlabeled pi ( oval 3 ) .
adp ( ado - p - p , p - p - ado ) supports entry of interior
pi ( oval 1 ) , adp plus pi are translocated
( white arrow ) to the exterior .
pi is displaced by
radioactive pi ( pi ) , whereas adp remains at
the binding center ( oval 2 ) .
the substrate complex is translocated
( white arrow ) to the interior and radioactive pi is
displaced by nonlabeled pi ( oval 3 ) .
the fact that atp import was highly reduced , whereas atp export still
occurred ( adpim / atpex transport ) in the mutant protein
pamntt1-k446e might argue against a single binding center for atp
import and export .
however , the vmax of
adpim / atpex exchange was remarkably low when compared
with that of adp homo - exchange ( table
1 ) . this characteristic could be explained by an impaired atp
export capacity leading to a return transport of previously imported adp .
this
would be in agreement with the characteristics of the mutated plastidial
atp / adp transporter , which exhibited a highly reduced capacity for both atp
import and atp export
( 34 ) .
it becomes clear that additional studies are required to get more insights
into structure / function relationships of ntts , and it will be interesting to
focus on this topic in the future .
furthermore , we wish to analyze transport
parameters under conditions mimicking the energy state of intracellular living
bacteria in their host cells . however , for this it is a precondition to
determine pi , atp , and adp concentrations in the bacterium and the
infected cell .
| chlamydiales and rickettsiales as metabolically impaired ,
intracellular pathogenic bacteria essentially rely on energy
parasitism by the help of nucleotide transporters ( ntts ) .
also in plant
plastids ntt - type carriers catalyze atp / adp exchange to fuel metabolic
processes .
the uptake of atp4- , followed by energy consumption and
the release of adp3- , would lead to a metabolically disadvantageous
accumulation of negative charges in form of inorganic phosphate
( pi ) in the bacterium or organelle if no interacting pi
export system exists .
we identified that pi is a third substrate of
several ntt - type atp / adp transporters . during adenine
nucleotide
hetero - exchange , pi is cotransported with adp in a one - to - one
stoichiometry .
additionally , pi can be transported in exchange with
solely pi .
this pi homo - exchange depends on the presence
of adp and provides a first indication for only one binding center involved in
import and export .
furthermore , analyses of mutant proteins revealed that
pi interacts with the same amino acid residue as the
-phosphate of atp .
import of atp in exchange with adp plus
pi is obviously an efficient way to couple energy provision with
the export of the two metabolic products ( adp plus pi ) and to
maintain cellular phosphate homeostasis in intracellular living energy
parasites and plant plastids .
the additional pi transport
capacity of ntt - type atp / adp transporters makes the existence of an
interacting pi exporter dispensable and might explain why a
corresponding protein so far has not been identified . |
limbic encephalitis is believed to be a disorder affecting the medial temporal lobe of the brain
. it could be either a paraneoplastic or autoimmune ( non - paraneoplastic ) origin .
the most common non - paraneoplastic antibodies associated with limbic encephalitis are voltage - gated potassium channel antibodies ( vgkc ) , n - methyl - d - aspartate ( nmda ) receptor antibodies , and glutamic acid decarboxylase ( gad ) receptor antibodies [ 1 , 2 ] . the myriad symptoms of limbic encephalitis make correct diagnosis challenging
. symptoms of limbic encephalitis could range from seizures , memory loss , dementia , confusion , to psychosis .
furthermore , besides limbic encephalitis , gad antibody is reported to be associated with intractable seizures with focus from frontal lobes , cerebellar ataxia , palatal myoclonus , stiff person syndrome and type i diabetic mellitus [ 2 , 3 ] .
gad is the major enzyme in the synthesis of gamma - amino butyric acid ( gaba ) which is a potent inhibitory neurotransmitter and a critical component of neurophysiologic function .
one isoform has a molecular size of 65 kda and is termed gad65 , while the second one , of 67 kda size , is termed gad67 .
it is proposed that these gad65 antibodies reduce the synthesis of gaba or interfere with exocytosis of gaba in the nervous system .
gad65 antibodies act like gaba antagonists and could produce seizures [ 5 , 6 ] .
we present a case of a 21-year - old female with limbic encephalitis associated with the gad65 antibody .
the case is unique because the clinical course suggests that the non - convulsive seizures are the major cause of this patient s clinical manifestations and magnetic resonance imaging ( mri ) signal abnormalities .
we present a case of a 21-year - old female , who started having complex partial seizures 1 week prior to first hospital admission .
she felt nauseous about half an hour after waking - up on a daily basis . during the first admission , electroencephalogram ( eeg ) confirmed independent seizures from bilateral temporal areas .
initial mri of the brain showed a slight abnormal increase of t2 signal intensity in the bilateral hippocampus , amygdala , fornix , and mammillary body ( fig .
1 ) . initial mri of brain showed subtle increased signal intensity on coronal and axial flair mr sequences in the bilateral hippocampus , amygdala , fornix and mammillary body . despite the seizure medicine ,
the follow - up mri of the brain showed significant stepwise interval increase in the extent of t2 signal intensity in the bilateral hippocampus , amygdala , fornix , and mammillary body on fluid - attenuated inversion recovery ( flair ) sequences ( figs . 2 and 3 ) .
the follow - up mri of brain showed interval increase in the extent of t2 signal intensity in the bilateral hippocampus , amygdala , fornix and mammillary body on fluid - attenuated inversion recovery ( flair ) sequences .
the follow - up mri of brain showed significant interval increase in the extent of t2 signal intensity in the bilateral hippocampus , amygdala , fornix and mammillary body on fluid - attenuated inversion recovery ( flair ) sequences .
the differential diagnosis of these mri findings included post - ictal edema , autoimmune limbic encephalitis , paraneoplastic limbic encephalitis , and herpes encephalitis .
the possibility of a tumor was considered unlikely due to the symmetric exclusive involvement of limbic system without any associated temporal lobe involvement .
she repetitively asked the same question , which was answered a couple of minutes prior , on the first day of admission .
prolonged video eeg documented the clusters of non - convulsive seizures ( figs . 4 and 5 ) .
( a , b , c ) the electrical seizures started with sudden appearance of repetitive epileptiform sharp waves over the left hemisphere displayed on a bipolar montage , which evolved in amplitude and frequency over the left hemisphere .
( d ) the onset of the same seizure displayed on an average reference montage . ( a , b , c ) electrical seizure in the sleep .
( d ) the onset of the same seizure displayed on an average reference montage . on the first day of video eeg monitoring , every 15 - 30 min ,
these electrical seizures started with the sudden appearance of repetitive epileptiform sharp waves over the left hemisphere , which evolved in amplitude and frequency over the left hemisphere .
these epileptiform discharges did not propagate to the right hemisphere in the majority of seizures .
the electrical seizures lasted around 20 - 30 s each ( figs . 4 and 5 ) .
in fact , the patient was able to answer simple questions during the seizures and did not show any major motor manifestations .
non - convulsive seizures continued in sleep ( figs . 4 and 5 ) . between seizures ,
the awake eeg background was well preserved between seizures . these non - convulsive seizure clusters , amnesia and mri signal abnormality improved after aggressive anti - seizure medication management and intravenous solumedrol ( fig .
follow - up mr examination showed significant interval decrease in the extent of t2 signal .
no tumor was found on the whole body ct scan [ 8 , 9 ] .
routine complete blood cell ( cbc ) count , basic metabolic chemistry and routine csf results were within normal range : sedimentation rate ( westergren ) 6 mm / h , csf wbc 9 cells/l , rbc < 1 cells/l , glucose 60 mg / dl , total protein 35 mg / dl , and igg csf 4 mg / dl ( reference range 0 - 6 mg / dl ) .
serum gad65 ab was more than 250 iu / ml ( reference range 0.0 - 5.0 iu / ml ) and csf gad65 ab assay was 53.0
the other antibodies in the encephalopathy - autoimmune panel of mayo clinic laboratories were negative including nmda - r ab cba , vgkc - complex ab ipa , gaba - b - r ab cba , ampa - r ab cba , anna-1 ( anti - neuronal nuclear ab , type 1 ) , anna-2 ( anti - neuronal nuclear ab , type 2 ) , anna-3 ( anti - neuronal nuclear ab , type 3 ) , agna-1 ( anti - glial nuclear ab , type 1 ) , pca-1 ( purkinje cell cytoplasmic ab type 1 ) , pca-2 ( purkinje cell cytoplasmic ab type 2 ) , pca - tr ( purkinje cell cytoplasmic ab type tr ) , amphiphysin ab , and crmp-5-igg negative titer < 1:2 ( collapsin response mediator protein 5 ) .
three months after onset , the patient was re - admitted . in this admission ,
the patient s non - convulsive seizures and amnesia reappeared during a transition of seizure medicines .
weeks afterward , the patient was re - admitted because of a skin rash assumed to be secondary to the seizure medicine . both amnesia and non - convulsive seizures were again prominent on the first day .
amnesia and non - convulsive seizures improved with modification of anti - seizure medicine and intravenous solumedrol . in this particular case , the mri of the brain , eeg monitoring , and the clinical course suggested that the non - convulsive seizures , amnesia , the abnormal t2 signal changes of the bilateral hippocampus , amygdala , fornix and mammillary body in mri of the brain were positively correlated .
the case we present here is unique because the patient s fluctuating course allows us to reach a conclusion that non - convulsive seizures , amnesia , the abnormal t2 signal changes of bilateral hippocampus and fornix in mri of the brain are correlated in this case .
this positive correlation supports the hypothesis that these non - convulsive seizure clusters are the primary cause of memory problems and mri abnormalities .
the mri abnormalities of reported cases of limbic encephalitis in the literature vary because the extent and severity of seizure vary .
the myriad of symptoms of limbic encephalitis can be explained by clusters of non - convulsing seizures .
the symptoms of limbic encephalitis vary because the extent and severity of non - convulsing seizure vary .
this hypothesis predicts that patients with gad antibodies may develop seizures from brain regions other than the limbic areas . in this case , the temporal lobe seizure clusters are triggered by a systemic autoimmune disease process .
temporal lobes happen to be a vulnerable area of the brain in which seizures develop .
the non - convulsive seizure clusters could also potentially lead to hippocampal sclerosis and intractable seizures afterward .
high - dose intravenous solumedrol and aggressive anti - seizure management are of help [ 12 , 13 ] . in this case
, the majority of seizures were not convulsing , and intensive video eeg monitoring would be imperative to clarify the diagnosis and guide the treatment . aggressive seizure control in this stage potentially could modify the clinical course and prevent probable hippocampal sclerosis in the future .
| with the increased availability of laboratory tests , glutamic acid decarboxylase ( gad ) antibody - positive limbic encephalitis has become an emerging diagnosis .
the myriad symptoms of limbic encephalitis make the diagnosis challenging .
symptoms range from seizures , memory loss , dementia , confusion , to psychosis .
we present a case of a 21-year - old female with gad65 antibody - positive limbic encephalitis .
the case is unique because the clinical course suggests that non - convulsive seizures are the major cause of this patient s clinical manifestations .
the following is the thesis : systemic autoimmune disease , associated with the gad65 antibody , gives rise to seizures , in particular , non - convulsive seizures . temporal lobes
happen to be the most susceptible sites to develop seizures .
the greater part of these seizures can be non - convulsive and hard to recognize without electroencephalogram ( eeg ) monitoring .
the variable symptoms mirror the severity and locations of these seizures . the magnetic resonance imaging ( mri )
signal abnormities in the bilateral hippocampus , fornix , and mammillary body correlate with the density of these seizures in the similar manner , which suggests it is secondary to post - ictal edema . |
world health organization ( who ) defines unsafe abortion as a procedure for terminating unwanted pregnancy either by people lacking the necessary skills or in an environment lacking minimal medical standards or both .
according to who , worldwide unsafe abortions were estimated to be 21.6 million , almost all in developing countries . each year
, throughout the world , approximately 210 million women became pregnant and around one in 10 pregnancies ends in an unsafe abortion .
the estimated annual number of unsafe abortions in sub - saharan africa is 5.5 million .
although unsafe abortions are preventable , they continue to pose undue risks to a woman 's health .
millennium development goal ( mdg ) 5 , announced in 2001 , is an internationally agreed - upon imperative to reduce maternal mortality by 75% from its 1990 level by the year 2015 .
as a significant proportion of mortality is due to unsafe abortion , this goal probably can not be met without specific and direct programmatic efforts to reduce the impact of unsafe abortion .
unsafe abortion accounts for 13% of maternal deaths worldwide and as much as 25% in some countries . over 40% of the total deaths due to unsafe abortion have occurred in africa making it the leading cause of maternal mortality in the region . the maternal mortality ratio ( mmr ) in ethiopia
it is estimated that there are 3.27 million pregnancies in ethiopia every year , of which approximately 500,000 end in either spontaneous or induced abortion . in 2005 , ethiopia expanded its abortion law , which had previously allowed the procedure only to save the life of a woman or protect her physical health . currently abortion is legal in ethiopia under certain preconditions that include cases of rape , incest or fetal impairment , if the pregnancy endangers her or her child 's life , or if continuing the pregnancy or giving birth endangers her life .
a woman may also terminate a pregnancy if she is unable to bring up the child , owing to her status as a minor or to a physical or mental infirmity . despite the implementation of the new law ,
reasons for resorting to unsafe abortion after unwanted or unplanned pregnancy were well established and reflected the status of women in the society . some of the reasons are inability to support self and family , having already enough children , being very young , and being in school [ 911 ] .
lack of access to adequate family planning services is a major contributor to the global problem of unsafe abortion .
a woman seeking treatment for incomplete abortion already may have experienced an unwanted pregnancy either as a result of not using contraception or method failure . a nationally representative survey conducted in ethiopia in 2008 revealed that an estimated 382,000 induced abortions were performed and 52,600 women were treated for complications of abortion .
the annual abortion rate was 23 per 1,000 women aged 1544 years and the abortion ratio was 13 per 100 live births , about 42% of pregnancies were unintended , and the unintended pregnancy rate was 101 per 1,000 women .
fifty - eight percent of the women who induced abortion terminated the current pregnancy either by seeking the help of untrained personnel or by themselves with no assistance . a survey conducted in harar , eastern ethiopia , showed 33.3% unintended pregnancies while induced abortion was found to be 14.4% .
unsafe abortion is one of the greatest neglected problems of health care in developing countries and is a major medical and public health problem in ethiopia .
nevertheless , lack of up - to - date and reliable information on induced abortion distribution and its determinant factors has partly hindered the progress towards alleviating the problem in the country .
thus , the aim of this study was to assess induced abortion and associated factors in health facilities of guraghe zone , southern ethiopia .
guraghe zone is one of the 13 zones in the southern nations , nationalities , and peoples region ( snnpr ) .
its capital welkite is located 156 km south west of addis ababa along the road from addis ababa to jimma .
the zone is divided into 13 administrative woredas with an estimated total population of 1.7 million . according to the zonal health bureau
, the zone has 26 health centers , one zonal public hospital , two nongovernmental hospitals , and one family guidance association ( fga ) clinic .
eleven health facilities have already initiated post - abortion care services ( two hospitals and nine health centers ) in the zone .
eight health facilities ( two hospitals and six heath centers ) were included in the study .
facility based study was preferred because health facility is the most logical , cost - effective , and convenient place to conduct research on unsafe abortion where women with complications of unsafe abortion are treated despite having its own limitations .
all women who received post - abortion care service in health facilities of guraghe zone were included .
all women who received post - abortion care during the study period in the 8 selected health facilities were included in the study .
those patients who were unable to hear , mentally disabled , seriously sick , and were not volunteer to participate and patients whose gestational age was greater than 28 weeks were excluded from the study .
the sample size was calculated based on the following assumptions : proportion of patients who seek post - abortion care ( p = 50% ) taken to get the maximum sample size , z = 1.96 at 95% confidence interval , d = the level of precision ( 0.05 ) , and non - response rate = 10% ; then the total sample size for this study became 422 .
all consecutively discharged cases from the selected health facilities during the study period were included in the study until the required sample was obtained for each heath facility as determined based on their patient load as recommended by who .
the questionnaire was developed after extensive review of similar studies done previously and adapted to the purpose of the study .
sociodemographic data , reproductive health history , reproductive intentions , and fertility control .
the questions were translated into local language and administered by the interviewers in a careful way so as to keep the patient at ease and in a manner showing respect to their opinion in a nonjudgmental approach .
information about induced abortion was elicited from the study participants by using the question is the current abortion after an attempt to terminate the pregnancy ? and asking them to explain about why ( reason ) they resort to induced abortion , the condition under which the induced abortion was performed , and so forth .
the clients were interviewed after discharge was decided and just before the client left the respective health institution as exit interview .
induced abortion which refers to post - abortion patients who admitted interference to terminate their pregnancy intentionally either on their own or by another person .
independent variables include the following : sociodemographic variables;reproductive history ( current pregnancy ( wanted versus unwanted ) , previous abortions , and number of pregnancies ) .
sociodemographic variables ; reproductive history ( current pregnancy ( wanted versus unwanted ) , previous abortions , and number of pregnancies ) .
after collection of the data the responses were coded and entered into epi info version 3.5.1 .
then , the frequency distribution of dependent and independent variables was worked out . a crude and adjusted odds ratio from bivariate and multivariate analyses was used to measure association between dependent and independent variables .
those variables which were found to be significant in the bivariate analysis at p value < 0.05 were taken to logistic regression model for further multivariate analysis .
then , logistic regression analysis was done to control confounding variables and to identify independent factors associated with induced abortion .
participants were allocated to cases ( those having induced abortion ) based on their response , so those who have admitted that the abortion is because of interference were allocated to cases and those who deny it as noncases in the logistic regression analysis .
the english version questionnaire was translated into local language amharic and again back to english so as to ensure its consistency .
the translated amharic version was pretested in a health facility other than the selected facilities in 5% of the sample size that were not part of the main study .
the data collectors and supervisors were trained by the principal investigator on the objectives of the study and how to make the interview , fill the questionnaire , and handle questions asked by clients during interviewing for three days .
a total of 422 patients were identified and 400 women participated in the study making the response rate 95% . majority , 193 ( 48.25% ) , of the respondents were aged between 20 and 29 years .
the mean age of the respondents was 25.3 years ( sd 6.4 ) . about half , 227 ( 56.8% ) , of the participants were married , 130 ( 32.5% ) were illiterate , and 185 ( 46.3% ) were housewives ( table 1 ) .
about 207 ( 51.8 ) of the respondents have history of one or two pregnancies including the current pregnancy that ended in abortion .
the majority , 185 ( 46.5% ) , of the patients have no history of delivery .
sixty - eight ( 17% ) of the respondents had previous history of abortion , which was experienced once in 86.8% , twice in 11.7% , and three times in 1.5% of the respondents .
the majority , 302 ( 75.5% ) , of the post - abortion patients revealed that the current pregnancy which ended in abortion was unwanted while the remaining ninety - eight ( 24.5% ) of the patients said it is wanted .
forty - nine ( 12.3% ) of the patients admitted interference with the current pregnancy ( table 2 ) . the majority of those who reported their last pregnancy as unwanted stated partner pressure and negligence to take contraceptives regularly as main reasons for the pregnancy . among those who admitted interference to the current pregnancy reported place of interference as health institutions , patient 's home , and inducer 's house , 25 ( 51% ) , 22 ( 45% ) , and 2 ( 4% ) , respectively .
materials used to induce the pregnancy were herbs , 18 ( 36.7% ) ; plastics , 15 ( 30.6% ) ; and different medications in 11 ( 22.4% ) of the patients .
twenty ( 40.8% ) reported that interference was done by themselves followed by health workers , 25 ( 51% ) ( table 3 ) .
main reasons given for resorting to induced abortion by those who admitted interference were to complete education , 20 ( 40.8% ) , and for economic reasons , 18 ( 36.7% ) ( figure 1 ) .
two hundred eighty - two ( 70.5% ) of the study participants knew at least one family planning method , the most commonly known being oral contraceptive pills .
two hundred forty three ( 60.8% ) of the participants have used contraceptive at least once in their life time . when seen by type of abortion , 33 ( 67.3% ) of those who admitted interference and 210 ( 69.5% ) of those with spontaneous abortion reported use of contraceptive at least once in their life time .
majority of the clients , 123 ( 30.8% ) , stated that fertility will return within one month , 162 ( 40.5% ) never want to be pregnant in the future , only 226 ( 56.5% ) got post - abortion family planning method , and the most common reason for not getting family planning method was , 96 ( 24% ) , refusal and , 47 ( 11.8% ) , not counseled family planning ( table 4 ) . in the bivariate analysis , educational status and number of pregnancies are associated with induced abortion . in multivariate analysis ,
those patients who have greater than four pregnancies were more likely to have induced abortion than those who have less than four pregnancies ( aor = 4.28 , ci = ( 1.2414.71 ) ) and those patients who reported that they want the current pregnancy were less likely to have induced abortion ( aor = 0.44 , ci : ( 0.140.65 ) ) than those who do not want their current pregnancy .
in addition , those patients who were in primary school and age group 3034 were less likely to have induced abortion ( aor = 0.26 , ci : ( 0.130.88 ) ) and ( aor = 0.15 , ci : ( 0.040.55 ) ) ( table 5 ) .
the main reasons for induced abortion were to complete education , 40.8% , and economic reason , 36.7% .
partner pressure and forgetting to take contraceptives were the main reasons mentioned for unwanted pregnancy .
more than half , 51% , of the induced abortions took place in health institution by health workers .
age 3034 years , primary education , number of pregnancies , and wanted pregnancy had association with induced abortion .
even though , 75.5% reported current pregnancy was unwanted in this study ; only 12.3% admitted that the pregnancy was interfered . according to who , distinguishing between spontaneous and induced abortion among women who received care in health facility is difficult .
since women fear retribution or inadequate care , they are likely to deny unsafe procedures even in the face of the most obvious evidence . therefore , who classified those women who admitted the pregnancy interference in the certainly induced abortion category and those women who states that the pregnancy was unwanted but denied interference in the category of possibly induced abortion . as a result the magnitude of induced abortion may be underestimated . in general this is a sensitive area that respondents do not want to disclose . due to this fact results from different studies show varying number of proportions between spontaneous and induced abortions .
a study conducted in isfahan , central iran , revealed a similar result ; 12% of the patients reported induced abortion .
another study conducted in northwest ethiopia revealed 4.8% prevalence rate of induced abortion which is much lower than our study , implying that induced abortion is a hidden public health problem affecting the reproductive health outcome status of women .
the current study finding shows partner pressure and negligence to take contraceptives as a main cause for the unwanted pregnancies which is similar to other studies [ 11 , 19 ] in that contraceptive misuse or poor knowledge are the main cause for unwanted pregnancies .
these facts show how the low socio - economic status of women in the community affects their decision making that risks their life temporarily or permanently .
as is the case with many other areas of reproductive health , husbands appear to be the primary decision - makers with regard to pregnancy .
furthermore , the main reasons for resorting to induced abortion in this study ( economic reason and to complete education ) were not different from the work of previous investigators [ 11 , 20 , 21 ] . in our study ,
40.5% of the clients never want to be pregnant which is higher than study conducted in addis ababa where only 31.7% never want to become pregnant at all .
the number of women who want to limit pregnancy is still high in both studies which imply that there is a high family planning demand to space or limit their next pregnancy calling that post - abortion setting continues to be an important opportunity to provide post - abortion family planning counseling and initiate family planning methods .
the fact that 51% of the induced abortion was undertaken in health institutions by health workers might be due to the flareup of private clinics which leads to management in substandard settings and poor professional competence of health workers . according to a study in mekelle , northern ethiopia , health professionals undertook the inductions in 82.7% which is even much higher than this study . a study in dare
selaam showed that most ( 79% ) abortions had been induced at clinic or hospital ( 43.6% ) by health care workers .
this study showed that being in primary school , age 3034 years , having more than 4 children , and wanted pregnancy had association with induced abortion .
a study in north west ethiopia showed a different result in that as the level of education increased there was an increase in the number of mothers who had induced abortion , particularly those who had a high school education were highly exposed to induced abortion .
this may be due to the fact that children in primary school were living with their family under strict parental supervision which prevents them from unwanted pregnancy which ends up with induced abortion .
age 3034 years [ aor = 0.15 , ( ci 0.040.55 ) ] was negatively associated with induced abortion .
a study done in ghana also showed the same result in which women with induced abortions were significantly within younger age groups .
this might be due to the fact that women at this age group will be economically independent , in a stable marital relationship , and have completed their education unlike the younger age groups .
clients having greater than 4 children [ aor = 4.28 , ( ci 1.2414.71 ) ] were 4 times more likely to commit induced abortion .
this finding is in line with a study in spain in which those clients who have some children were more likely to have induced abortion than those who have no children .
this may be true because women who had several pregnancies would have the tendency not to have additional children so they may tend to avoid unwanted pregnancies by inducing their current pregnancy . those patients who want the pregnancy [ aor = 0.44 , ( ci 0.140.65 ) ] were less likely to have induced abortion .
the immediate explanation for this is that a woman seeking induced abortion usually have unplanned or unwanted pregnancy which in turn is related to lack of wide spread information about family planning and its utilization . strength of this study is that it used primary data by directly interviewing the owners of the problem , whereas the limitation of the study is that since abortion is a sensitive issue and legally restricted in ethiopia , patient interviews may introduce social desirability bias which may result in under reporting .
. educational level , age of the patient , number of pregnancy , and whether or not the pregnancy is wanted were factors significantly associated with induced abortion . increased access to high - standard family planning service , post - abortion care , strict counseling about family planning method reminder , and partner involvement in family planning service should be emphasized . | unsafe abortion is one of the major medical and public health problems in developing countries including ethiopia .
however , there is a lack of up - to - date and reliable information on induced abortion distribution and its determinant factors in the country .
this study was intended to assess induced abortion and associated factors in health facilities of guraghe zone , southern ethiopia .
institution based cross - sectional study was conducted in eight health facilities in guraghe zone .
client exit interview was conducted on 400 patients using a structured questionnaire .
bivariate and multivariate logistic regression analysis was performed to identify factors associated with induced abortion . out of 400 women
, 75.5% responded that the current pregnancy that ended in abortion is unwanted .
however , only 12.3% of the respondents have admitted interference to the current pregnancy .
having more than four pregnancies ( aor = 4.28 , ci : ( 1.2414.71 ) ) , age of 3034 years ( aor = 0.15 , ci : ( 0.040.55 ) ) , primary education ( aor = 0.26 , ci : ( 0.130.88 ) ) , and wanted pregnancy ( aor = 0.44 , ci : ( 0.140.65 ) ) were found to have association with induced abortion .
the study revealed high level of induced abortion which is underpinned by high magnitude of unwanted pregnancy .
there is requirement for widespread expansion of increased access to high quality family planning service and post - abortion care . |
older adults ( 65 + years ) are among the most rapidly growing segments in the united states population , and projections predict this trend to continue into the future [ 1 , 2 ] . despite modern advancements in medicine and technology , there are continual health concerns in the older adult population .
the prevalence of chronic conditions , such as diabetes , osteoporosis , hypertension , hyperlipidemia , obesity , and cancer , remains high , having a detrimental effect on an older adult 's overall health and quality of life and placing excessive economic strain on our nation 's health care system [ 3 , 4 ] .
accordingly , there is an increased emphasis on exploring the effectiveness of preventative efforts to ameliorate the burden of such adverse health outcomes in older adults .
regular physical activity and exercise have long been promoted as a means to treat and prevent a multitude of health conditions , yet the number of older adults who are regularly active is staggeringly low .
based on objective physical activity assessments , it is estimated that only 3.510% of older adults are meeting physical activity recommendations [ 6 , 7 ] .
furthermore , the amount of physical activity performed across the older adult years steadily decreases as sedentary behaviors begin to dominate everyday life [ 8 , 9 ] .
a key barrier to physical activity for the older adult population is access to resources that promote regular physical activity and exercise [ 1012 ] .
community - based fitness resources ( cbfr ) can provide older adults a wealth of opportunities to promote increases in physical activity levels , such as removing / minimizing barriers to physical activity , including the availability , supervision , and instruction on use of exercise equipment , and developing a supportive network of peers .
such factors have been shown to be critical in influencing physical activity levels in older adults .
proximity to cbfr is likely to be important , as it further reduces a potential transportation barrier [ 1013 ] and may also result in a greater awareness of programming opportunities and their associated benefits .
senior centers offer an excellent conduit in which to promote cbfr and could serve as an organizational mediator to physical activity behavior in older adults . to date , it remains unclear whether proximity to senior centers with cbfr has an impact on awareness and utilization of resources and ultimately overall physical activity levels of older adults .
thus , the purpose of the current study was to assess awareness and utilization of cbfr , based on residential spatial tiers of increasing distance from said resources .
it was hypothesized that individuals living in closer proximity to cbfr would have greater awareness , utilization rate , and overall higher physical activity levels , compared to those residing further away from cbfr .
this cross - sectional study involved gathering a series of information regarding awareness and utilization of cbfr and current physical activity levels , based on proximity to the facilities .
participation consisted of the completion of a series of questionnaires mailed to participants , which included a health history questionnaire , a community - based resources questionnaire , and the champs physical activity questionnaire .
in addition to the questionnaires , a cover letter was enclosed to orient the participant on completing the forms , as well as a preaddressed , stamped envelope for the questionnaires to be returned to the investigative team .
the surrounding areas of seven local senior centers with cbfr throughout a large metropolitan area were included in the current study .
these lists were designated to include all older adults residing within 5 miles of targeted senior centers , obtained through marketing companies .
calling lists were then segmented by geographic information systems software into those who resided 1.6 , > 1.6 to 3.2 , and > 3.2 to 8.0 km from targeted senior centers . within the stratified calling lists ,
a random sample of potential participants was contacted via telephone to inquire if they would be interested in participating in this study . upon receiving verbal consent to participate , as approved by the university 's institutional review board
all data collection was conducted within a single season , thus reducing the confounding of seasonality on responses .
inclusion criteria for participating in the study consisted of being between 60 and 90 years and willingness to complete and return all questionnaires . by nature of the study design ,
all participants contacted were previously stratified to be residing within 8.0 km of a targeted senior center .
an 11-point questionnaire was developed by the investigators to amass descriptive data pertaining to cbfr awareness , utilization , transportation , and barriers to utilization .
, participants checked a box either yes or no to the following question : are you aware of any exercise / fitness programs or classes at your local senior center ?
community - based fitness resource utilization . to assess utilization of cbfr
, participants checked a box either yes or no to the following question : do you currently attend or participate in any of the exercise / fitness programs or classes at your local senior center ?
barriers to community - based fitness resource utilization .
additionally , participants were given the opportunity to identify what barriers pertaining to cbfr use were applicable to them from the following question : what barriers prevent you from attending and participating in any exercise / fitness programs or classes at your local senior center more often / if at all ?
a list of common barriers was provided , including knowledge of services , time , transportation , work / other commitments , health , lack of interest , and distance from resources , prompting participants to check a box adjacent to each applicable barrier that contributed to limiting their engagement .
there was no limit to how many barriers could be marked as influencing cbfr utilization .
the champs physical activity questionnaire was used to collect information on physical activity engagement , targeting frequency ( days / week ) and weekly duration spent engaging in various exercise behaviors , everyday activities , and leisure - time activities common to older adults . for the current study ,
the outcome measurement from the champs questionnaire was weekly caloric expenditure in moderate to vigorous intensity activities , using adapted met values for older adults .
calculating energy expenditure from the champs questionnaire requires calculating weekly duration engaged in each activity , which has been shown to have acceptable measures of reliability , with r values ranging from 0.67 to 0.76 [ 14 , 15 ] .
the champs questionnaire has also been shown to appropriately demarcate varying physical activity levels with a level of precision similar to more intensive physical activity interviewing assessments ( f2,246 = 20.85 , p < 0.001 ) , providing evidence for the champs questionnaire to be a valid physical activity assessment tool .
chi - square tests were performed to examine if awareness and utilization rates of cbfr differed across spatial tiers .
kruskal - wallis tests were performed to examine if engagement in moderate to vigorous physical activity ( mvpa ) differed across spatial tiers .
multinomial logistic regression analyses were performed to identify which mediators to physical activity ( age , gender , income , car ownership , and cbfr utilization ) were significant predictors to overall activity levels , represented by caloric expenditure .
the dependent physical activity categories included sedentary ( 0 kcals / wk ; referent category ) , low levels of physical activity ( > 06710 kcals / wk ) , and high levels of physical activity ( > 6710 kcals / wk ) .
the cut point used to delineate low and high physical activity levels was based on the median energy expenditure values among all nonsedentary ( > 0 kcals / wk ) participants in the current sample .
binary logistic regression analyses were performed to identify which barriers significantly inhibited cbfr utilization , including knowledge of services , time , transportation , work / other commitments , health , lack of interest , and distance from resources .
all statistical analyses were performed utilizing spss 19.0 for windows ( chicago , il ) .
figure 1 depicts the recruitment flow , leading to the final sample of 417 older adults . of the final sample , 161
were included in the 1.6 km radius group , 114 in the > 1.6 to 3.2 km radius , and 142 in the > 3.2 to 8.0 km radius .
the successful return rates of complete questionnaires for the aforementioned spatial tiers were 61.5% , 63.0% , and 62.6% , respectively .
there was an even distribution of female ( n = 208 ) and male ( n = 206 ) respondents , and participants were primarily caucasian , had at least a high school education , and owned a car .
no clear trend was discernable between education and income levels with car ownership across spatial tiers , although little variation was evident among these variables to allow such a distinction to be made .
body mass index ( bmi ) for all participants averaged just below the threshold ( 30 kg / m ) to define obesity .
the responses for awareness and utilization of cbfr are reported in figure 2 . among all respondents in the 1.6 km , > 1.6 to 3.2 km , and the > 3.2 to 8.0 km radii ,
respondents ' overall utilization rates of cbfr , however , were extremely low ( 2.9% ) , with no differences across the spatial tiers ( = 2.37 , df = 2 , p = 0.306 ) . among those residing in the 1.6 km , > 1.6 to 3.2 km , and the > 3.2 to 8.0 km radii , only 4.3% , 2.6% , and 1.4% of participants responded positively to utilizing cbfr , thus exhibiting a weak trend of decreased utilization with increasing distance from cbfr . among the barriers
listed that had a negative influence on cbfr utilization , lack of interest in cbfr was the most frequently cited barrier ( 51.6% of participants ) , followed by time ( 18.2% ) , work ( 16.1% ) , health ( 14.1% ) , transportation ( 9.1% ) , and distance ( 2.9% ) . including all participants across all spatial tiers ,
only health ( = 1.408 , p = 0.004 ) and lack of interest ( = 2.302 , p = 0.002 ) were significant predictors of individuals not utilizing cbfr .
specific to spatial tiers , the only significant barriers were transportation ( = 5.47 , p = 0.002 ) in the > 1.6 to 3.2 km radius and health ( = 2.27 , p < 0.05 ) in the > 3.2 to 8.0 km radius .
the average energy expenditure in mvpa for all participants across all sites was 1601 2293
engagement in mvpa differed across spatial tiers ( = 15.74 , df = 2 , p = 0.000 ) , with mean caloric expenditures rising in conjunction with increasing distance from cbfr : from 1263 2177 kcals / wk ( n = 146 ) to 1555 1793 kcals / wk ( n = 101 ) to 2013 2680 kcals / wk ( n = 131 ) , respectively .
overall , 27.8% reported an energy expenditure of 0 kcals / wk ( n = 105 ) , 29.1% from > 0 to 999 kcals / wk ( n = 110 ) , 8.7% from 1000 to 1499 kcals / wk ( n = 33 ) , 8.5% from 1500 to 1999 kcals / wk ( n = 32 ) , 5.8% from 2000 to 2499 kcals / wk ( n = 22 ) , and 20.6% > 2500 kcals / wk ( n = 78 ) .
including participants from all spatial tiers , the multinomial regression model accounted for 16.7% of variability in mvpa values , with age and income being significant predictors of low ( = 0.04 , p < 0.05 ) and high ( = 0.92 , p < 0.05 ) levels of physical activity , respectively .
specific to spatial tiers , age was a significant predictor of low levels of physical activity within the 1.6 km radius ( = 0.062 , p < 0.05 ) and > 25 mile radius ( = 0.07 , p < 0.05 ) . no other independent variables were significant predictors of low or high levels of physical activity .
national data suggest only a small percentage of older adults are active enough to receive the health benefits of physical activity , raising the susceptibility to poor overall health with increasing age .
one approach to promoting physical activity and exercise specific to older adults is through local senior centers , providing an environment conducive to support physical activity and exercise by way of exercise equipment / rooms and supervised fitness classes . such cbfr aim to reduce the influence of barriers that negatively impact regular physical activity , including lack of access to facilities , guidance , and social support .
still , other factors remain potentially unresolved by cbfr that contribute to their utilization ( or lack thereof ) . mainly , the influence of the availability of transportation and lack of time constraints remain unaffected and are heavily governed by one 's residence distance from such resources . however , it is unclear how awareness and utilization of cbfr are thus impacted by distance from centers promoting and providing resources for active lifestyles .
the main findings of this study show that , among spatial tiers of increasing distance surrounding cbfr , there were no statistical differences in awareness or utilization of cbfr . moreover , despite approximately one - half of participants being currently aware of cbfr , utilization rates were less than 5% among those surveyed .
an estimated 25% of older adults report utilizing senior centers , providing a promising setting for physical activity promotion efforts . despite this
, the results of the current study suggest that cbfr have a negligible impact on physical activity .
there was a substantial decline in the number of individuals who used cbfr , relative to those who were aware of the resources ( only 3% utilized cbfr , out of approximately 50% who were aware ) .
similar awareness - to - active engagement statistics are also available at the national level , were one to consider that an estimated 36% of us adults are aware of physical activity recommendations , with only 10% meeting such benchmarks [ 18 , 19 ] .
there is evidence to suggest that increasing the density of community resources positively relates to physical activity levels , specifically with increasing overall exercise frequency .
therefore , implementing such resources is a first step in promoting exercise in older adults , given the noted positive relationships between the presence of fitness resources within the community and physical activity levels
. still , awareness of the benefits of exercise towards health is a critical determinant of exercise adoption and adherence . given that 50% of the current study population was aware of cbfr , it is plausible that many did not favorably view cbfr or were possibly not aware of specific opportunities within community centers to tailor physical activity and exercise to their preferences . provided the disconnect between awareness of cbfr and the use of these available resources , other factors pertaining to facility use are likely more influential .
barriers , both personal and environmental , represent factors that inhibit cbfr utilization . among barriers measured in the current study across all spatial tiers ,
health and interest were the only significant predictors of not utilizing cbfr , although interest was the most commonly reported barrier .
health , in the context of reporting multiple chronic conditions , has been shown to decrease the amount of adults meeting physical activity guidelines by up to 30% . in the current study ,
34% reported multiple chronic conditions , suggesting that this sample population may have been in poor health overall , which resulted in low cbfr utilization rates .
although health status has been noted as a critical barrier , it has also been viewed as a powerful physical activity motivator among older adults . accordingly
, this sample population also represents the massive potential benefit to promote health as a theme to increase interest in cbfr .
thus , intervening and educating on health is a logical first step towards bolstering interest rates , targeting both barriers reported for not utilizing cbfr . among other barriers ,
only transportation and health were significant barriers in the > 1.6 to 3.2 km and > 3.2 to 8.0 km radii , respectively .
the term transportation is one of the most influential barriers to physical activity in older adults [ 2729 ] and includes multiple contexts , spanning financial , health , distance , time , and built environment factors [ 30 , 31 ] . among factors related to transportation that require extended time and/or monetary investments and thus are less feasible to modify in the short term , are environmental aesthetics , safety , and walkability ( sidewalks , traffic lights ) .
conversely , factors more easily modified are often specific to each individual . given that both health and transportation were significant barriers in the current study , there is more evidence to suggest a poor level of health in the population that would inhibit transportation to cbfr and , therefore , result in low levels of utilization .
based on current physical activity recommendations , the current sample population can be classified as , on average , sufficiently active with an average energy expenditure exceeding 1500 kcals / wk , assuming 100 kcals per 10 minutes of moderate intensity activity .
although this is higher compared to other reported activity levels in older adults , there were large variations in energy expenditure , from sedentary to extremely active .
only 132 participants ( 35.0% ) reported actually engaging in over 1500 kcals / wk , providing evidence that bolsters the potential for cbfr to increase physical activity among those insufficiently active .
in particular , such resources have been shown to be linked to increased participation in more intense , exercise - type behaviors , which is increasingly important , given the combination of low utilization rates of such resources and overall inactivity of the sample population ( two - thirds not meeting recommended activity levels ) .
despite low utilization rates of cbfr , there was a marginal trend of increased utilization of exercise bikes , aerobic machines , and strength training equipment ( in general ) with increased distance from cbfr .
coupled with the observation that overall physical activity increased the further one resided from cbfr , this evidence reinforces the potential of cbfr to increase overall physical activity levels via exercise equipment should utilization rates increase .
the study was cross - sectional , so one is not able to glean causation between awareness and use of cbfr and overall physical activity levels . however , there is benefit to the random sampling of participants from surrounding neighborhoods , providing a large and diverse population to draw conclusions from .
another limitation is that physical activity data were obtained from subjective methodologies , specifically pertaining to the risk of participant bias based on expectant outcomes and recall error .
considering such weaknesses , though , self - report questionnaires have been shown to accurately rank individuals across varying levels of physical activity .
overall , closer proximity to cbfr did not impact awareness or utilization rates of such resources , while physical activity levels marginally increased the further one resided from cbfr . future work in objectively assessing physical activity while utilizing cbfr is warranted to explore the utility of such resources to promote meaningful increases in energy expenditure in older adults , while investigating strategies to increase awareness and utilization of such resources . | introduction .
it is unclear if community - based fitness resources ( cbfr ) translate to heightened activity levels within neighboring areas .
the purpose of this study was to determine whether awareness and utilization of fitness resources and physical activity differed depending on residential distance from cbfr .
methods .
four hundred and seventeen older adults ( 72.9 7.7 years ) were randomly recruited from three spatial tiers ( 1.6 , > 1.6 to 3.2 , and > 3.2 to 8.0 km ) surrounding seven senior centers , which housed cbfr .
participants completed questionnaires on health history , cbfr , and physical activity , gathering data on cbfr awareness , utilization , and barriers , overall levels , and predictors to engagement in moderate to vigorous physical activity ( mvpa ) .
results . across spatial tiers
, there were no differences in positive awareness rates of cbfr or cbfr utilization .
engagement in mvpa differed across spatial tiers ( p < 0.001 ) , with the > 3.2 to 8.0 km radius having the highest mean energy expenditure . across all sites , age and income level ( p
< 0.05 ) were significant predictors of low and high amounts of mvpa , respectively , and current health status and lack of interest represented barriers to cbfr utilization ( p < 0.05 ) .
conclusion .
closer proximity to cbfr did not impact awareness or utilization rates and had an inverse relationship with physical activity . |
pancreatic neuroendocrine neoplasms ( pnens ) are relatively rare tumors with an estimated incidence of less than 1 per 100,000 according to the national cancer institute surveillance , epidemiology and end results program and similar findings from studies derived from europe and asia .
the 2010 who classification divides neuroendocrine neoplasms ( nens ) according to their site of origin dismissing prior categorization based on their embryological origin .
in addition , the same classification has also introduced the number of mitoses and ki67 labeling index ( li ) to characterize their biological behavior , dividing them into grades 1 ( g1 ) and 2 ( g2 ) well differentiated neoplasms and grade 3 ( g3 ) small - cell or large - cell carcinomas ( neuroendocrine carcinomas ) .
based on their clinical presentation , pnens are divided into functioning pnens , which present with symptoms related to the specific type of hormone secreted and nonfunctioning pnens ( nf - pnens ) which are not associated with a specific clinical syndrome and mainly present with symptoms of mass effects .
nf - pnens are currently the most frequently encountered pnens , whereas the most common types of functioning pnens are insulinomas and gastrinomas with an incidence of 1 to 3/million population / y and 0.5 to 2/million population / y , respectively . among the remaining , rare functioning tumors that represent overall less than 10% of all pnens , pancreatic tumors causing the carcinoid syndrome ( cs ) due to secretion of serotonin or other tachykinins ,
these tumors are believed to originate from either enterochromaffin cells ( ecs ) ( kulchitsky cells ) or from multipotent precursor cells that are diffusely scattered along the epithelium of pancreatic ducts and among the islet cells , and are frequently referred to as
however , the term carcinoid has been used for a wide spectrum of endocrine neoplasms arising from different endocrine cell types and does not adequately convey the morphological , biological , clinical , and molecular features of such tumors .
further confusion is created by the frequent use of this term to describe clinically functioning serotonin - producing ec neoplasms of the small intestine associated with the cs .
alternatively , the terms serotonin - producing pnen and serotoninoma have been proposed , reflecting positive immunoreactivity to serotonin , elevated serum serotonin levels or urine levels of its metabolite 5-hydroxyindoleacetic acid ( 5-hiaa ) .
up to date , approximately 100 cases of such tumors have been reported in the literature either as small case series or in literature reviews . in epidemiological analyses of early series of gastroenteropancreatic nens from 2 cancer registries , pancreatic tumors likely corresponding to serotonin - producing neoplasms accounted for 0.58% to 1.4% ( 235 cases in total ) and represented approximately 1% of all pnens . however , because enrollment was made on the basis of registered diagnostic codes without revision of the clinical / histological features and given the lack of standardized terminology before 2000 , there is concern regarding the potential contamination of those series by tumors of a different phenotype not secreting serotonin . herein , we present a series of 5 patients , from our database of 138 pnens ( 5/138 , 3.62% ) , with advanced well differentiated serotonin - producing pnens associated with elevated levels of urinary 5-hiaa , treated according to the standard clinical guidelines . the clinical , morphological , and histological features of these patients are summarized in table 1 .
clinicopathological characteristics of patients are presented anonymously without any images or interventions , and therefore ethical approval as well as informed consent of patients was considered not necessary .
a 79-year - old male , presented with weakness , progressive body weight loss without diarrhea , or flushing . a computed tomography ( ct ) scan revealed a 35 15 mm mass in the head of the pancreas along with multiple lesions in both hepatic lobes .
immunohistochemical ( ihc ) staining of tissue obtained from hepatic biopsies was positive for synaptophysin , chromogranin a ( cga ) , and the ki67 li was 5% . based on these findings , the tumor was classified as a well differentiated ( g2 ) nf - pnen .
somatostatin receptor scintigraphy ( srs ) revealed uptake in the head of the pancreas and multiple hepatic sites .
cga levels ( 3940 ng / ml ; normal range : 1098 ) and 24-hour urine 5-hiaa ( 27 mg/24 h ; normal range <
following treatment with everolimus 10 mg / d orally in combination with the long - acting repeatable ( lar ) somatostatin analog octreotide , cga and 5-hiaa levels were significantly reduced to 2000 ng / ml and 18 mg/24 h , respectively , while central necrosis was noticed in some lesions . a 55-year - old male patient with a 7-year history of intermittent atypical abdominal pain presented with episodic flushing and diaphoresis without diarrhea .
a ct scan confirmed the presence of metastatic liver deposits and a hypervascular 30 35 mm tumor in the pancreatic tail ; a biopsy from a liver lesion revealed a well differentiated nen with a ki67 li of 10% ( grade 2 ) and positive staining for serotonin .
srs showed avid uptake in both primary pancreatic tumor and the liver metastases , whereas urine 5-hiaa levels and serum cga were elevated to 50.5 mg/24 h and 910 ng / ml , respectively ; cardiac echocardiography did not reveal carcinoid heart disease . following treatment with everolimus 10 mg daily and octreotide lar 30 mg every 4 weeks , 3 months later
, cga and 5-hiaa levels were reduced to 250 ng / ml and 21.7 mg/24 h , respectively , but still remained 3 times above normal .
the patient subsequently underwent a distal pancreatectomy and radiofrequency ablation of the larger metastatic lesion and continued treatment with everolimus and octreotide showing further reduction of 5-hiaa levels ( 12.3 mg/24 h ) . a 60-year - old female patient presented with malaise and excessive vomiting , mild anemia , and abnormal liver function tests .
abdominal mri showed diffuse bilobar liver metastases and a 38 34 mm tumor at the pancreatic body ; biopsy of the hepatic lesions revealed metastases from a g2 well differentiated nen ( ki67 li 5% ) .
although the patient did not report any symptoms related to hormonal hypersecretion , increased cga ( 320 ng / ml ) and 5-hiaa ( 42.7 ng / ml ) levels were found . following treatment with everolimus in combination with octreotide lar , cga (
230 ng / ml ) and 5-hiaa ( 14.5 mg/24 h ) levels were substantially reduced , whereas imaging studies confirmed stable disease . a 68-year - old male patient with abdominal pain
was found to harbor a 25 mm tumor at the pancreatic body with multiple liver lesions in both hepatic lobes .
biopsy of a liver lesion showed a well differentiated nen with a ki67 li of 2% .
srs revealed uptake in all lesions , and biochemical markers ( cga and 5-hiaa ) were found to be 1200 ng / ml and 40.9 mg/24 h , respectively .
apart from the abdominal pain , the patient did not report any symptoms suggestive of cs .
the patient was initially treated with 10 cycles of indium - dtpa octreotide that was followed by a distal pancreatectomy ; histological examination showed a ki67 li of 2% , with hotspots up to 12% .
following treatment with octreotide lar , cga and 5-hiaa levels were reduced to 242.8 ng / ml and 7.5 mg/24 h , respectively . a large pnen ( 9 7 4.5 cm )
was diagnosed in a 54-year - old female patient during investigation for chronic dyspepsia without any secretory symptoms .
the tumor was surgically removed and proven to be a well differentiated pnen with a ki67 li of < 2% .
after a 10-year follow - up period , she was found to have a large left ovarian tumor ( 4.1 2.8 2.5 cm ) that was histologically confirmed to be a metastasis of the previously excised pnen , along with further nonresectable peritoneal metastases . following stabilization of her disease after 12 cycles of streptozotocin and 5-fluorouracil chemotherapy and octreotide lar ,
she was found to have a breast lesion 3 years later that was shown to be a pnen metastasis with a ki67 li of 51% .
she underwent surgery , local radiation therapy , and 2 cycles of platinum - based chemotherapy ; however , 1 year later , developed secretory diarrheas and liver metastases .
her 5-hiaa levels were found to be increased ( 63.2 mg/24 h ) and a biopsy from a liver lesion stained positive for serotonin .
a 79-year - old male , presented with weakness , progressive body weight loss without diarrhea , or flushing . a computed tomography ( ct ) scan revealed a 35 15 mm mass in the head of the pancreas along with multiple lesions in both hepatic lobes .
immunohistochemical ( ihc ) staining of tissue obtained from hepatic biopsies was positive for synaptophysin , chromogranin a ( cga ) , and the ki67 li was 5% . based on these findings , the tumor was classified as a well differentiated ( g2 ) nf - pnen .
somatostatin receptor scintigraphy ( srs ) revealed uptake in the head of the pancreas and multiple hepatic sites .
cga levels ( 3940 ng / ml ; normal range : 1098 ) and 24-hour urine 5-hiaa ( 27 mg/24 h ; normal range <
following treatment with everolimus 10 mg / d orally in combination with the long - acting repeatable ( lar ) somatostatin analog octreotide , cga and 5-hiaa levels were significantly reduced to 2000 ng / ml and 18 mg/24 h , respectively , while central necrosis was noticed in some lesions .
a 55-year - old male patient with a 7-year history of intermittent atypical abdominal pain presented with episodic flushing and diaphoresis without diarrhea .
a ct scan confirmed the presence of metastatic liver deposits and a hypervascular 30 35 mm tumor in the pancreatic tail ; a biopsy from a liver lesion revealed a well differentiated nen with a ki67 li of 10% ( grade 2 ) and positive staining for serotonin .
srs showed avid uptake in both primary pancreatic tumor and the liver metastases , whereas urine 5-hiaa levels and serum cga were elevated to 50.5 mg/24 h and 910 ng / ml , respectively ; cardiac echocardiography did not reveal carcinoid heart disease . following treatment with everolimus 10 mg daily and octreotide lar 30 mg every 4 weeks , 3 months later
, cga and 5-hiaa levels were reduced to 250 ng / ml and 21.7 mg/24 h , respectively , but still remained 3 times above normal .
the patient subsequently underwent a distal pancreatectomy and radiofrequency ablation of the larger metastatic lesion and continued treatment with everolimus and octreotide showing further reduction of 5-hiaa levels ( 12.3 mg/24 h ) .
a 60-year - old female patient presented with malaise and excessive vomiting , mild anemia , and abnormal liver function tests .
abdominal mri showed diffuse bilobar liver metastases and a 38 34 mm tumor at the pancreatic body ; biopsy of the hepatic lesions revealed metastases from a g2 well differentiated nen ( ki67 li 5% ) .
although the patient did not report any symptoms related to hormonal hypersecretion , increased cga ( 320 ng / ml ) and 5-hiaa ( 42.7 ng / ml ) levels were found . following treatment with everolimus in combination with octreotide lar , cga ( 230 ng / ml ) and 5-hiaa ( 14.5 mg/24 h ) levels were substantially reduced , whereas imaging studies confirmed stable disease .
a 68-year - old male patient with abdominal pain was found to harbor a 25 mm tumor at the pancreatic body with multiple liver lesions in both hepatic lobes .
biopsy of a liver lesion showed a well differentiated nen with a ki67 li of 2% .
srs revealed uptake in all lesions , and biochemical markers ( cga and 5-hiaa ) were found to be 1200 ng / ml and 40.9 mg/24 h , respectively .
apart from the abdominal pain , the patient did not report any symptoms suggestive of cs .
the patient was initially treated with 10 cycles of indium - dtpa octreotide that was followed by a distal pancreatectomy ; histological examination showed a ki67 li of 2% , with hotspots up to 12% .
following treatment with octreotide lar , cga and 5-hiaa levels were reduced to 242.8 ng / ml and 7.5 mg/24 h , respectively .
a large pnen ( 9 7 4.5 cm ) was diagnosed in a 54-year - old female patient during investigation for chronic dyspepsia without any secretory symptoms .
the tumor was surgically removed and proven to be a well differentiated pnen with a ki67 li of < 2% .
after a 10-year follow - up period , she was found to have a large left ovarian tumor ( 4.1 2.8 2.5 cm ) that was histologically confirmed to be a metastasis of the previously excised pnen , along with further nonresectable peritoneal metastases . following stabilization of her disease after 12 cycles of streptozotocin and 5-fluorouracil chemotherapy and octreotide lar ,
she was found to have a breast lesion 3 years later that was shown to be a pnen metastasis with a ki67 li of 51% .
she underwent surgery , local radiation therapy , and 2 cycles of platinum - based chemotherapy ; however , 1 year later , developed secretory diarrheas and liver metastases .
her 5-hiaa levels were found to be increased ( 63.2 mg/24 h ) and a biopsy from a liver lesion stained positive for serotonin .
the diagnosis of a serotonin - producing pancreatic tumor is based on the presence of a pnen plus a positive finding of at least one of the following , without demonstration of another dominant hormone : elevation of 5-hydroxytryptamine ( 5-ht ) ( serotonin ) in the serum or detected in tumor tissue and/or elevation of 5-hiaa in the urine . however , not all pathologists and clinicians investigate pnens for serotonin expression via ihc or secretion via determination of its plasma levels or its urine metabolite 5-hiaa , in the absence of symptoms suggestive of cs .
furthermore , symptoms related to serotonin secretion from these tumors may be subtle , nonspecific or intermittent due to fluctuations in the secretory rate , or even atypical and thus elude clinical detection .
routine measurement of urine 5-hiaa in patients with apparently nf - pnen is not recommended by either the european neuroendocrine tumor society ( enets ) or the north american tumor society .
thus , it has been suggested that the real incidence and prevalence of pancreatic serotonin - producing tumors may be underestimated . in our series ,
only 2 patients ( cases 2 and 5 ) reported symptoms suggestive of the cs , while in the remaining 5-hiaa measurement was ordered incidentally from the referring physicians even without the presence of secretory symptoms .
serotonin ( 5-ht ) is mainly considered responsible for producing the classic symptoms of cs .
serotonin is synthesized and stored in the ecs of the gastrointestinal tract from tryptophan through its precursor , 5-hydroxytryptophan ( 5-htp ) , and subsequently metabolized to 5-hydroxyindoleacetic acid ( 5-hiaa ) , which is excreted in the urine . in order for the systemic features of cs to develop ,
serotonin must circumvent the portal and pulmonary arterial circulation and escape liver and lung metabolism , as in the case of liver metastases or primary tumors of the ovaries and bronchus .
gastric , bronchial carcinoids , and pnens have been found to have a low content of serotonin and often secrete the serotonin precursor 5-htp as they lack the appropriate decarboxylase ( l - aromatic amino acid decarboxylase ) .
markedly increased 5-hiaa levels in these patients can be explained by 5-htp decarboxylation elsewhere in the intestine and other tissues or by the rare expression of decarboxylase in these tumors . in some patients with pancreatic carcinoid tumors ,
an atypical form of cs develops consisting of pain , diarrhea , weight loss , and less frequently flushing ( only in 30% of these patients ) , and it is thought to be mediated by 5-htp , histamine , and other biogenic amines .
similarly in our small cohort , 2 patients presented with symptoms of atypical cs , while abdominal pain was the most common initial symptom .
ihc detection of serotonin in tumor tissue is more specific for the diagnosis of carcinoid tumors and has replaced the classic silver staining ( argyrophilic and/or argentaffin reaction ) . among the patients included in our series , only 2 patients ( cases 2 and 5 ) showed positive immunoreactivity for serotonin ( table 1 ) .
earlier case series and systematic reviews of pancreatic carcinoids have reported substantially higher rates of positive serotonin ihc .
this difference possibly reflects a selection bias in those pathological studies and can also be influenced by the small number of patients in our cohort and the lack of central ihc assessment , as in only 3 cases histopathological material was available for central assessment .
furthermore , in the present study , we included patients found to have elevated 5-hiaa in the setting of a pnen , in which the presence of positive serotonin ihc has not been systematically studied .
5-hiaa is the serotonin metabolite excreted through the urine and represents a useful clinical marker in the diagnosis and follow - up of patients with cs .
midgut carcinoids are most likely to produce the typical cs with 5-hiaa elevation compared to foregut and hindgut nens that produce substantially less serotonin .
tumor burden and fluctuating release of serotonin can influence the levels of urinary 5-hiaa as they may be normal in patients with low tumor burden even in the presence of liver metastases .
furthermore certain food , medication , and medical conditions such as renal impairment and malabsorption may affect 5-hiaa urinary levels , producing false positive or false negative results .
in the patients of the present series , there were no comorbidities , food , or drug interaction that could falsely elevate urine levels of 5-hiaa , which were consistently found to be elevated in many instances in each patient 's history .
furthermore , clinical and imaging response following medical or surgical treatment correlated with similar changes in 5-hiaa levels .
another feature of pancreatic carcinoids that has been shown to correlate with serotonin immunoreactivity is extensive stromal fibrosis . in a study that retrospectively examined serotonin immunoexpression in 52 pancreatic tumors characterized by gross stromal fibrosis ,
14 were at least focally positive to serotonin stain , and this significantly correlated with trabecular or trabecular glandular architecture and involvement of a large pancreatic duct .
pancreatic duct stenosis and marked dilatation of the main pancreatic duct represents a finding recently recognized in 5 other reports ( 12 total cases ) .
in our series , only patient 1 presented with duct stenosis and consequent peripheral dilatation .
abdominal pain frequently reported as a presenting symptom in these patients , maybe due to episodes of pancreatitis subsequent to ductal stenosis .
earlier reports on serotonin - producing pnens suggested that these tumors were associated with higher incidence of metastatic disease on diagnosis and worse prognosis , due to late onset of symptoms and delayed diagnosis .
some patients have been anecdotally diagnosed early in the context of marked dilatation of the main pancreatic duct , leading to a curative surgical excision at an early stage .
no clear - cut data exist to date to compare patients with serotonin - producing pnens with patients harboring nf - pnens or islet cell tumors , in terms of survival and outcome .
such data could justify the routine screening of all patients with apparently nf - pnens for serotonin production via ihc or via measurement of urine 5-hiaa . nowadays , according to enets guidelines , the only potential pnens predictive biomarker of response to treatments is ki-67 li .
recent evolving biomarkers are currently under investigation to identify potential predictors of response to various therapies besides tumor grading taking into consideration the individual characteristics of the patients and tumor pathology and genetics such as daxx , atrx , p53 , rb , smad4 , and so on .
however , all these possible predictive pnens biomarkers for response to treatments have not been validated until now , are not included in any clinical guidelines , and therefore are not performed routinely in daily clinical practice .
in summary , we reported a small case series of well differentiated pnens with high levels of urinary 5-hiaa from our database of 138 pnens .
such tumors are considered rare , although this may be an underestimate as systematic screening for the presence of serotonin in tissue or elevated urinary 5-hiaa levels in patients with apparently nf - pnens is not currently recommended . in order to reach such a consensus ,
data from large prospective studies are needed in order to evaluate the impact of this type of tumors in survival and clinical outcome , since some studies have suggested a worse prognosis . | abstractintroduction : pancreatic neuroendocrine neoplasms ( pnens ) are relatively rare tumors representing 1% to 2% of all pancreatic neoplasms .
these tumors can secrete a variety of biologically active substances giving rise to distinct clinical symptoms or can be clinically nonfunctioning .
apart from insulinomas and gastrinomas , which constitute the majority of functioning pnens , some tumors may secrete serotonin presenting with the features of the carcinoid syndrome .
these so - called pancreatic carcinoids are considered relatively rare tumors and are associated with increased urinary levels of 5-hydroxyindoleacetic acid ( 5-hiaa ) .
it has recently been suggested that the prevalence of such tumors might be underestimated.cases:we present a series of 5 patients from our database of 138 pnens ( 5/138 , 3.62% ) , harboring serotonin - producing pnens and describe their distinctive clinical , biochemical , histopathological features , and response to treatment along with a review of the relevant available literature.conclusion:such tumors are considered rare , although this may be an underestimate as systematic screening for the presence of serotonin in tissue or elevated urinary 5-hiaa levels in patients with apparently nonfunctioning pnens is not currently recommended . in order to reach such a consensus ,
data from large prospective studies are needed in order to evaluate the impact of this type of tumors in survival and clinical outcome , since some studies have suggested a worse prognosis . |
we developed a web - based content management system that enables the delivery of content to mobile devices .
the main purpose of creating this system was to enable users with average computing skills to build flexible and customizable computer - based learning packages for distribution to mobile devices via direct internet links or as a downloaded program .
users of the system can create and manage the structure and layout of the content , links to other content within the program , and links to multimedia and external programs .
this content management system differs from computer programming languages in that extensive technical expertise is not a prerequisite to producing web content .
it is designed to make content distribution easy , so that it can be viewed and interacted with using microsoft windows - based mobile devices and , more recently , the iphone and ipod touch . because the system has all its data stored in a central location , updating and viewing content in real - time is possible .
the system was built on the microsoft.net framework , and , because it is web - based , it is widely accessible . we have previously reported our experience with this tool in distributing 19 of the core training problems from the clerkship directors in internal medicine ( cdim ) curriculum guide , and with using this tool as a patient - encounter log ( 9 , 10 ) .
we recently equipped this software with a tool for the development of real - time assessment of student competencies related to these 19 cdim training problems .
after adding the training problem ( e.g. , abdominal pain ) in the root directory , we added the competencies to be assessed ( e.g. , communication skills , history taking , and physical examination skills ) in subdirectories . within each of the specific competency sub - directories
, we added line items ( e.g. , for abdominal pain history - taking we added lines for pain characteristics , associated symptoms , past history of gi disease , family history of gi disease , medications , social history ) . within each line , using an html editor , we added the specific items to be assessed ( e.g. , for abdominal painhistory takingassociated symptoms , added items included weight change , fever , dysuria , and gi - related symptoms , such as diarrhea , melena , etc . , mostly derived from the cdim curriculum ) . when displayed on a mobile device , these items appear as checkboxes .
the tool allows the author to define the user to define the minimum number of checked items that constitutes a
the faculty evaluators , however , have the option to override the grade at their discretion .
we allowed the students to choose the problem - specific performance objectives on which they wished to be assessed ( e.g. , the physical examination objectives in a patient with abdominal pain ) .
they , then , displayed the line items for that objective on their device and handed it to a faculty member ( including residents ) .
the faculty preceptors subsequently used the checklist to assess the students interacting with a real patient .
we have termed this electronic problem - specific clinical evaluation tool the ecex ( fig .
, the faculty preceptors electronically signed the assessment record and this record of the students ' performance was then stored in a central server for tracking purposes .
the college of human medicine at michigan state university trains third - year medical students in six communities throughout michigan . during the academic year 20082009
, we implemented the ecex as a required element of the internal medicine clerkship in a convenience sample of 56 students in three of our six community training sites .
fifty - six students in the remaining three training sites did not use the ecex and constituted the control group .
for the grading incentive , students using the ecex were to arrange 10 ecex evaluations with their clinical preceptors as part of the requirements to pass the clerkship .
students chose both the specific competency ( e.g. , abdominal examination in a patient with abdominal pain ) and the specific resident or attending evaluator at their discretion .
they were also responsible for orienting and assisting the evaluator in the use of the ecex software .
students at the control sites were required to complete a single observed complete history and physical examination on a patient chosen for them by the evaluator , in order to pass the clerkship .
all additional direct observations in the clerkship were discretionary . at the end of each eight - week clerkship rotation ,
all students and preceptors were surveyed at the ecex sites on the educational utility and technical aspects of the ecex ( appendices 1 and 2 ) .
additionally , students in both study groups were surveyed on the number of times they were directly observed by either faculty or residents during their eight weeks on the clerkship .
all data were obtained by using the online survey service survey monkey ( www.surveymonkey.com ) .
the number of directly observed encounters was analyzed with a one - tailed t - test between the communities using the ecex and those not using the ecex , with significance set at a p - value of < 0.05 .
we developed a web - based content management system that enables the delivery of content to mobile devices .
the main purpose of creating this system was to enable users with average computing skills to build flexible and customizable computer - based learning packages for distribution to mobile devices via direct internet links or as a downloaded program .
users of the system can create and manage the structure and layout of the content , links to other content within the program , and links to multimedia and external programs .
this content management system differs from computer programming languages in that extensive technical expertise is not a prerequisite to producing web content .
it is designed to make content distribution easy , so that it can be viewed and interacted with using microsoft windows - based mobile devices and , more recently , the iphone and ipod touch . because the system has all its data stored in a central location , updating and viewing content in real - time is possible .
the system was built on the microsoft.net framework , and , because it is web - based , it is widely accessible . we have previously reported our experience with this tool in distributing 19 of the core training problems from the clerkship directors in internal medicine ( cdim ) curriculum guide , and with using this tool as a patient - encounter log ( 9 , 10 ) .
we recently equipped this software with a tool for the development of real - time assessment of student competencies related to these 19 cdim training problems .
after adding the training problem ( e.g. , abdominal pain ) in the root directory , we added the competencies to be assessed ( e.g. , communication skills , history taking , and physical examination skills ) in subdirectories . within each of the specific competency sub - directories
, we added line items ( e.g. , for abdominal pain history - taking we added lines for pain characteristics , associated symptoms , past history of gi disease , family history of gi disease , medications , social history ) . within each line , using an html editor , we added the specific items to be assessed ( e.g. , for abdominal painhistory takingassociated symptoms , added items included weight change , fever , dysuria , and gi - related symptoms , such as diarrhea , melena , etc . , mostly derived from the cdim curriculum ) . when displayed on a mobile device , these items appear as checkboxes .
the tool allows the author to define the user to define the minimum number of checked items that constitutes a
the faculty evaluators , however , have the option to override the grade at their discretion .
we allowed the students to choose the problem - specific performance objectives on which they wished to be assessed ( e.g. , the physical examination objectives in a patient with abdominal pain ) .
they , then , displayed the line items for that objective on their device and handed it to a faculty member ( including residents ) .
the faculty preceptors subsequently used the checklist to assess the students interacting with a real patient .
we have termed this electronic problem - specific clinical evaluation tool the ecex ( fig .
, the faculty preceptors electronically signed the assessment record and this record of the students ' performance was then stored in a central server for tracking purposes .
the college of human medicine at michigan state university trains third - year medical students in six communities throughout michigan . during the academic year 20082009
, we implemented the ecex as a required element of the internal medicine clerkship in a convenience sample of 56 students in three of our six community training sites .
fifty - six students in the remaining three training sites did not use the ecex and constituted the control group . for the grading incentive ,
students using the ecex were to arrange 10 ecex evaluations with their clinical preceptors as part of the requirements to pass the clerkship .
students chose both the specific competency ( e.g. , abdominal examination in a patient with abdominal pain ) and the specific resident or attending evaluator at their discretion .
they were also responsible for orienting and assisting the evaluator in the use of the ecex software .
students at the control sites were required to complete a single observed complete history and physical examination on a patient chosen for them by the evaluator , in order to pass the clerkship .
all additional direct observations in the clerkship were discretionary . at the end of each eight - week clerkship rotation ,
all students and preceptors were surveyed at the ecex sites on the educational utility and technical aspects of the ecex ( appendices 1 and 2 ) .
additionally , students in both study groups were surveyed on the number of times they were directly observed by either faculty or residents during their eight weeks on the clerkship .
all data were obtained by using the online survey service survey monkey ( www.surveymonkey.com ) .
the number of directly observed encounters was analyzed with a one - tailed t - test between the communities using the ecex and those not using the ecex , with significance set at a p - value of < 0.05 .
all 56 students in the ecex group successfully uploaded 10 or more completed directly observed encounters .
the average number of ecex evaluations per faculty evaluator was 4.6 ( range = 119 ) .
twenty - seven faculty evaluators ( 20.9% ) participated in a single ecex whereas the remainder performed two or more ecex evaluations ( table 1 ) .
student and faculty ecex use data note : summer 2008 through february 2009 ; 129 separate faculty provided at least one ecex evaluation .
student were observed , no by an average , by of 3.64 separate faculty evaluators depending on the community ( range = 17 evaluators per student ) .
fifty - three of 56 ( 91% ) ecex students and 71 of 125 faculty evaluators ( 55% ) responded to an online survey of their perception of the educational utility and technical aspects of the ecex ( table 2 ) .
faculty and student survey on the utility of ecex note : summer 2008 through february 2009 ; neutral or na not counted ; 56 students used the ecex , 53 ( 91% ) responded to the survey on the ecex utility ; 70 out of 129 ( 54% ) faculty / residents . needing to know and demonstrate for students , and
students and faculty both agreed that the ecex helped them understand the specific history and physical examination competencies being assessed during the encounters ( 75.588.4% agree / strongly agree ) .
faculty felt that the ecex improved their assessments ( 74.7% ) and their ability to give feedback ( 88.7% ) .
they did not perceive accommodating the ecex into their workday as difficult ( 84.2% ) .
however , only 11.620.7% of the students agreed or strongly agreed that arranging for either residents or faculty to observe them was easy .
all 112 students ( 100% ) reported the number of times they were directly observed by a resident or attending .
students in the ecex group identified residents as observers of their focused history and focused physical examinations an average of 7.7 ( 8.3 ) and 11.5 ( 11.3 ) times , respectively .
corresponding numbers for the control group were 4.63.9 ( p<0.05 ) and 6.97.4 ( p<0.05 ) .
students were free to choose the specific training problem from our pre - specified list of problems and the competency in which they would be evaluated .
all the students chose at least one physical examination competency on which to be evaluated , whereas 20% of the students chose not to be evaluated on communication skills or history taking ( table 3 ) .
note : the average number of patients that students were directly observed interacting with was 7.8 .
all 56 students in the ecex group successfully uploaded 10 or more completed directly observed encounters .
the average number of ecex evaluations per faculty evaluator was 4.6 ( range = 119 ) .
twenty - seven faculty evaluators ( 20.9% ) participated in a single ecex whereas the remainder performed two or more ecex evaluations ( table 1 ) .
student and faculty ecex use data note : summer 2008 through february 2009 ; 129 separate faculty provided at least one ecex evaluation .
student were observed , no by an average , by of 3.64 separate faculty evaluators depending on the community ( range = 17 evaluators per student ) .
fifty - three of 56 ( 91% ) ecex students and 71 of 125 faculty evaluators ( 55% ) responded to an online survey of their perception of the educational utility and technical aspects of the ecex ( table 2 ) .
faculty and student survey on the utility of ecex note : summer 2008 through february 2009 ; neutral or na not counted ; 56 students used the ecex , 53 ( 91% ) responded to the survey on the ecex utility ; 70 out of 129 ( 54% ) faculty / residents . needing to know and demonstrate for students , and improved ability to identify the specific competencies needing to be evaluated for faculty .
students and faculty both agreed that the ecex helped them understand the specific history and physical examination competencies being assessed during the encounters ( 75.588.4% agree / strongly agree ) .
faculty felt that the ecex improved their assessments ( 74.7% ) and their ability to give feedback ( 88.7% ) .
they did not perceive accommodating the ecex into their workday as difficult ( 84.2% ) .
however , only 11.620.7% of the students agreed or strongly agreed that arranging for either residents or faculty to observe them was easy .
all 112 students ( 100% ) reported the number of times they were directly observed by a resident or attending .
students in the ecex group identified residents as observers of their focused history and focused physical examinations an average of 7.7 ( 8.3 ) and 11.5 ( 11.3 ) times , respectively .
corresponding numbers for the control group were 4.63.9 ( p<0.05 ) and 6.97.4 ( p<0.05 ) .
students were free to choose the specific training problem from our pre - specified list of problems and the competency in which they would be evaluated .
all the students chose at least one physical examination competency on which to be evaluated , whereas 20% of the students chose not to be evaluated on communication skills or history taking ( table 3 ) .
note : the average number of patients that students were directly observed interacting with was 7.8 .
there are several unique aspects to our study . to our knowledge , this is the first electronic mobile assessment tool linked to problem - specific performance objectives from a nationally recognized curriculum .
additionally , we believe this is the first comparative study assessing an intervention aimed at increasing the number of directly observed student patient encounters .
a number of studies have noted the difficulty encountered by faculty members in identifying the specific performance criteria upon which medical students should be evaluated . in a study of the implementation of a mini - cex with 326 medical students ,
hill commented that some examiners in the study were unsure of the standard to expect from medical students and problems existed in trying to ensure that everyone was working to the same or similar standards ( 5 ) . in another study of 396 mini - cex assessments of 176 students ,
fernando concluded that faculty evaluators were unsure of the level of performance expected of the learners ( 11 ) . in a study of 172 family medicine clerks , on the role of direct observation in clerkship ratings ,
hasnain suggested that the high level of faculty - rater disagreement concerning clinical performance of students might have been due to the fact that standards for judging clinical competence were not explicit ( 4 ) .
for example , the cdim curriculum used in this study specifies that , in patients with copd , students should be able to demonstrate the ability to obtain at least 17 specific historical items , demonstrate at least 10 separate physical examination steps , and be able to counsel and encourage patients about smoking cessation , peak flow monitoring , environmental issues , and asthma action plans when indicated .
this volume of information makes knowing and remembering what a medical student should be able to do at a particular point in time and with a particular patient in a specific clinical situation very difficult .
our results demonstrate that delivery of expected performance criteria just before planned assessments using the ecex fostered an understanding among faculty evaluators and students of what to expect during the evaluation ; specifically 88% of faculty agreed that the ecex helped them understand the physical examination and history competencies that they needed to evaluate , and 74% of students agreed that the ecex helped understand the specific history and physical examination competencies that they needed to know and demonstrate .
in addition , 7589% of the faculty respondents felt that the ecex improved their assessments and their ability to give feedback .
our second objective was to test whether or not a grading incentive increased the number of directly observed patient - encounters .
the use of a grading incentive to accomplish cex evaluations is not a new concept .
kogan used it as an incentive for students to return their paper - based mini - cex booklets that were handed out at the beginning of their internal medicine clerkship .
however , the incentive was not tied to the number of forms completed , unlike in our study where a grade was given only if the student completed 10 evaluations ( 8) . as in our study , the actual results of the mini - cex evaluations in kogan 's study were not tied to the final grade . using a handheld device - based mini - cex ,
torre required students to complete two electronic mini - cex evaluations during a two - month clerkship and attained 100% compliance . the presence or absence of a specific grading incentive
again , the results of the actual cex evaluations did not impact a student 's final clerkship evaluation in torre 's study .
neither kogan 's nor torre 's study used a comparison group to assess the effect of their mini - cex intervention on the number of directly observed encounters . in a study of 173 medical students ,
fernando noted that only 41% of students met the requirement of three mini - cex evaluations in their final year of medical school .
fernando speculated that since these assessments were formative and did not count toward the students ' final grade , the perceived importance of the mini - cex may have been diminished ( 11 ) .
it has been estimated that adequate reliability of the mini - cex can be achieved with 815 encounters ( 5 ) .
therefore , it is educationally important to ensure that a minimum number of cex encounters occur .
we have previously demonstrated that a grading incentive is associated with a very high rate of compliance with students meeting patient - log requirements ( 10 ) . in this study
, we also demonstrated that a grading incentive was associated with 100% compliance in all 56 students completing 10 ecex evaluations . in their study ,
fernando et al . commented : providing formal training ( on the use of the mini - cex ) to all clinical staff who are likely to encounter students , or who may be approached by students for assessment , is probably unrealistic ( 11 ) .
the results of our study strongly suggest that at least when it comes to the faculty 's understanding of what to assess , formal training may not be necessary .
giving students the responsibility to orient faculty to the process and to provide faculty with a detailed electronic checklist just prior to the assessment may be an answer to the dilemma posed by fernando .
several limitations , however , need to be kept in mind when interpreting the findings of our study .
first , this is a single institution study and the results may not be generalized to other institutions .
second , the three intervention sites in the study were assigned based upon convenience and not via randomization .
it is possible that site - specific characteristics , not our intervention , may have explained the results .
third , we did not require our students to complete a full cex but only a focused history or focused aspect of the physical examination .
thus our results may not be applied to situations in which a full cex is required .
fourth , although we had an excellent survey response from the students , only 55% of the faculty responded , and those who did not respond may have been less enthusiastic about the value of the ecex .
the use of structured checklists in the assessment of clinical performance is known to at least double the accuracy of trainee assessment by identifying more performance errors ( 12 ) .
the global assessments in our study were generated automatically , and several of the faculty commented that they appreciated this aspect of the ecex .
we aim to study the inter - rater reliability of the ecex over the next academic year .
sixth , perhaps our study confirms a self - evident outcome that a grading incentive provides a strong stimulus for students . although the expectations were quite different for the two groups , students in each group met their respective requirement for directly observed encounters .
we can not say with certainty which intervention increased the number of directly observed encounters .
it is quite possible that the use of the ecex , and not the grading incentive , stimulated more frequent direct observation .
finally , we do not know how allowing students to select the patient and specific competency assessment in the ecex group influenced the study 's outcome .
we conclude that the use of a problem - specific electronic mobile checklist increased the students ' and evaluators ' understanding of the specific objectives of the observation and the evaluators ' self - reported ability to give feedback and provide assessments .
we further conclude that a grading incentive increased the number of times a student reported being observed by a resident physician while performing a focused history and physical examination .
this study was funded in part by a grant 5 d56hp05217 from the division of medicine , bureau of health professions , health services and resources administration . | backgroundfacilitating direct observation of medical students ' clinical competencies is a pressing need.methodswe developed an electronic problem - specific clinical evaluation exercise ( ecex ) based on a national curriculum .
we assessed its feasibility in monitoring and recording students ' competencies and the impact of a grading incentive on the frequency of direct observations in an internal medicine clerkship .
students ( n = 56 ) at three clinical sites used the ecex and comparison students ( n = 56 ) at three other clinical sites did not .
students in the ecex group were required to arrange 10 evaluations with faculty preceptors .
students in the second group were required to document a single , faculty observed full history and physical encounter with a patient .
students and preceptors were surveyed at the end of each rotation.resultsecex increased students ' and evaluators ' understanding of direct - observation objectives and had a positive impact on the evaluators ' ability to provide feedback and assessments .
the grading incentive increased the number of times a student reported direct observation by a resident preceptor.conclusionsecex appears to be an effective means of enhancing student evaluation . |
isolated dislocation of the radial head in adults is a rare injury with less than 30 cases reported in literature [ 1 - 9 ] .
if neglected , these can cause restriction of forearm supination and pronation , secondary degenerative arthritis of the elbow and distal radio - ulnar joints later on .
this important injury can easily be missed in the presence of other major associated injuries . also since with this injury there is no limitation of elbow flexion - extension and limitation of prono
supination is relatively less disabling , the patient may tend to ignore the disability and present late .
lastly the mushrooming of unlawful and uncontrolled bone setters can make the simple injuries complicated and problematic later on .
a 26-year old man presented to us 8 weeks after injury to his right elbow with a motorbike accident .
the patient complained of pain in the right elbow immediately after accident though he was able to flex and extend the elbow .
thinking it to be a trivial injury he went to a bone setter for treatment , who applied wooden splints after oil massage to the elbow .
however , the patient had pain in the elbow and presented to us after further 4 weeks with gross limitation of supination and pronation .
there was no previous history of elbow injury or arthritis . on examination , there was minimal swelling over the elbow and tenderness over the radial head , which appeared displaced backwards .
although he had good flexion and extension of the elbow , forearm pronation and supination were grossly restricted and painful .
radiographs showed posterior dislocation of the radial head with no associated fractures of the radius , ulna or disruption of the distal radio - ulnar joint .
immobilisation was done in flexion and supination and continued for three weeks with serial radiographs done at week 1 and 2 to make sure the superior radio - ulnar joint was in reduced position ( fig .
2 ) . elbow mobilisation was started after removal of the plaster , under supervision of the physiotherapist .
the patient was followed up at 3 and 6 months . at 6 months he had no residual pain at the elbow and movements of elbow
26 year old man presenting with 8 weeks old isolated posterior dislocation of right radial head with limitation of prono - supination at right elbow .
post operative radiographs showing excision of the radial head three months follow up showing good range of prono supination
isolated dislocation of the radial head with no other associated fracture or dislocation of elbow is rare but not unknown injury in adults is a rare injury [ 1 - 5 ] .
reduction in such cases is easily achieved by a pronation / supination manoeuvre and most authors propose immobilization of the elbow in flexion and supination in a plaster cast .
the proximal radio - ulnar joint is most stable in supination : in this position , the contact between radius and ulna is maximal and the interosseous membrane , the annular ligament , and the anterior fibres of the quadrate ligament are all taut , thus drawing the radial head snugly against its notch in the ulna , cadaveric studies have shown that posterior dislocation of the radial head can not occur without the rupture of the annular ligament ; in addition , partial tear of the quadrate ligament and the proximal interosseous membrane takes place .
most acute cases can be reduced closed and the functional outcome seems to be good post reduction .
if missed or neglected , an open reduction has to be done with either an annular ligament reconstruction or a radial head excision deemed as the procedure of choice . in this case radial head excision
the period of immobilisation was 21 days . in the presence of major distracting injuries like long bone fractures , pelvic fractures , chest and abdominal injuries ,
an isolated radial head dislocation can be easily missed as pain is masked by the presence of major distracting injuries and flexion and extension of the elbow is almost normal . even if it is an isolated injury patient
may take it as a minor injury because of normal flexion and extension at elbow .
therefore occurrence of such an injury must be kept in mind in all cases of elbow trauma and supination and pronation of the forearm must be assessed .
this injury if missed can have serious repercussions later on , such as degenerative arthritis of the elbow and the distal radio - ulnar joints .
there is also need for awareness amongst the public about the dangers of going to bonesetters , even for small injuries after any accident . | a 26 year old male reported with gross limitation of pronation and supination of right forearm 8 week after a vehicular accident .
x - ray of the elbow showed isolated radial head dislocation without any associated fracture .
it was treated with radial head excision with almost full restoration of movements postoperatively .
the case is presented because of rarity of this injury and the tendency to miss this rare injury especially in the presence of other distracting injuries .
however its early diagnosis and proper management is required to prevent chronic pain in future due to ensuing osteoarthritic changes and the disability of pronation and supination . |
hesc lines ( wa-09 , passages 32 - 50 ; shef1 , ) and hipsc lines ( c14 , c72 ; passages 10 - 20 ) were cultured with mouse embryonic fibroblasts ( mefs , globalstem ) pre - plated at 12 - 15,000 cells / cm .
medium containing dmem / f12 , 20% knockout serum replacement , 1 mm l - glutamine , 100 m mem non - essential amino acids , and 0.1 mm -mercaptoethanol was made .
6ng / ml fgf-2 was added after sterile filtration and cells were fed daily and passaged weekly using 6u / ml dispase .
briefly , cells were rendered to single cells using accutase plated on gelatin for 30 minutes to remove mefs .
non - adherent cells were collected and plated on matrigel treated dishes at a density of 20 - 40,000 cells / cm in the presence of mef - conditioned hesc media containing 10 ng / ml fgf-2 and 10 m y-27632 .
neural differentiation was initiated when the cells were confluent using ksr media containing 820 ml of knockout dmem , 150 ml knockout serum replacement , 1 mm l - glutamine , 100 m mem non - essential amino acids , and 0.1 mm -mercaptoethanol .
to inhibit smad signaling , 100 nm ldn-193189 and 10 m sb431542 were added on days 0 through 5 .
cells were fed daily , and n2 media was added in increasing 25% increments every other day starting on day 4 ( 100% n2 on day 10 ) .
nociceptor induction was initiated with the addition of the three inhibitors 3 m chir99021 , 10 m su5402 , 10 m dapt on days 2 through 10 .
cell passage to lower density can promote maturation of sox10 + progenitors , and long - term culture media consisted of n2 containing 25ng / ml human - b - ngf , bdnf , and gdnf .
cells were fixed with 4% paraformaldehyde for 20 minutes , washed with pbs , permeabilized using 0.5% triton in pbs , and blocked using 1% bsa in pbs . for glutamate staining , .05% glutaraldehyde was added to the fixative .
primary antibodies used for microscopy included pax6 ( covance ) , tuj1 ( covance ) , ki67 ( sigma ) , isl1 ( dshb , abcam ) , brn3a ( millipore ) , ret ( r&d ) , runx1 ( sigma ) , glutamate ( sigma ) , peripherin ( santa cruz ) , trpv1 ( neuromics ) , substance p ( neuromics ) , cgrp ( neuromics ) , sox10 ( santa cruz ) . for flow cytometry
, cells were fixed using the bd cytofix / cytoperm kit ( bd ) , including the optional 4% paraformaldehyde fixation step .
primary conjugated antibodies for flow cytometry were ntrk1-apc ( r&d ) , nestin - alexa647 ( bd pharmingen ) , tuj1-alexa488 ( bd pharmingen ) .
total rna was isolated at days 2 , 3 , 5 , 7 , 9 , and 15 of differentiation of lsb or lsb3i treated hpscs using trizol ls .
all samples were processed by the mskcc genomics core facility and hybridized to the illumina human 12 oligonucleotide array .
normalization and model - based expression measurements were calculated using the illumina analysis package ( lumi ) from the bioconductor project ( www.bioconductor.org ) within the statistical programming language r ( http://cran.r-project.org/ ) .
pair - wise comparison cut - off was significant if the multiple test corrected p - value was < 0.05 .
patch - clamp experiments were performed in whole - cell configuration at room temperature ( 20 - 22c ) using a multiclamp 700b patch - clamp amplifier controlled by pclamp 10 software ( molecular devices ) .
patch pipettes had resistances between 1.5 and 2 m. extracellular solution contained ( mm ) : 140 nacl , 4.7 kcl , 2.5 cacl2 , 1.2 mgcl2 , 10 hepes and 10 glucose ; ph was adjusted to 7.4 with naoh .
the intracellular ( pipette ) solution for voltage - clamp contained ( mm ) 100 csf , 45 cscl , 10 nacl , 1 mgcl2 , 10 hepes , and 5 egta ; ph was adjusted to 7.3 with csoh . for current - clamp
the intracellular ( pipette ) solution contained ( mm ) : 130 kcl , 1 mgcl2 , 5 mgatp , 10 hepes , and 0.5 egta ; ph was adjusted to 7.3 with koh .
series resistance was compensated by 80 - 90% to reduce voltage errors , however space - clamp artifacts occurred due to the presence of processes emanating from the cell bodies .
a current - voltage protocol was applied using a 20 ms pulse from -120 mv to + 50 mv in 10 mv increments from a holding potential of -120 mv .
the voltage protocol used to assess pharmacological block of voltage - gated sodium channels consisted of a voltage step to -85 mv for 8 seconds , a subsequent voltage step to -120mv for 100 millisecond followed by a test voltage at 0 mv for 20 ms , from a holding potential of -120 mv ( fig .
action potentials were recorded by holding the membrane potential at -60 mv and injecting increasing currents to elicit action potentials .
whole - cell patch - clamp data were analyzed using clampfit 10 ( molecular devices ) and origin 8.1 ( originlab ) .
tests for statistical significance were performed using student t test or nonparametric anova as noted . for calcium imaging studies ,
differentiated cells at 3 - 4 weeks following addition of growth factors were loaded with calcium 5 dye ( molecular devices ) for 1 hour at 37c/5% co2 .
calcium 5 dye was added at 2 stock to 100l dmem / f12 1:1 medium in well .
calcium flux was monitored using the bd pathways 855 bioimager platform to quantify calcium flux at the single cell level . to monitor stimulation by capsaicin or ,-methyleneatp ,
5 basal images were taken , then compounds were added at 10 directly to medium in well to give the final stimulation concentration ( capsaicin 1m , ,-methyleneatp 30m ) .
calcium flux was monitored for 30 seconds , with 0.3s exposure time and 0.25s delay between exposures . in a subset of wells ,
kcl ( 20 mm ) was added as a second stimulation to confirm the neural identity of responsive cells . for experiments using the selective p2rx3 antagonist a-317491 ,
compound was added to wells 15 minutes prior to calcium imaging , and then the protocol was followed as above . following data collection ,
segmentation was carried out on one control image , and held constant across all wells and images in each experiment .
calcium flux was determined by subtracting the mean basal intensity value per cell from the peak intensity following stimulation to determine the change in intensity for each individual cell .
hesc lines ( wa-09 , passages 32 - 50 ; shef1 , ) and hipsc lines ( c14 , c72 ; passages 10 - 20 ) were cultured with mouse embryonic fibroblasts ( mefs , globalstem ) pre - plated at 12 - 15,000 cells / cm .
medium containing dmem / f12 , 20% knockout serum replacement , 1 mm l - glutamine , 100 m mem non - essential amino acids , and 0.1 mm -mercaptoethanol was made .
6ng / ml fgf-2 was added after sterile filtration and cells were fed daily and passaged weekly using 6u / ml dispase .
briefly , cells were rendered to single cells using accutase plated on gelatin for 30 minutes to remove mefs .
non - adherent cells were collected and plated on matrigel treated dishes at a density of 20 - 40,000 cells / cm in the presence of mef - conditioned hesc media containing 10 ng / ml fgf-2 and 10 m y-27632 .
neural differentiation was initiated when the cells were confluent using ksr media containing 820 ml of knockout dmem , 150 ml knockout serum replacement , 1 mm l - glutamine , 100 m mem non - essential amino acids , and 0.1 mm -mercaptoethanol .
to inhibit smad signaling , 100 nm ldn-193189 and 10 m sb431542 were added on days 0 through 5 .
cells were fed daily , and n2 media was added in increasing 25% increments every other day starting on day 4 ( 100% n2 on day 10 ) .
nociceptor induction was initiated with the addition of the three inhibitors 3 m chir99021 , 10 m su5402 , 10 m dapt on days 2 through 10 .
cell passage to lower density can promote maturation of sox10 + progenitors , and long - term culture media consisted of n2 containing 25ng / ml human - b - ngf , bdnf , and gdnf .
cells were fixed with 4% paraformaldehyde for 20 minutes , washed with pbs , permeabilized using 0.5% triton in pbs , and blocked using 1% bsa in pbs . for glutamate staining , .05% glutaraldehyde was added to the fixative .
primary antibodies used for microscopy included pax6 ( covance ) , tuj1 ( covance ) , ki67 ( sigma ) , isl1 ( dshb , abcam ) , brn3a ( millipore ) , ret ( r&d ) , runx1 ( sigma ) , glutamate ( sigma ) , peripherin ( santa cruz ) , trpv1 ( neuromics ) , substance p ( neuromics ) , cgrp ( neuromics ) , sox10 ( santa cruz ) . for flow cytometry , cells were fixed using the bd cytofix / cytoperm kit ( bd ) , including the optional 4% paraformaldehyde fixation step .
primary conjugated antibodies for flow cytometry were ntrk1-apc ( r&d ) , nestin - alexa647 ( bd pharmingen ) , tuj1-alexa488 ( bd pharmingen ) .
total rna was isolated at days 2 , 3 , 5 , 7 , 9 , and 15 of differentiation of lsb or lsb3i treated hpscs using trizol ls .
all samples were processed by the mskcc genomics core facility and hybridized to the illumina human 12 oligonucleotide array .
normalization and model - based expression measurements were calculated using the illumina analysis package ( lumi ) from the bioconductor project ( www.bioconductor.org ) within the statistical programming language r ( http://cran.r-project.org/ ) .
pair - wise comparison cut - off was significant if the multiple test corrected p - value was < 0.05 .
patch - clamp experiments were performed in whole - cell configuration at room temperature ( 20 - 22c ) using a multiclamp 700b patch - clamp amplifier controlled by pclamp 10 software ( molecular devices ) .
patch pipettes had resistances between 1.5 and 2 m. extracellular solution contained ( mm ) : 140 nacl , 4.7 kcl , 2.5 cacl2 , 1.2 mgcl2 , 10 hepes and 10 glucose ; ph was adjusted to 7.4 with naoh .
the intracellular ( pipette ) solution for voltage - clamp contained ( mm ) 100 csf , 45 cscl , 10 nacl , 1 mgcl2 , 10 hepes , and 5 egta ; ph was adjusted to 7.3 with csoh . for current - clamp
the intracellular ( pipette ) solution contained ( mm ) : 130 kcl , 1 mgcl2 , 5 mgatp , 10 hepes , and 0.5 egta ; ph was adjusted to 7.3 with koh .
series resistance was compensated by 80 - 90% to reduce voltage errors , however space - clamp artifacts occurred due to the presence of processes emanating from the cell bodies .
a current - voltage protocol was applied using a 20 ms pulse from -120 mv to + 50 mv in 10 mv increments from a holding potential of -120 mv .
the voltage protocol used to assess pharmacological block of voltage - gated sodium channels consisted of a voltage step to -85 mv for 8 seconds , a subsequent voltage step to -120mv for 100 millisecond followed by a test voltage at 0 mv for 20 ms , from a holding potential of -120 mv ( fig .
action potentials were recorded by holding the membrane potential at -60 mv and injecting increasing currents to elicit action potentials .
whole - cell patch - clamp data were analyzed using clampfit 10 ( molecular devices ) and origin 8.1 ( originlab ) .
tests for statistical significance were performed using student t test or nonparametric anova as noted .
for calcium imaging studies , differentiated cells at 3 - 4 weeks following addition of growth factors were loaded with calcium 5 dye ( molecular devices ) for 1 hour at 37c/5% co2 .
calcium 5 dye was added at 2 stock to 100l dmem / f12 1:1 medium in well .
calcium flux was monitored using the bd pathways 855 bioimager platform to quantify calcium flux at the single cell level . to monitor stimulation by capsaicin or ,-methyleneatp ,
5 basal images were taken , then compounds were added at 10 directly to medium in well to give the final stimulation concentration ( capsaicin 1m , ,-methyleneatp 30m ) .
calcium flux was monitored for 30 seconds , with 0.3s exposure time and 0.25s delay between exposures . in a subset of wells ,
kcl ( 20 mm ) was added as a second stimulation to confirm the neural identity of responsive cells . for experiments using the selective p2rx3 antagonist a-317491 ,
compound was added to wells 15 minutes prior to calcium imaging , and then the protocol was followed as above . following data collection ,
segmentation was carried out on one control image , and held constant across all wells and images in each experiment .
calcium flux was determined by subtracting the mean basal intensity value per cell from the peak intensity following stimulation to determine the change in intensity for each individual cell .
| there has been considerable progress in identifying signaling pathways directing the differentiation of human pluripotent stem cells ( hpscs ) into specialized cell types including neurons . however , extrinsic factor - based differentiation of hpscs is a slow , step - wise process mimicking the protracted timing of normal human development.using a small molecule screen we identified a combination of five small molecule pathway inhibitors sufficient to yield hpsc - derived neurons at > 75% efficiency within 10 days of differentiation .
the resulting neurons express canonical markers and functional properties of human nociceptors including ttx - resistant , scn10a - dependent sodium currents and response to nociceptive stimuli including atp and capsaicin .
neuronal fate acquisition occurs three - fold faster than during in vivo1 development suggesting that use of small molecule pathway inhibitors could develop into a general strategy for accelerating developmental timing in vitro .
the quick and high efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain . |
adamantinoma of the tibia is a rare slow - growing primary tumor of the bone .
there has been much controversy on the origins of this tumor with one major focus being the relationship between osteofibrous / fibrous dysplasia and adamantinoma .
there have been approximately 300 cases of tibial adamantinoma reported in the published english language literature . of these cases , 1520%
the most common site of metastasis is to the lungs . as metastases to the regional lymph nodes
are rare , there is limited information regarding the best practice plan for their management in the literature .
we report 2 cases of tibial adamantinoma , which were problematic from a diagnostic point of view .
case 2 is of a tibial adamantinoma that presented with groin metastases which was initially diagnosed as a cutaneous adnexal carcinoma .
a review of inguinal nodal metastatic adamantinoma is outlined in addition to the clinic - pathological data on these cases .
in 2007 , a 29-year - old female presented with a swelling in her left inguinal area .
investigations including a computed tomography ( ct ) scan showed an extremely large heterogeneous , hyperdense , lobular lymph node centered in the left inguinal region measuring 6.54.6 cm .
histopathological examination revealed the presence of a malignant neoplasm that was composed of sheets of basaloid cells with round vesicular nuclei and minimal cytoplasm .
this lesion was interpreted as a cutaneous eccrine carcinoma ( figure 1a c ) . due to the unusual diagnosis ,
an external consultation and a request for additional clinical history were obtained . on detailed review ,
the pathological report was reinterpreted as metastatic adamantinoma in conjunction with a confirmed past clinical history of surgical excision of a left tibial adamantinoma three years previously in 2004 .
the specimen consisted of a firm rubbery mass of fibro - fatty tissue that measured 18.511.37.2 cm with an ellipse of skin measuring 820.3 cm .
the cut surface revealed a multi - lobulated light brown well - circumscribed mass that measured 10.475.5 cm . on microscopic examination ,
the neoplastic cells were composed of predominantly basaloid and tubular nests of malignant cells with some peripheral palisading ( figure 1d f ) .
the histomorphological appearance of these cells was identical to the previous histology in this case ( figure 1a c ) confirming the presence of inguinal lymph nodal metastases from adamantinoma of the tibia .
biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) .
lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) .
the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) .
biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) .
lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) .
the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . in april 2009 ,
a new node along the left external iliac in the retroperitoneum was detected and biopsied under radiological guidance .
histopathological examination showed similar findings confirming the presence of metastatic adamantinoma ( figure 2a and b ) .
ct scans of the chest showed the presence of two nodules in the right lung , which remained stable on follow up over the following year .
follow up in early 2011 shows stable residual disease with no additional findings of note .
photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm .
the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 .
photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm .
the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 . in 1999 , a 58-year - old female presented with a painful swelling on the mid shaft of her left tibia .
histopathological examination showed the presence of islands of woven bone in a background of bland spindled fibrous tissue , which was interpreted as fibrous dysplasia ( figure 3a and b ) .
nine years later , in 2008 , she was seen for a painful swelling in the same region , which had been increasing in size since her previous operation .
a wide excision of the lesion with a tibial allograft and intramedullary nail fixation was performed in october 2009 .
there was a predominant fibrous dysplasia like pattern consisting of cellular bland , spindled fibroblastic proliferation with scattered islands of woven bone . embedded within the spindle cells were occasional clusters of plump epitheliod appearing cells that had a corded arrangement in focal regions ( figure 3c ) .
immunohistochemistry confirmed the epithelial nature of these cells with strong positive staining to pankeratin antibodies ( figure 3d ) .
such areas were not identified even on retrospective detailed examination of the previous biopsy from 1999 .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) .
medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma .
( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) .
medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma .
( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) .
the patient underwent revision surgery for non - union of the left tibia in 2010 .
in 2007 , a 29-year - old female presented with a swelling in her left inguinal area .
investigations including a computed tomography ( ct ) scan showed an extremely large heterogeneous , hyperdense , lobular lymph node centered in the left inguinal region measuring 6.54.6 cm .
histopathological examination revealed the presence of a malignant neoplasm that was composed of sheets of basaloid cells with round vesicular nuclei and minimal cytoplasm .
this lesion was interpreted as a cutaneous eccrine carcinoma ( figure 1a c ) . due to the unusual diagnosis ,
an external consultation and a request for additional clinical history were obtained . on detailed review ,
the pathological report was reinterpreted as metastatic adamantinoma in conjunction with a confirmed past clinical history of surgical excision of a left tibial adamantinoma three years previously in 2004 .
the specimen consisted of a firm rubbery mass of fibro - fatty tissue that measured 18.511.37.2 cm with an ellipse of skin measuring 820.3 cm .
the cut surface revealed a multi - lobulated light brown well - circumscribed mass that measured 10.475.5 cm . on microscopic examination ,
the neoplastic cells were composed of predominantly basaloid and tubular nests of malignant cells with some peripheral palisading ( figure 1d f ) .
the histomorphological appearance of these cells was identical to the previous histology in this case ( figure 1a c ) confirming the presence of inguinal lymph nodal metastases from adamantinoma of the tibia .
biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) .
lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) .
the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) .
biopsy of left inguinal mass ( a , b , c ) and left inguinal lymphadenopathy ( d , e , f ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a malignant neoplasm in the subcutaneous fat ( a ) .
lesional cells are composed predominantly of basaloid cells with round vesicular nuclei and minimal cytoplasm as seen at medium power ( b ) ( lens objective 4 ) and at high power ( c ) ( lens objective 10 ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of a metastatic malignant neoplasm in the lymph node ( ) ( d ) .
the neoplastics cells were composed of basaloid and tubular nests of malignant cells with some peripheral palisading as seen at medium power ( e ) ( lens objective 4 ) and at high power ( f ) ( lens objective 10 ) . in april 2009 ,
a new node along the left external iliac in the retroperitoneum was detected and biopsied under radiological guidance .
histopathological examination showed similar findings confirming the presence of metastatic adamantinoma ( figure 2a and b ) .
ct scans of the chest showed the presence of two nodules in the right lung , which remained stable on follow up over the following year .
follow up in early 2011 shows stable residual disease with no additional findings of note .
photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm .
the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 .
photomicrographs of hematoxylin and eosin stained slide at low power ( a ) ( lens objective 2 ) shows the presence of a malignant neoplasm .
the neoplastic cells ( b ) at high power ( lens objective 10 ) shows a similar morphology of basaloid cells as seen in figure 1 .
in 1999 , a 58-year - old female presented with a painful swelling on the mid shaft of her left tibia .
histopathological examination showed the presence of islands of woven bone in a background of bland spindled fibrous tissue , which was interpreted as fibrous dysplasia ( figure 3a and b ) .
nine years later , in 2008 , she was seen for a painful swelling in the same region , which had been increasing in size since her previous operation .
a wide excision of the lesion with a tibial allograft and intramedullary nail fixation was performed in october 2009 .
there was a predominant fibrous dysplasia like pattern consisting of cellular bland , spindled fibroblastic proliferation with scattered islands of woven bone . embedded within the spindle cells were occasional clusters of plump epitheliod appearing cells that had a corded arrangement in focal regions ( figure 3c ) .
immunohistochemistry confirmed the epithelial nature of these cells with strong positive staining to pankeratin antibodies ( figure 3d ) .
such areas were not identified even on retrospective detailed examination of the previous biopsy from 1999 .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) .
medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma .
( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) .
photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of islands of woven bone amidst a background of bland fibrous spindle cells ( a ) .
medium power examination ( b ) ( lens objective 4 ) shows the presence of a rich fibro - collagenous stroma .
( c and d ) 2009 histopathological examination of the tibial lesion photomicrographs of hematoxylin and eosin stained slides at low power ( lens objective 2 ) shows the presence of occasional clusters of plump epitheliod appearing cells with a corded arrangement in focal regions amidst a fibrous spindle background as seen ( c ) which are strongly positive to pankeratin antibodies on immunohistochemical examination ( d ) .
the patient underwent revision surgery for non - union of the left tibia in 2010 .
a literature search limited to the english language using the search terms adamantinoma of tibia and tibial adamantinoma and metastases and inguinal lymph node metastases was conducted .
research engines used included pubmed , medline , scopus , embase , and google / google scholar .
table 1 provides a comprehensive chronological review of 10 cases with inguinal lymph node metastases with details of reference number , year reported , author , sex , age at diagnosis , bone involved , location in bone , character and duration of symptoms + / history of trauma , latent period , treatment , and outcome .
, six cases with lymph node metastases as described in keeney 's study are not included in this table due to the lack of supporting clinical information .
similarly , cases with inguinal lymph node metastases as described in moon 's review have been excluded from this table as the original reports are not in english .
table 1inguinal lymph node metastases from adamantinoma of the tibia as reported in the english literature ( pub med , medline , scopus , embase , google / google scholar ) since 1930-search terms included adamantinoma of the tibia , and tibial adamantinoma and metastases , and inguinal lymph node metastases.reference numberyear reportedauthorsexage at diagnosisbone involvedlocation in bonecharacter and duration of symptoms + /history of traumalatent periodtreatmentoutcome#101938dunnem32left tibiaupper and middle thirdsswelling , 4yr ; pain later months severe contusionnine monthshigh voltage roentgen , ak amputation 9 months laterdied , 8 years inguinal lymph nodes*#111942dockerty , meyerdingf24left tibiamiddlerecurrent pain , 8 yr . , swelling , 4yr .
no history of traumanonelocal excision , ak amputation 15 months laterdied , 1 year , inguinal lymph node metastases#121952mangalik , lal mehrotram40femur , tibia , fibuladistal proximal middleduration of symptoms for 12 months .
previous injury to leg1 yearak amputation , irradiation of metastatic nodes#131962knappf14left tibiamiddlemass for 4 years no history of traumaak amputationtotal survival 16 years .
pulmonary and inguinal lymph node metastasis#141976winter wgm30left tibiadistalnoneresection , disarticulation of lk , inguinal node dissection , pleurectomydied with inguinal nodes , pulmonary , brain - right occipital metsm37tibiamiddlenoneresection and bone grafting very late knee disarticulationdied , 8 years after initial surgery inguinal lymph nodes and pulmonary metastasis#151990de keyserf13tibiapathological fractureintramedullary nailing several curettages , en bloc resection bk amputation , ak amputationdied 4 years after ak amputation had pulmonary and lymph node metastasis#61994hazelbagm26left tibiadistalnoneamputation metastasis to groin lymph node , pelvisdied of diseasem15left tibiadistalnonechemotherapy , amputationdied of disease .
lung / lymph metastases#162008freym20tibianadnadtotal tibia allograft knee disarticulation polychemotherapypulmonary and inguinal lymph node metastases amputation2011tharmabala et al.f24left tibiaswelling left tibia swelling in left groin4 yearssurgical excision in left tibia 2004 .
left retroperitoneal mass excision 2009left inguinal nodal metastasis , stable pulmonary nodules with routine follow up in 2011*this is reported in reference # 17 by moon in 1965 .
it was first described by maier in 1900 as a primary bone tumor with epithelial characteristics . later in 1913 , fischer named the tumor primary adamantinoma of the tibia because of its resemblance to adamantinoma ( also known as ameloblastoma ) of the jaw and the pituitary gland .
although , adamantinoma of the tibia histologically resembles adamantinomas of the jaw and pituitary , there has been no evidence to suggest that these tumors are of similar origin .
adamantinoma has also been associated with osteofibrous / fibrous dysplasia and in this regard is considered by some authors to represent a spectrum of disease with evidence of progression from osteofibrous / fibrous dysplasia to adamantinoma in some cases . due to the rarity of this lesion and the varied histological phenotypes , adamantinoma of the tibia
can be a diagnostic dilemma leading to misdiagnosis , delay , and suboptimal treatment , as seen in our cases .
it can present at any age but mostly occurs in the second to fourth decade .
they are most commonly ( 8590% ) localized in the tibial diaphysis rarely , it has been described in other bones of the appendicular skeleton including the fibula , ulna , femur , humerus , radius , and short bones of the feet .
, , adamantinoma of the tibia can be divided into two main groups based on their histology and biological behavior .
classical adamantinoma refers to the prototype cases with a predominance of epithelial tumor cells and are associated with a destructive growth pattern on radiology .
patients with classical adamantinoma are usually of an older age group with a reported range of 1565 years .
this variant of classical adamantinoma has a stronger predisposition to local recurrences and a higher potential for distant metastases .
in contrast , differentiated adamantinoma of the tibia has a predominance of a osteofibrous / fibrous dysplasia like pattern with scant epithelial tumor elements and is commonly seen in an intracortical location .
many authors suggest that osteofibrous/ fibrous dysplasia , a benign lesion occurring commonly in the tibia of children under 10 years of age , is related to adamantinomas and may even be a precursor lesion for adamantinomas .
, in such scenarios , tibial adamantinomas have often been misdiagnosed as fibrous dysplasia , as seen in our case .
the association of fibrous dysplasia with adamantinoma was first described by cohen et al . in 1962 who proposed that the fibrous pattern seen in such cases was different from that seen in classical fibrous dysplasia .
this variant of differentiated adamantinoma is considered by some authors as a secondary reparative process that is associated with a more benign behavior and better prognosis .
distant metastases have been reported in adamantinoma of the long bones in about 1520% of cases this may be falsely low due to the slow growing nature of the tumor as long term follow up is required to evaluate its true metastatic potential .
regional or other distant metastases as seen in the lung are found more frequently in patients who have a history of repeated local recurrences linked to inadequate primary excision of this lesion .
however , in our case ( case 1 ) inguinal nodal metastases occurred with no evidence of local recurrence .
risk factors for metastases include male sex , persistent pain , symptoms of less than five years duration , and inadequate initial treatment by biopsy curettage / excision , or resection .
the only histological feature associated with an increased recurrence rate was lack of squamous differentiation . in view of the relationship between adamantinoma and osteofibrous dysplasia , czerniak et al . proposed two biological courses that a tibial adamantinoma may follow .
one is by the differentiated adamantinomas , which are predominantly intracortical and behave as a reparative process with a low potential to metastasize .
the second , such as that seen in our case ( case # 2 ) of classical adamantinoma , is one of progressive tumor growth with a higher potential to metastasize .
early metastases within the first two years of initial diagnosis is commonly seen to occur to the regional lymph nodes , whereas delayed metastases is almost always pulmonary or osseous .
given that lymph node metastasis usually occurs within the first two years , this is suggestive that these were present at the time of initial diagnosis . in this context , we recommend ultrasound imaging of regional lymph nodes as an essential component in the follow - up examinations of these cases .
the current practice recommends surgical extirpation as the primary mode of treatment for localized regional lymph node metastases with radiotherapy and chemotherapy as suggested alternatives .
the exact role for sentinel node biopsy and its cost / benefit ratio is unknown due to limited clinical experience in these rare lesions . in conclusion ,
tibial adamantinoma is a rare primary bone tumor that can cause diagnostic dilemmas especially with clinical presentations of the uncommon pattern of regional nodal metastases .
increased clinical awareness of such rare neoplasms with recognition of uncommon patterns of metastases is essential for accurate diagnosis and optimal patient management .
additionally , due to the slow - growing nature of tibial adamantinomas we recommend continuous long - term follow up for these patients . | adamantinoma is a rare primary bone tumor that commonly arises in the jaw and has also been described in the appendicular skeleton such as the tibia .
we report 2 cases of tibial adamantinomas that were originally misdiagnosed ; one as fibrous dysplasia of the tibia and the other as a cutaneous eccrine carcinoma in a groin mass , which was metastatic adamantinoma to the inguinal lymph nodes .
such metastatic adamantinoma to the groin lymph nodes is extremely rare .
the clinical and pathological data with a review of the available literature on inguinal lymph node metastases from primary tibial adamantinoma are reported .
increased clinical awareness and accurate recognition of such uncommon patterns of inguinal nodal metastases are imperative for appropriate planning of therapeutic strategies and risk management in these patients . |
granulomatous lobular mastitis ( glm ) is a rare inflammatory disease of the breast that was first described by kessler and wolloch in 1972 .
this disease usually affects women of childbearing age or those with a history of oral contraceptive use .
although glm is a benign condition , its clinical and radiological features may mimic breast cancer .
the diagnosis is made only after excluding breast cancer and other infective and non - infective causes of granulomatous inflammation , such as tuberculous , parasitic and fungal infections , sarcoidosis , wegener s granulomatosis , giant cell arteritis , polyarteritis nodosum , and foreign - body reactions .
the treatments for glm include corticosteroids , immunosuppressants , antibiotics , abscess drainage , and surgical excision .
although a preferred therapeutic modality has not been established because of the rarity of this disease , corticosteroid therapy has been shown to be effective for shrinking the granulomatous mass . recently , ogura et al .
categorized glms into two groups : immunoglobulin g ( igg ) 4-related glms and non - igg4-related glms .
they speculated that patients with igg4-related glm might benefit from steroid therapy , as do those with igg4-related autoimmune disorders .
however , the effectiveness of steroid therapy for patients with non - igg4-related glm is unknown .
we evaluated the effectiveness of corticosteroid treatment for non - igg4-related glms and discussed the strategy of the treatment for glm .
we performed a retrospective study of the records from nagoya university hospital and kato surgery - gynecology - obstetrics - breast clinic for patients treated between october 2005 and october 2012 . during this period ,
all of the women underwent a clinical breast examination to identify palpable lumps , skin thickening , or axillary lymphadenopathy .
mammography ( mmg ) and ultrasonography ( us ) were the initial imaging methods used for evaluation , although mmg was not performed in one case due to pain caused by drainage of the sinus tracts .
additional radiological examinations , such as computed tomography ( ct ) and magnetic resonance imaging ( mri ) , were performed as needed .
definitive diagnosis was obtained by core needle biopsy ( 14-gauge ) , vacuum - assisted biopsy ( 11-gauge ) , or surgical excision .
the obtained specimens were fixed overnight in 10% formalin , and the residual tissues were cultured for bacteria .
paraffin - embedded specimens were stained with hematoxylin and eosin ( he ) , ziehl - neelsen , and the periodic acid - schiff reaction with or without diastase digestion .
other possible causes of granulomatous inflammatory lesions of the breast were excluded by serological tests , histological analysis , and tests of the affected tissue for aerobic and anaerobic bacteria , mycobacteria , and fungi .
the expression levels of igg4 and igg in the glm lesion were determined immunohistochemically in the paraffin - embedded tissue specimens .
the primary antibodies used were an anti - igg mouse monoclonal antibody ( 1:2 , histofine , nichirei bioscience , tokyo , japan ) and an anti - igg4 sheep polyclonal antibody ( 1:500 , the binding site , birmingham , uk ) . before incubation with the primary antibodies , antigen retrieval ( microwave pretreatment for 10 min at 100c in ph 6 citrate buffer )
igg+ or igg4 + plasma cells were counted in high power fields ( hpf , magnification 300 ) with intense inflammation , and 4 fields were analyzed per case .
the ratio ( percentage ) of igg4 + plasma cells / igg+ plasma cells was calculated in each case .
previous studies had shown that an elevated igg4 + to igg+ plasma cell ratio ( igg4/igg ratio ) is helpful in distinguishing igg4-related from non - igg4-related inflammatory conditions .
those studies revealed that most patients with igg4-related disease had an igg4+/igg+ plasma cell ratio of more than 30% .
in contrast , the ratio was less than 10% in cases of non - igg4-related disease .
therefore , the diagnosis of igg4-related disease was made in patients having characteristic h&e findings , diffuse igg4 + plasma cell infiltration , and an igg4+/igg+ cell ratio greater than 30% .
seven patients were diagnosed within 5 years of their most recent pregnancy , 2 patients had not been pregnant within 5 years prior to the diagnosis , and 1 patient did not list the date of her most recent childbirth .
the residual 2 patients who had not experienced childbirth were taking psychotropic medications , such as risperidone or aripiprazole .
the common presenting symptoms were breast pain ( 11/12 , 92% ) and breast masses ( 9/12 , 75% ) . the lesions detected as palpable masses or indurations ranged from 2.4 to 10.0 cm in size ( median size of lesion ; 6.0 cm ) .
four lesions were located at the periphery of the breast ( 4/12 , 33.3% ) , and a residual lesion was in a subareolar location ( 1/12 , 8.3% ) .
none of the women had any systemic disorder or history of a specific granulomatous infection .
d / m : diffuse and/or multiple ; l : localized ; s : subareolar location ; v : vacuum - associated biopsy ; c : core needle biopsy ; and e : excisional biopsy .
diagnosis of glm was established histologically in 9 patients by a core needle biopsy , in 2 patients by a vacuum - assisted biopsy , and in one patient by an excisional biopsy .
all patients showed non - caseating granulomas involving the mammary lobules , with variable numbers of langhans - type multinucleated giant cells and inflammatory infiltrates such as neutrophils , polymorphs , lymphocytes , plasma cells , and eosinophils ( fig .
specific staining for tuberculosis ( ziehl - neelsen ) and fungal infections ( periodic acid - schiff ) were all negative .
the representative images of immunohistochemical staining for igg and igg4 are shown in figure 2a and 2b , respectively .
histological findings showing granulomatous inflammation of the lobules without caseation necrosis ( 1a , magnification 40 and the inserted bar 250m ) and the presence of multinucleated giant cells ( langhans cells ) and plasma cells ( 1b , magnification 400 and the inserted bar 25m ) .
a ) immunohistochemical findings for igg expression in the plasma cells from patients with glm ( magnification 300 and the inserted bar 25m ) .
b ) immunohistochemical findings for igg4 expression in the plasma cells from patients with glm ( magnification 300 and the inserted bar 25m ) .
eleven of the 12 patients had been administered antibiotics , but their glms had not improved . after the initial treatment with antibiotics ,
the median initial dosage was 30 mg / day ( range : 1560 mg / day ; mean weight were 63.8 kg ) , and the initial dosage was tapered weekly or biweekly according to clinical and image improvements .
the median duration of corticosteroid treatment was 5 months ( range : 112 months ) . although the corticosteroid dosage was increased in 6 of 10 patients because the focus sizes of their glms increased during treatment , all 10 cases eventually improved with the corticosteroid treatment .
findings showed residual glm lesions , the steroid treatment was terminated if the glms became smaller and did not cause pain .
complications due to steroid treatment were observed in only one patient who developed a moon face and acne .
surgical excision was performed for 1 patient with peripheral , localized disease , and another patient was treated with incision and drainage alone .
the therapeutic durations of the two patients were 5 months after local excision and 4 months after incision and drainage , respectively .
because these patients refused steroid treatment , they did not receive corticosteroids after the recurrence .
in contrast , no patient receiving the corticosteroid treatment experienced a recurrence during the follow - up period .
seven patients were diagnosed within 5 years of their most recent pregnancy , 2 patients had not been pregnant within 5 years prior to the diagnosis , and 1 patient did not list the date of her most recent childbirth .
the residual 2 patients who had not experienced childbirth were taking psychotropic medications , such as risperidone or aripiprazole .
the common presenting symptoms were breast pain ( 11/12 , 92% ) and breast masses ( 9/12 , 75% ) . the lesions detected as palpable masses or indurations ranged from 2.4 to 10.0 cm in size ( median size of lesion ; 6.0 cm ) .
four lesions were located at the periphery of the breast ( 4/12 , 33.3% ) , and a residual lesion was in a subareolar location ( 1/12 , 8.3% ) .
none of the women had any systemic disorder or history of a specific granulomatous infection .
d / m : diffuse and/or multiple ; l : localized ; s : subareolar location ; v : vacuum - associated biopsy ; c : core needle biopsy ; and e : excisional biopsy .
diagnosis of glm was established histologically in 9 patients by a core needle biopsy , in 2 patients by a vacuum - assisted biopsy , and in one patient by an excisional biopsy .
all patients showed non - caseating granulomas involving the mammary lobules , with variable numbers of langhans - type multinucleated giant cells and inflammatory infiltrates such as neutrophils , polymorphs , lymphocytes , plasma cells , and eosinophils ( fig .
specific staining for tuberculosis ( ziehl - neelsen ) and fungal infections ( periodic acid - schiff ) were all negative .
the representative images of immunohistochemical staining for igg and igg4 are shown in figure 2a and 2b , respectively .
histological findings showing granulomatous inflammation of the lobules without caseation necrosis ( 1a , magnification 40 and the inserted bar 250m ) and the presence of multinucleated giant cells ( langhans cells ) and plasma cells ( 1b , magnification 400 and the inserted bar 25m ) .
a ) immunohistochemical findings for igg expression in the plasma cells from patients with glm ( magnification 300 and the inserted bar 25m ) .
b ) immunohistochemical findings for igg4 expression in the plasma cells from patients with glm ( magnification 300 and the inserted bar 25m ) .
eleven of the 12 patients had been administered antibiotics , but their glms had not improved . after the initial treatment with antibiotics , 10 patients received corticosteroid treatment . the initial dosage of corticosteroid ( prednisolone ) was determined by the physician .
the median initial dosage was 30 mg / day ( range : 1560 mg / day ; mean weight were 63.8 kg ) , and the initial dosage was tapered weekly or biweekly according to clinical and image improvements .
the median duration of corticosteroid treatment was 5 months ( range : 112 months ) . although the corticosteroid dosage was increased in 6 of 10 patients because the focus sizes of their glms increased during treatment , all 10 cases eventually improved with the corticosteroid treatment .
findings showed residual glm lesions , the steroid treatment was terminated if the glms became smaller and did not cause pain .
complications due to steroid treatment were observed in only one patient who developed a moon face and acne .
surgical excision was performed for 1 patient with peripheral , localized disease , and another patient was treated with incision and drainage alone .
the therapeutic durations of the two patients were 5 months after local excision and 4 months after incision and drainage , respectively .
because these patients refused steroid treatment , they did not receive corticosteroids after the recurrence .
in contrast , no patient receiving the corticosteroid treatment experienced a recurrence during the follow - up period .
we investigated the involvement of igg4 in glm and found that there was no patient with igg4-related glm in our study .
.
demonstrated that igg4 positive cells were scarce in 6 of 7 cases of glm .
although all 12 patients had non - igg4-related glm in our study , 10 patients were treated with corticosteroids and all of them improved .
it should be noted that steroid treatment was effective for non - igg4-related glms .
kok et al .
reported that only 17% of patients ( 4 of 23 cases ) in their study had been diagnosed with glm using fine needle aspiration cytology ( fnac ) .
because the definitive diagnosis of glm requires enough specimens to rule out breast cancer , a core needle biopsy or vacuum - assisted biopsy should be necessary .
the optimum initial dosage has not been established ; however , in this study , 5 of the 10 patients were administered 30 mg of prednisolone for several weeks , which was then tapered weekly or biweekly . although long therapeutic periods were necessary , all patients improved and did not experience recurrence after completion of the steroid treatment .
mg / kg / day according to the severity of clinical symptoms during 4.5 1.3 ( sd ) months and demonstrated a significant decrease in the number of recurrences after prednisone treatment compared with that before treatment .
in addition , hovanessian et al .
showed that treatment of glm with corticosteroids before surgical management appears to be beneficial , with 77% of patients showing improvement .
in contrast , no patient who received corticosteroid treatment experienced recurrence during the follow - up period .
this result suggests that the effectiveness of the corticosteroid treatment may be greater than that of surgical treatment . because most cases involved large , diffuse , or multiple lesions , the cosmetic result after the surgical treatment should be considered .
recommended a wide surgical excision or mastectomy for glm with persistent symptoms because of a lower rate of recurrence . in this case , a delay of subsequent plastic surgical procedures for breast asymmetry might be necessary .
therefore , it would be appropriate to treat glm with a corticosteroid before excision , except in patients whose systemic complications are too severe to administer steroid therapy . in other report , it has been reported that medical treatment with corticosteroids provided significant regression of the inflammatory disease and allowed more conservative surgery .
several presumptive causes of glm , including alpha-1-antitrypsin deficiency , oral contraceptive usage , pregnancy , lactation , and hyperprolactinemia , have been reported .
it has been postulated that glm results from a localized autoimmune response to the retained or extravasated secretions in the breast ducts during the childbearing period due to previous hyperprolactinemia . because the occurrence of glm without a history of recent pregnancy is uncommon , the serum prolactin level might play an important role in the pathogenesis of glm in non - pregnant women .
lin et al .
reported that glm was associated with risperidone - induced hyperprolactinemia and discussed the development of glm associated with high serum prolactin levels in non - pregnant women . in our cases
, two non - pregnant women were administered antipsychotic drugs ; one woman had been treated with aripiprazole and presented a high level of serum prolactin ( 179.14 ng / m , normal range : 1.2029.93 ) and the other woman was not tested for serum prolactin levels but had received risperidone .
these findings support the argument that hyperprolactinemia , whether primary or secondary , might contribute to the pathogenesis of glm in non - pregnant women .
therefore , the roles of prolactin in the pathogenesis of glm should be studied in more detail . in summary
although corticosteroid treatment requires a lengthy therapeutic period , there was no recurrence in patients during observation .
however , if glm resists steroid treatment , other treatments , such as surgical excision or immunosuppressants , should be considered .
the authors thank dr . koji oda , department of breast oncology , aichi cancer center aichi hospital , and dr .
yukihiro yokoyama , division of surgical oncology , department of surgery , nagoya graduate school of medicine , for advice on our investigation . | abstractgranulomatous lobular mastitis ( glm ) is a rare inflammatory pseudotumor .
no therapeutic modality for this disease has been established because of its rarity .
the purpose of this study is to evaluate the treatment strategies of glm .
twelve women who met the histological criteria for glm were retrospectively studied .
the clinical data and the presentation , histopathology , and management of the disease were analyzed by reviewing the patients medical records .
the diagnosis of glm was confirmed histologically by core needle biopsy in 9 cases , by vacuum - assisted biopsy in 2 cases , and by excisional biopsy in 1 case .
ten patients received corticosteroid treatment and another two patients were treated with local excision or incision and drainage .
the median initial dosage of corticosteroid ( prednisolone ) was 30 mg / day ( range : 1560 mg / day ) , and the dosages were tapered according to improvement .
the median duration of corticosteroid treatment was 5 months ( range : 112 months ) .
the median follow - up period was 22 months ( range : 6104 months ) , and no patient treated with corticosteroid demonstrated recurrence .
however , patients treated with excision or incision and drainage had recurrences .
these results suggest that steroid treatment may be the first choice in treatment strategies for glm . |
brain computed tomography ( ct ) is commonly used to assess intracranial abnormalities in the emergency department .
possibilities that should be considered in the differential diagnosis of a hyperdense basal ganglion lesion on brain ct include benign calcification , hypertensive intracerebral hemorrhage ( ich ) , presence of a foreign body , tay - sachs disease , and metabolic diseases such as hyperglycemia .
the basal ganglion is a common anatomic site for hypertensive ich ; thus , emergency physicians are familiar with these ct findings .
several studies have reported patients with nonketotic hyperglycemia whose ct findings mimic those of patients with ich .
these patients mainly present with a contralateral movement disorder referred to as diabetic hemichorea - hemiballism ( hc - hb ) [ 1 - 4 ] .
patients with diabetic hc - hb usually present with involuntary , continuous unilateral movement abnormalities in both the proximal and distal extremities .
however , not all patients with hyperglycemia and a hyperdense basal ganglion lesion present with an abnormal movement disorder .
for instance , hansford et al . reported a patient with these imaging abnormalities without a movement disorder .
similarly , the present report details a patient with nonketotic hyperglycemia and a hyperdense basal ganglion lesion with an unusual clinical presentation .
a 77-year - old woman lost consciousness 6 hours before being admitting to a local clinic where she underwent brain ct .
the patient was promptly referred to our department with suspected basal ganglion ich based on the brain ct .
her premorbid eastern cooperative oncology group score was 3 due to dementia but she had normal cognitive functioning .
the patient had stopped taking her diabetic medication 2 months earlier , on her own volition , and had been suffering progressive debilitation .
the initial vital signs were blood pressure 180/98 mmhg , pulse rate 110/min , respiratory rate 18/min , and body temperature 36.8c .
a neurological examination revealed stupor , intact brainstem signs , and greater than grade iii motor strength in all four extremities without lateralizing signs .
the initial serum laboratory test results were blood glucose 607 mg / dl , hemoglobin 12.8 g / dl , white blood cells 6,720/mm , platelets 193,000/mm , aspartate aminotransferase 32 iu / l , alanine aminotransferase 38
iu / l , blood urea nitrogen 17 mg / dl , serum creatinine 1.2 mg / dl , sodium 120 meq / l , potassium 5.0 meq / l , chloride 89 meq / l , serum osmolarity 280 mosm , and lactate 12.5 mg / dl . no serum ethanol was detected , and an arterial blood gas analysis revealed the following : ph 7.454 , pco2 37.0 mmhg , po2 78.1 mmhg , hco3 25.4 mmol / l , and base excess 1.6 mmol / l . no ketones were detected in the urinalysis .
barbiturates , benzodiazepines , and tricyclic antidepressants were not found during a toxin - screening test .
the initial brain ct showed a hyperdense lesion in the right basal ganglion ( fig .
1 ) . on magnetic resonance imaging , t1-weighted images revealed high signal intensity indicative of changes due to nonketotic hyperglycemia ; diffusion weighted imaging ( dwi ) and an apparent diffusion coefficient map showed restricted diffusion ( fig .
blood glucose level was controlled with regular insulin , and conservative treatment and fluid resuscitation were provided .
electroencephalography revealed findings compatible with diffuse encephalopathy , but no signs of seizure were detected .
after admission to the intensive care unit , the patient gradually recovered consciousness and was alert after 24 hours , as blood glucose and sodium were controlled .
diabetic hc - hb is a rare complication of nonketotic hyperglycemia in which patients usually present with continuous unilateral involuntary movement .
the predominant ct findings are hyperattenuation of the putamen with or without hyperattenuation of the caudate nucleus , sparing the internal capsule .
magnetic resonance imaging findings include high signal intensity on t1-weighted images , variable signal intensity on t2-weighted images , and restricted diffusion on dwi [ 1,8 - 10 ] .
emergency physicians can misdiagnose this condition as acute ich if not familiar with these radiographic abnormalities , considering the acute nature of the clinical symptoms and similarities on ct findings .
acute basal ganglion hemorrhage usually involves peripheral edema and cerebral parenchymal displacement due to mass effect .
petechial hemorrhage was initially considered as the main mechanism underlying hyperdensity on ct and high signal intensity on t1-weighted images .
an alternative hypothesis is that hyperviscosity induced by hyperglycemia and vasogenic edema play a role in radiological abnormalities as they induce restricted diffusion on dwi and apparent diffusion coefficient maps .
similarly , hyperviscosity may cause partial ischemic injury to the striatum , which results in high signal intensity on t1-weighted images .
proposed that metabolic derangements related to hyperglycemia and cerebral vascular insufficiency constitute the main underlying mechanism .
they found that fluorodeoxyglucose uptake in the basal ganglion decreased based on results from a positron emission tomography analysis .
another possible hypothesis is that excess formation of gemistocytes , which are reactive swollen forms of astrocytes , result from ischemic changes and metabolic derangement . in line with this theory , shan et al .
found several gemistocytes in a stereotactic putamen biopsy specimen , claiming that these changes led to high signal intensity on t1-weighted images .
hence , while various hypotheses have been suggested , the main mechanisms underlying radiographic abnormalities remain unclear . with the exception of two documented patients , all patients with nonketotic hyperglycemia and radiographic abnormalities
have also presented with an involuntary movement disorder . specifically , hansford et al . reported a patient who complained of vague stroke - like symptoms , and hsu et al . reported a patient who had unilateral limb weakness , gait disturbance , and speech difficulties .
similar to these two cases , the present report observed the same radiographic abnormalities without a movement disorder , but the clinical presentation only included diminished consciousness .
although altered consciousness in a patient with hyperglycemia can be caused by increased serum osmolarity , electrolyte imbalance , or prolonged metabolic acidosis , such results did not apply to the present patient .
we concluded that the patient developed a form of metabolic encephalopathy , considering the electroencephalography results and gradual improvement in consciousness in accordance with corrected blood glucose and sodium .
the importance of this case is that radiographic abnormalities observed in a patient with diabetic hc - hb can also be observed in patients presenting only with altered consciousness .
although radiographic changes caused by nonketotic hyperglycemia are uncommon , emergency physicians should consider typical radiographic features .
in addition , emergency physicians should remember that clinical features likely involve various signs and symptoms apart from a movement disorder . | a 77-year - old woman was admitted to a local clinic for altered consciousness and presented with a suspected basal ganglion hemorrhage detected on brain computed tomography .
the patient was stuporous , but her vital signs were stable .
her initial blood glucose was 607 mg / dl , and a hyperdense lesion was found in the right basal ganglion on brain computed tomography .
t1-weighted magnetic resonance imaging revealed high signal intensity in the right basal ganglion .
electroencephalography showed no seizure activity .
the patient was treated with a fluid infusion , and serum glucose level was controlled with insulin .
the patient gradually recovered consciousness and was alert within 24 hours as serum glucose level normalized .
the basal ganglion lesion caused by hyperglycemia was not accompanied by involuntary limb movement .
this is the first report of a patient presenting with decreased consciousness and typical neural radiographic changes associated with nonketotic hyperglycemia but without movement abnormalities . |
all patients in this study had previously been published as cases of meier - gorlin syndrome or fulfilled diagnostic criteria for meier - gorlin syndrome ( microtia , absent / small patellae and short stature ) .
no cytogenetic abnormalites were observed on routine karyotyping of these meier - gorlin syndrome cases ( n=10 patients ) .
furthermore , no significant increase of either spontaneous , or radiation - induced chromosome breaks was observed ( 2 cases versus 2 control patients , 100 metaphases assessed per patient ; p 0.7 , x - test ) .
informed consent was obtained from all participating families and the studies were approved by the scottish multicentre research ethics committee ( 04:mre00/19 ) , regional committee on research involving human subjects nijmegen - arnhem ( 0006 - 0119 ) ; or university of texas southwestern medical center at dallas ( irb # 032008 - 066 ) .
coding exons and exon - intron boundaries of each gene were screened by bidirectional capillary sequencing on an abi 3730 gene sequencer .
sequence analysis and mutation detection performed using mutation surveyor v. 2.61 ( softgenetics llc ) .
amplicons were separated by capillary electrophoresis on an abi3730 sequencer , and allele sizes obtained using genemapper ( applied biosystems ) .
all patients in this study had previously been published as cases of meier - gorlin syndrome or fulfilled diagnostic criteria for meier - gorlin syndrome ( microtia , absent / small patellae and short stature ) .
no cytogenetic abnormalites were observed on routine karyotyping of these meier - gorlin syndrome cases ( n=10 patients ) .
furthermore , no significant increase of either spontaneous , or radiation - induced chromosome breaks was observed ( 2 cases versus 2 control patients , 100 metaphases assessed per patient ; p 0.7 , x - test ) .
informed consent was obtained from all participating families and the studies were approved by the scottish multicentre research ethics committee ( 04:mre00/19 ) , regional committee on research involving human subjects nijmegen - arnhem ( 0006 - 0119 ) ; or university of texas southwestern medical center at dallas ( irb # 032008 - 066 ) .
primers were designed using exonprimer ( supplementary table 2 ) . coding exons and exon - intron boundaries of each gene
sequence analysis and mutation detection performed using mutation surveyor v. 2.61 ( softgenetics llc ) .
amplicons were separated by capillary electrophoresis on an abi3730 sequencer , and allele sizes obtained using genemapper ( applied biosystems ) .
| meier - gorlin syndrome ( ear , patella , short stature syndrome ) is an autosomal recessive primordial dwarfism syndrome characterised by absent / hypoplastic patellae and markedly small ears1 - 3 .
both pre and post - natal growth are impaired in this disorder and although microcephaly is often evident , intellect is usually normal .
we report here that this disorder shows marked locus heterogeneity and we identify mutations in five separate genes : orc1 , orc4 , orc6 , cdt1 and cdc6 .
all encode components of the pre - replication complex , implicating defects in replication licensing as the cause of a genetic syndrome with distinct developmental abnormalities . |
currently , hypertension is increasingly growing in the most of developing countries due to rapid changes in lifestyle and the increase in urbanization stress ( 1 ) . as reported by world health statistics ( 2012 ) ,
28.1% of men and 25% of women suffer from systolic blood pressure ( bp ) more than 140 or diastolic one above 90 across the world .
such statistics are estimated 26.1% for males and 22.4% for females in iran . in this case , a growth of cardiovascular disease for about 3% , from 2002 to 2008 , has been reported in iran ( 2 ) , while the age of hypertension is decreasing . in iran , 4.7% of total
investigated 1318 years old students had a bp higher than normal level . furthermore , hypertension was more in females compared to males ( 3 ) . in a study , systolic bp of females was significantly higher than males ( 4 ) . in line with these findings , new views about the prevention of hypertension
emphasize on prevention and treatment of hypertension during the adolescence , since the risk factors for cardio - vascular disease are formed during adolescence period .
hence , it is reasonable to start a healthy lifestyle to improve health level from adolescence period ( 5 ) .
considering the fact that adolescents usually do not put any limitation for their nutrition and do not consider it , inappropriate nutrition is observed in this age group more than any other group ( 6 ) . in order to increase the impact of health education , community
one is the theory of planned behavior ( tpb ) which provides a systematic framework for the health education . according to this theory ,
the intention to conduct a behavior is related to an individual 's positive or negative evaluation of self - performance of the particular behavior ( attitude ) , an individual 's perception about the particular behavior , which is influenced by the judgment of significant others , such as parents , spouse , friends and , teachers ( subjective norms ) , and an individual 's perceived ease or difficulty of performing the particular behavior ( perceived behavioral control ) ( 8 , 9 ) .
this theory presents an appropriate framework to study behaviors associated with nutrition , such behaviors are those activities conducted to confront competitive motivations and twofold attitude , mentioning that they are influenced by social factors .
therefore , the theory that pays attention to the impact of attitudes , subjective norms , and perceived behavioral control is appropriate to conduct nutritional behaviors ( 10 ) . in a study investigating
the application of tpb in healthy nutritional behaviors in american adolescents , the tpb was effective to predict affecting factors of nutritional behaviors ( 11 ) .
all components of the tpb had a positive correlation with behavioral intention where the theory could predict the intention of physical activities accomplishment up to 58% among australian adolescents ( 12 ) .
activities of the workshop are based on the process of problem solving , aim to provide a condition for the participants to make decisions on a health problem or behavior , and then provide their own solutions ( 13 ) .
school , as the focal center of education , is a suitable place , making adolescents accessible assumed as a place to acquire health information from health assistants . in this
examined nutrition - based health improvement in schools ; leading to the increase of student s attitudes in school using educational intervention ( 14 ) .
further , applying the education based on tpb led to the increase of attitude , perceived behavioral control and intentional behavior in physical activity dimension ( 15 , 16 ) . considering that educational program about risk factors of hypertension rather than reinforcing tendency to improvement of sport and physical activity in schools
is emphasized by health experts ( 17 ) , this study designed an educational program based on tpb to investigate intention to change of nutrition and activity related behaviors among female adolescents of schools .
in this quasi - experimental study 160 , girl adolescent were participated . for this , four schools in the central region of tehran were selected based on cluster sampling method during two stages . according to studies related to the impact of education on the hypertension related behaviors ( 11 ) , and also with regard to the alfa error 5% , and study power 90% , the sample size equals 40 people in each group , however , by considering the loss and coefficient of cluster sampling , and also with the purpose of decreasing error , sample size was considered 80 people in each group . as the quasi - experimental studies , we had not any blinding .
first , each of the eight central regions of tehran ( districts number six with 30 schools , 10 with 16 schools , 11 with 21 schools and , 12 with 31 schools ) was considered as a cluster , and then one school was selected from each cluster at random via table of random numbers .
all students of these schools were almost 18000 and 752 students were selected in this stage . as randomly , two schools were allocated as the case group and two schools as the control group .
finally , in each school , one class and in each class 40 students were selected randomly ( 80 girls in the case group and 80 girls in the control group ) .
since the south and north regions of tehran have considerable cultural , social , and economic differences , the comparison between the participants of these regions could make bias in the study results , therefore , the central regions of tehran selected .
overall , 1216 year old girls who had not educated in prevention of hypertension over the recent three months and had not had a special nutritional regimen participated in the study .
not participating in even one of the class sessions , participating in the other similar educational classes , and loosing tendency to take part in the study are the criteria to get out of the study . according to the mentioned criteria
the data were gathered in two stages ( before education and after education ) through a self - report two - part questionnaire :
the questionnaire of demographic character - ristics : the first part entailed 13 demographic items including age , parents ' educational status , family members , birth order , history of specific disease , history of specific medication use , family history of hypertension and its complications , family history of certain diets use , source of acquiring health information , history of participating in sport class or team , and history of bp measurement.the questionnaire of investigating components of tpb : the second part consisted of 49 phrases in likert scale related to prevention of hypertension and based on tpb involving the attitude in nutritional dimension ( 10 phrases ) , attitude in physical activity dimension ( 8 phrases ) , subjective norms in nutrition dimension ( 6 phrases ) , subjective norms in physical activity dimension ( 5 phrases ) , perceived behavioral control in nutrition dimension ( 5 phrases ) , perceived behavioral control in nutrition physical activity ( 5 phrases ) , behavioral intention in nutrition dimension ( 5 phrases)and , behavioral intention in physical activity dimension ( 5 phrases ) .
each dimension of nutrition and physical activity was designed using the guidance of questionnaire analysis based on theory of planned behavior ( 18 ) . according to the available standard instruction , the 5 point likert - style scale
is used to measure the questions ( 0=strongly disagree , 1=disagree , 2= having no idea , 3=agree , 4= strongly agree ) .
the questionnaire of demographic character - ristics : the first part entailed 13 demographic items including age , parents ' educational status , family members , birth order , history of specific disease , history of specific medication use , family history of hypertension and its complications , family history of certain diets use , source of acquiring health information , history of participating in sport class or team , and history of bp measurement .
the questionnaire of investigating components of tpb : the second part consisted of 49 phrases in likert scale related to prevention of hypertension and based on tpb involving the attitude in nutritional dimension ( 10 phrases ) , attitude in physical activity dimension ( 8 phrases ) , subjective norms in nutrition dimension ( 6 phrases ) , subjective norms in physical activity dimension ( 5 phrases ) , perceived behavioral control in nutrition dimension ( 5 phrases ) , perceived behavioral control in nutrition physical activity ( 5 phrases ) , behavioral intention in nutrition dimension ( 5 phrases)and , behavioral intention in physical activity dimension ( 5 phrases ) .
each dimension of nutrition and physical activity was designed using the guidance of questionnaire analysis based on theory of planned behavior ( 18 ) . according to the available standard instruction , the 5 point likert - style scale
is used to measure the questions ( 0=strongly disagree , 1=disagree , 2= having no idea , 3=agree , 4= strongly agree ) .
the validity of questionnaire was determined by two methods of face validity and content validity , which confirmed by 10 experts of cardiologist and 10 nurses . the total content validity index ( cvi ) in the relevancy
, simplicity , and clarity respectively equals 82.5 , 92.94 , and 90.71 .
the reliability of the questionnaire further evaluated through internal consistency ( = 0.91 ) and test - retest ( r = 0.82 ) . after allocating the participants into two groups of case and control , participants , their parents and deans of schools
so after a week , a pretest was administered for both case and control groups using the questionnaire .
once more , after a week , the educational classes with the content of teaching accurate nutrition and physical activities to prevent hypertension were held for the case group using the workshop method during four weeks , one session a week and two hours each session ( two sessions for nutrition and two sessions for physical activity ) .
, separate times were allocated for attitude change , subjective norms change , perceived control change , and finally for question / answer . at the end of each session
, participants were provided with an educational pamphlet with the summative content of the educational program due to making the opportunity to solve any question . to follow up the case group , six weeks later than the intervention , one review session held for the group .
three months after the reviewing session , a post - test was held for both groups using the same questionnaire ( 13 ) .
because students were repeatedly recommended to complete all items of questionnaire , the study had not missed data .
a confounding variable was the researchers inability to control completely the participants not to receive educational information from other educational resources during the study .
anyhow , the researchers recommended the adolescents and their families to avoid same educational programs .
the data were analyzed using descriptive and inferential statistics ( independent t - test , k - s test , paired t - test , and chi - square test ) through spss version 17 ( chicago , il , usa ) .
the present paper is confirmed by the ethics committee of tehran university of medical sciences .
the research objectives were explained for all of the adolescents participating in the study , deans , and adolescents parents , and then the adolescents written consents were received .
moreover , a similar educational session presented to the control group after collecting post - test data .
in this quasi - experimental study 160 , girl adolescent were participated . for this , four schools in the central region of tehran were selected based on cluster sampling method during two stages . according to studies related to the impact of education on the hypertension related behaviors ( 11 ) , and also with regard to the alfa error 5% , and study power 90% , the sample size equals 40 people in each group , however , by considering the loss and coefficient of cluster sampling , and also with the purpose of decreasing error , sample size was considered 80 people in each group . as the quasi - experimental studies , we had not any blinding .
first , each of the eight central regions of tehran ( districts number six with 30 schools , 10 with 16 schools , 11 with 21 schools and , 12 with 31 schools ) was considered as a cluster , and then one school was selected from each cluster at random via table of random numbers .
all students of these schools were almost 18000 and 752 students were selected in this stage . as randomly , two schools were allocated as the case group and two schools as the control group .
finally , in each school , one class and in each class 40 students were selected randomly ( 80 girls in the case group and 80 girls in the control group ) .
since the south and north regions of tehran have considerable cultural , social , and economic differences , the comparison between the participants of these regions could make bias in the study results , therefore , the central regions of tehran selected .
overall , 1216 year old girls who had not educated in prevention of hypertension over the recent three months and had not had a special nutritional regimen participated in the study .
not participating in even one of the class sessions , participating in the other similar educational classes , and loosing tendency to take part in the study are the criteria to get out of the study . according to the mentioned criteria
the data were gathered in two stages ( before education and after education ) through a self - report two - part questionnaire :
the questionnaire of demographic character - ristics : the first part entailed 13 demographic items including age , parents ' educational status , family members , birth order , history of specific disease , history of specific medication use , family history of hypertension and its complications , family history of certain diets use , source of acquiring health information , history of participating in sport class or team , and history of bp measurement.the questionnaire of investigating components of tpb : the second part consisted of 49 phrases in likert scale related to prevention of hypertension and based on tpb involving the attitude in nutritional dimension ( 10 phrases ) , attitude in physical activity dimension ( 8 phrases ) , subjective norms in nutrition dimension ( 6 phrases ) , subjective norms in physical activity dimension ( 5 phrases ) , perceived behavioral control in nutrition dimension ( 5 phrases ) , perceived behavioral control in nutrition physical activity ( 5 phrases ) , behavioral intention in nutrition dimension ( 5 phrases)and , behavioral intention in physical activity dimension ( 5 phrases ) .
each dimension of nutrition and physical activity was designed using the guidance of questionnaire analysis based on theory of planned behavior ( 18 ) . according to the available standard instruction
, the 5 point likert - style scale is used to measure the questions ( 0=strongly disagree , 1=disagree , 2= having no idea , 3=agree , 4= strongly agree ) .
the questionnaire of demographic character - ristics : the first part entailed 13 demographic items including age , parents ' educational status , family members , birth order , history of specific disease , history of specific medication use , family history of hypertension and its complications , family history of certain diets use , source of acquiring health information , history of participating in sport class or team , and history of bp measurement .
the questionnaire of investigating components of tpb : the second part consisted of 49 phrases in likert scale related to prevention of hypertension and based on tpb involving the attitude in nutritional dimension ( 10 phrases ) , attitude in physical activity dimension ( 8 phrases ) , subjective norms in nutrition dimension ( 6 phrases ) , subjective norms in physical activity dimension ( 5 phrases ) , perceived behavioral control in nutrition dimension ( 5 phrases ) , perceived behavioral control in nutrition physical activity ( 5 phrases ) , behavioral intention in nutrition dimension ( 5 phrases)and , behavioral intention in physical activity dimension ( 5 phrases ) .
each dimension of nutrition and physical activity was designed using the guidance of questionnaire analysis based on theory of planned behavior ( 18 ) . according to the available standard instruction
, the 5 point likert - style scale is used to measure the questions ( 0=strongly disagree , 1=disagree , 2= having no idea , 3=agree , 4= strongly agree ) .
the validity of questionnaire was determined by two methods of face validity and content validity , which confirmed by 10 experts of cardiologist and 10 nurses . the total content validity index ( cvi ) in the relevancy , simplicity , and clarity
the reliability of the questionnaire further evaluated through internal consistency ( = 0.91 ) and test - retest ( r = 0.82 ) .
after allocating the participants into two groups of case and control , participants , their parents and deans of schools were justified with the aim and procedure of the .
so after a week , a pretest was administered for both case and control groups using the questionnaire .
once more , after a week , the educational classes with the content of teaching accurate nutrition and physical activities to prevent hypertension were held for the case group using the workshop method during four weeks , one session a week and two hours each session ( two sessions for nutrition and two sessions for physical activity ) .
, separate times were allocated for attitude change , subjective norms change , perceived control change , and finally for question / answer . at the end of each session
, participants were provided with an educational pamphlet with the summative content of the educational program due to making the opportunity to solve any question . to follow up the case group , six weeks later than the intervention , one review session held for the group .
three months after the reviewing session , a post - test was held for both groups using the same questionnaire ( 13 ) .
because students were repeatedly recommended to complete all items of questionnaire , the study had not missed data .
a confounding variable was the researchers inability to control completely the participants not to receive educational information from other educational resources during the study .
anyhow , the researchers recommended the adolescents and their families to avoid same educational programs .
the data were analyzed using descriptive and inferential statistics ( independent t - test , k - s test , paired t - test , and chi - square test ) through spss version 17 ( chicago , il , usa ) .
the present paper is confirmed by the ethics committee of tehran university of medical sciences .
the research objectives were explained for all of the adolescents participating in the study , deans , and adolescents parents , and then the adolescents written consents were received .
moreover , a similar educational session presented to the control group after collecting post - test data .
the result of the k - s test reveals that there was no significant difference between the case and control groups in terms of all demographic characteristics ( p < 0.05 ) .
demographic characteristics of participants the results of independent t - test indicate that there is no statically significant difference between two groups in terms of the components of the tpb including attitude , subjective norm , perceived behavioral control , and behavioral intention in terms of nutrition and physical activity ( p>0.05 ) ( table 2 ) .
comparison between mean and sd of attitudes , subjective norm , perceived behavioral control , and behavioral intention of participants in terms of nutrition and physical activity in case group with control group before intervention further , the results of paired t - test signified that after intervention , both in nutrition and physical activity , attitude , perceived behavior control , and behavioral intention significantly increased in the case group compared to the control group while subjective norm had a significant difference only in nutrition part .
there was no significant difference in components of tpb in paired t - test of the control group .
comparison between mean and sd of attitudes , subjective norm , perceived behavioral control , and behavioral intention of participants in terms of nutrition and physical activity in case and control groups before and after intervention moreover , according to the results of independent t - test signified in table 4 , the case group compared with control group indicate a significant difference in terms of nutrition in the all dimensions of tpb and a significant difference in the dimensions of attitude , perceived behavioral control , and behavioral intention in terms of physical activity ( p<0.05 ) .
comparison between mean and sd of attitudes , subjective norm , perceived behavioral control , and behavioral intention of participants in terms of nutrition and physical activity in case group with control group after intervention
the result of the k - s test reveals that there was no significant difference between the case and control groups in terms of all demographic characteristics ( p < 0.05 ) .
the results of independent t - test indicate that there is no statically significant difference between two groups in terms of the components of the tpb including attitude , subjective norm , perceived behavioral control , and behavioral intention in terms of nutrition and physical activity ( p>0.05 ) ( table 2 ) .
comparison between mean and sd of attitudes , subjective norm , perceived behavioral control , and behavioral intention of participants in terms of nutrition and physical activity in case group with control group before intervention further , the results of paired t - test signified that after intervention , both in nutrition and physical activity , attitude , perceived behavior control , and behavioral intention significantly increased in the case group compared to the control group while subjective norm had a significant difference only in nutrition part .
there was no significant difference in components of tpb in paired t - test of the control group .
comparison between mean and sd of attitudes , subjective norm , perceived behavioral control , and behavioral intention of participants in terms of nutrition and physical activity in case and control groups before and after intervention moreover , according to the results of independent t - test signified in table 4 , the case group compared with control group indicate a significant difference in terms of nutrition in the all dimensions of tpb and a significant difference in the dimensions of attitude , perceived behavioral control , and behavioral intention in terms of physical activity ( p<0.05 ) .
comparison between mean and sd of attitudes , subjective norm , perceived behavioral control , and behavioral intention of participants in terms of nutrition and physical activity in case group with control group after intervention
the obtained results indicate that the mean of attitude , subjective norm , perceived behavioral control , and behavioral intention regarding nutrition dimension increased after the intervention in the case group .
in other words , adolescent students showed higher tendency to have healthy nutritional behavior to prevent hypertension ( attitude ) , felt more expectations from who were important to her has a healthy diet ( subjective norm ) , and considered themselves more capable to accomplish healthy nutritional behaviors .
hence , it can conclude that the intention of healthy nutritional behaviors was increased in them .
meanwhile , attitude and behavioral intention had a more highlighted role among the components of the theory ( fig . 1 and 2 ) .
in fact , the results proved that educational intervention leads to reinforcing the beliefs and attitudes of adolescents and also the increase of mean score of behavioral intention in nutrition dimension ; revealing their tendency to have healthy diets .
further , adolescent participants , followed by receiving information about the positive effect of behaviors proposed by educational program in their nutritional behaviors , showed more control to have healthy nutritional behaviors . the components of tpb based on variables before education the components of tpb based on variables after education
high effect : low effect : in this regards , tavakkoli et al .
worked on nutritional behavior improvement , investigating the effect of education in nutritional behaviors in army personnel and found the findings consistent with the present paper 's results ( 19 ) .
they concluded that the attitude of mothers having young children was improved after an educational intervention using training in small groups .
however , the sample was inconsistent in terms of age with the current study samples , but the same educational method was used in intervention leading to the similar results . considering these findings
, it seems that educational intervention in group sessions can increase the probability of positive changes in deterministic dimensions of attitude by providing a field to exchange information and use of peers ' experiences .
attitude towards a subject is derived from the beliefs of an individual about that subject .
that is , the favorable consequence of a behavior will improve the individual 's attitude toward that behavior .
since the aim of the present study was to make adolescents familiar with the benefits of healthy nutritional behaviors through the proposed intervention , the obtained results were expected by the researcher .
the results obtained from physical activity dimension revealed that in the case group , all the components of theory , except subjective norm were significantly increased after the intervention .
this finding is consistent with other studies , as revealed , subjective norm could not be promoted in the physical activity dimension of their sample while attitude , perceived behavioral control and behavioral intention were increased in the sample ( 15 , 16 ) .
merely using a theoretical program may be the cause for lack of change in the subjective norm in physical activity so that in the study by quinn et al .
, attitude towards performing sport activities was increased in fat women after four practical educational sessions and four months after the intervention ( 21 ) . in our study
, it was better to prepare sport sessions at the schools to improve physical activities , accomplishment as a healthy behavior but sport facility was not possible for the researcher .
it seems that subjective norm , signifying the importance of the behavior accomplishment of individuals , groups and communities imposed on individual , is a value established in the individuals rooted in her culture , as well as family and social values . in iranian culture , the tendency of female adolescents to do physical activities is less than males compared with other age groups where there has been no obligation to participate in sport groups from childhood .
therefore , it can not be expected that subjective norm can be changed only through theoretical educational interventions .
another factor affecting subjective norm formation is family , which is undeniable . in this case
, it may be better to hold theoretical and practical educational sessions with the participation of family members and adolescents in family groups where on families are encouraged for sport activities in family environments .
indeed , the culture of sport and increasing physical activity should firstly promoted in families to achieve this objective . in this
regards , further studies are recommended . comparing the mean scores of the case and control groups after the intervention showed a significant difference between two groups in all components but the subjective norm in physical dimension ; among them , the perceived behavioral control in both nutrition and physical activity and attitude in physical activity dimension showed the most difference which can be due to presenting better education in this part .
the lack of statistically significant difference between the score of attitude , perceived behavioral control and behavioral intention both in nutrition and physical activity was expected with respect to the lack of any intervention in the control group .
the present study aimed to investigate the effect of education based on the tpb in preventive behaviors of hypertension in school female adolescent students . as found , using education based on tpb was effective in hypertension prevention and increased attitude , perceived behavioral control and behavioral intention in both nutrition and physical activity and subjective norms were perceived only in nutrition dimension . with regard to the results , tpb can be introduced as an appropriate theory to design and implement educational intervention in order to change healthy nutrition - related behaviors and physical activity with the aim of improving the intention of preventive behaviors of hypertension in adolescents .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | background : since risk factors of hypertension are formed during adolescent period and regarding that attitudes change occurs more easily in these ages , the present paper aimed to evaluate the impact of education based on the theory of planned behavior in hypertension prevention behaviors in female adolescent students.methods:in this quasi - experimental study , 160 girls of 1216 yr old
( 80 in each case and control group ) , who had not been educated in prevention of hypertension over the recent three months , participated .
four schools in tehran were selected based on cluster sampling method during two stages .
the education was provided based on the theory of planned behavior in two sections ( nutrition and physical activity ) in four sessions .
data gathered before and after education through a two - part valid and reliable questionnaire .
the results were analyzed based on spss software , version 17.results:the results of independent t - test showed in the nutrition section , attitude ( p=0.000 ) , subjective norm ( p=0.025 ) , perceived control ( p=0.016 ) and behavioral intention ( p=0.025 ) ; significantly increased . about physical activity , except subjective norm ( p=0.219 ) , the mean score of the attitude ( p=0.001 ) , perceived control ( p=0.000 ) and behavioral intention ( p=0.000 ) revealed a significant difference between two groups.conclusion:education based on the theory of planned behavior affects the intention of preventive behaviors of hypertension in female adolescents . |
ageing often leads to a loss of functional fitness in older people , reducing their ability to perform daily tasks.1 moreover , fallrelated injuries are serious problems in old age , as they often lead to prolonged , or even permanent , disability .
thus , maintenance of functional fitness and prevention of the associated injuries should reduce disability , improve the quality of life , and reduce the costs of health care .
exercise training has been shown to be an effective nonpharmacological way of reducing physical difficulties in older people .
recently , we demonstrated that exercise training contributes significantly to the maintenance of functional fitness in older healthy people2 and those with cardiac disease.3 furthermore , exercise is generally accepted as effective in preventing falls in old age .
a review of randomized controlled trials has demonstrated that exercise reduces the risk of falls in elderly people , and reduction in the incidence of fallrelated injuries is related to lower healthcare costs.4 waterbased exercise ( we ) has been widely promoted as the optimal type of exercise for older people as it reduces weightbearing stresses on the skeletal joints and provides therapeutic benefits for orthopedic conditions.5,6 in addition , weassociated benefits3,6 are in agreement with current recommendations stating that aerobic power , muscle strength , and body flexibility are important factors in maintaining functional independence in older people.7,8 the health benefits of any exercise depend on its regularity , but older people commonly have different chronic diseases , side effects of drugs , hospitalization , and disability , which may be compelling reasons to abandon exercise programs.9 most studies of detraining in older people have evaluated the effects after stopping landbased resistance training of low to high intensity10 - 12 or multicomponent programs.9,13 to the best of our knowledge , few studies have reported the effects of detraining after a we program , and they have evaluated functional ability only in people with neuromuscular and cardiovascular illness.12,14 despite some evidence of physiological decline after short detraining periods,9,15,16 there are insufficient data on functional fitness and qualityoflife changes in healthy older women when a we program is stopped . therefore , this study was designed to determine the effects of 12weeks of we training followed by a 6week detraining period on the functional fitness of older healthy women .
all participants underwent a clinical examination and answered questionnaires about their medical history ; the exclusion and inclusion criteria have been described in detail elsewhere.2,3 all participants read and signed the terms of consent before inclusion in the study in accordance with the declaration of helsinki .
the women were randomized into a trained ( tr ; n = 30 ) or untrained group ( un ; n = 20 ) . the tr group was directed to follow a we program , and the un group comprised women who could not undergo frequent training sessions .
subjects from both groups continued with their daily activities , and the tr group underwent no other physical training besides we .
a detailed description of the training process can be found in a recent study published by our laboratory.2 briefly , the we program consisted of 60min sessions , three times a week , over 12 weeks .
each training session included a 10min warmup stretching period ( stage 1 ) ; 45min endurance training ( stage 2 ) ; and a 5min cooldown / relaxation period with slow walking and stretching ( stage 3 ) .
this training protocol is in agreement with the exercise guidelines recommended by the american college of sports medicine for healthy adults and the elderly.7 to detrain , the we group were instructed to give details of any new prescribed medication , to not participate in any other type of physical activity , and finally , to continue only with their usual daily activities .
both groups were assessed by the same researcher at four time points as follows : before training ( baseline ) , after 12 weeks of training , and after 4 and 6 weeks of detraining . five sets of evaluations were obtained on different days to improve knowledge of the methods and robustness of the test technique .
body mass index was calculated by the equation : bmi = body mass ( kg)/height ( m ) .
a bruce treadmill protocol was carried out to determine the vo2max as previously described.2 a 1min warmup without treadmill inclination at 1.0 mph preceded the bruce treadmill protocol .
the borg scale with scores of between 6 and 20 was used for determination of subjective exertion level .
the test was considered a success if participants reached their agepredicted maximum heart rate that is , 220 age , with the result in beats per minute ) . for the functional aerobic test , we used the 800 meters test standardized for the brazilian population17 and previously used by bocalini et al.3 subjects were instructed to run or walk a distance of 800 meters as quickly as possible .
the arm curl test assessed upper body strength and was scored as the total number of hand weight curls through the full range of motion in 30 seconds .
test assessed lower body strength and was scored as the number of stands executed correctly within 30 seconds .
test that is , rising from a seated position , walking 8 feet , returning to the seated position .
the chair sit and reach test assessed lower body flexibility and was scored as the best distance achieved between the extended fingers and the tip of the toe .
static balance was assessed by asking subjects to stand on one leg for a maximum of 30 seconds on each side18 and was scored as the longest time of quiet standing , allowing only minimal fluctuations of ankle position or obvious toe clawing , and no hopping or arm movement .
the test was stopped after 30 seconds or whenever the raised foot touched the floor or contacted the other leg / foot , the ankle movement was excessive , or hopping occurred .
quality of life was evaluated by a shortened who qualityoflife questionnaire as previously used by bocalini et al.3 the questionnaire comprised 25 questions about several aspects of quality of life , including : physical domain , which concerns pain or discomfort , energy or fatigue , sleep , rest , mobility , daily activities , medicine dependency , and job performance ; psychological domain , which includes feelings , learning , memory and attention , selfesteem , aspect , spirituality , religiousness , and positive or negative thinking ; social domain , which deals with personal relationships , social support and sexuality ; environmental domain , which includes physical security , home environment , financial security , opportunity for information assessment , social or cultural event participation , and activities undertaken during spare time .
each domain was scored from 0 to 100 points , and higher scores represented improvement .
data ( means sem ) were analyzed using the graphpad prism software ( version 4.0 , san diego , ca , usa ) .
twoway analysis of variance for repeatedmeasures analysis was used to examine differences within and between groups over time , and the tukey post hoc test was used when appropriate .
all participants underwent a clinical examination and answered questionnaires about their medical history ; the exclusion and inclusion criteria have been described in detail elsewhere.2,3 all participants read and signed the terms of consent before inclusion in the study in accordance with the declaration of helsinki .
the women were randomized into a trained ( tr ; n = 30 ) or untrained group ( un ; n = 20 ) . the tr group was directed to follow a we program , and the un group comprised women who could not undergo frequent training sessions .
subjects from both groups continued with their daily activities , and the tr group underwent no other physical training besides we .
a detailed description of the training process can be found in a recent study published by our laboratory.2 briefly , the we program consisted of 60min sessions , three times a week , over 12 weeks .
each training session included a 10min warmup stretching period ( stage 1 ) ; 45min endurance training ( stage 2 ) ; and a 5min cooldown / relaxation period with slow walking and stretching ( stage 3 ) .
this training protocol is in agreement with the exercise guidelines recommended by the american college of sports medicine for healthy adults and the elderly.7 to detrain , the we group were instructed to give details of any new prescribed medication , to not participate in any other type of physical activity , and finally , to continue only with their usual daily activities .
both groups were assessed by the same researcher at four time points as follows : before training ( baseline ) , after 12 weeks of training , and after 4 and 6 weeks of detraining .
five sets of evaluations were obtained on different days to improve knowledge of the methods and robustness of the test technique .
body mass index was calculated by the equation : bmi = body mass ( kg)/height ( m ) .
a bruce treadmill protocol was carried out to determine the vo2max as previously described.2 a 1min warmup without treadmill inclination at 1.0 mph preceded the bruce treadmill protocol .
the borg scale with scores of between 6 and 20 was used for determination of subjective exertion level .
the test was considered a success if participants reached their agepredicted maximum heart rate that is , 220 age , with the result in beats per minute ) . for the functional aerobic test
, we used the 800 meters test standardized for the brazilian population17 and previously used by bocalini et al.3 subjects were instructed to run or walk a distance of 800 meters as quickly as possible .
the arm curl test assessed upper body strength and was scored as the total number of hand weight curls through the full range of motion in 30 seconds .
test assessed lower body strength and was scored as the number of stands executed correctly within 30 seconds .
test that is , rising from a seated position , walking 8 feet , returning to the seated position .
the chair sit and reach test assessed lower body flexibility and was scored as the best distance achieved between the extended fingers and the tip of the toe .
static balance was assessed by asking subjects to stand on one leg for a maximum of 30 seconds on each side18 and was scored as the longest time of quiet standing , allowing only minimal fluctuations of ankle position or obvious toe clawing , and no hopping or arm movement .
the test was stopped after 30 seconds or whenever the raised foot touched the floor or contacted the other leg / foot , the ankle movement was excessive , or hopping occurred .
quality of life was evaluated by a shortened who qualityoflife questionnaire as previously used by bocalini et al.3 the questionnaire comprised 25 questions about several aspects of quality of life , including : physical domain , which concerns pain or discomfort , energy or fatigue , sleep , rest , mobility , daily activities , medicine dependency , and job performance ; psychological domain , which includes feelings , learning , memory and attention , selfesteem , aspect , spirituality , religiousness , and positive or negative thinking ; social domain , which deals with personal relationships , social support and sexuality ; environmental domain , which includes physical security , home environment , financial security , opportunity for information assessment , social or cultural event participation , and activities undertaken during spare time .
each domain was scored from 0 to 100 points , and higher scores represented improvement .
body mass index was calculated by the equation : bmi = body mass ( kg)/height ( m ) .
a bruce treadmill protocol was carried out to determine the vo2max as previously described.2 a 1min warmup without treadmill inclination at 1.0 mph preceded the bruce treadmill protocol .
the borg scale with scores of between 6 and 20 was used for determination of subjective exertion level .
the test was considered a success if participants reached their agepredicted maximum heart rate that is , 220 age , with the result in beats per minute ) . for the functional aerobic test
, we used the 800 meters test standardized for the brazilian population17 and previously used by bocalini et al.3 subjects were instructed to run or walk a distance of 800 meters as quickly as possible .
the arm curl test assessed upper body strength and was scored as the total number of hand weight curls through the full range of motion in 30 seconds .
test assessed lower body strength and was scored as the number of stands executed correctly within 30 seconds .
test that is , rising from a seated position , walking 8 feet , returning to the seated position .
the chair sit and reach test assessed lower body flexibility and was scored as the best distance achieved between the extended fingers and the tip of the toe .
static balance was assessed by asking subjects to stand on one leg for a maximum of 30 seconds on each side18 and was scored as the longest time of quiet standing , allowing only minimal fluctuations of ankle position or obvious toe clawing , and no hopping or arm movement .
the test was stopped after 30 seconds or whenever the raised foot touched the floor or contacted the other leg / foot , the ankle movement was excessive , or hopping occurred .
quality of life was evaluated by a shortened who qualityoflife questionnaire as previously used by bocalini et al.3 the questionnaire comprised 25 questions about several aspects of quality of life , including : physical domain , which concerns pain or discomfort , energy or fatigue , sleep , rest , mobility , daily activities , medicine dependency , and job performance ; psychological domain , which includes feelings , learning , memory and attention , selfesteem , aspect , spirituality , religiousness , and positive or negative thinking ; social domain , which deals with personal relationships , social support and sexuality ; environmental domain , which includes physical security , home environment , financial security , opportunity for information assessment , social or cultural event participation , and activities undertaken during spare time .
each domain was scored from 0 to 100 points , and higher scores represented improvement .
data ( means sem ) were analyzed using the graphpad prism software ( version 4.0 , san diego , ca , usa ) .
twoway analysis of variance for repeatedmeasures analysis was used to examine differences within and between groups over time , and the tukey post hoc test was used when appropriate .
during the research period , three women from the tr group left the study and two women from the un group could not be traced at the end of the study for the reevaluation .
therefore , our data are based on the following subjects : un ( n = 18 ) and tr ( n = 27 ) .
there were no differences between the groups at baseline for age or body mass index ( table 1 ) , or for aerobic capacity ( figure 1 ) , neuromuscular fitness ( table 2 ) and quality of life ( figure 2 ) .
after the 12week we program , the body mass index was similar between the groups , and nor were any differences found between the groups after 4 and 6weeks of detraining ( table 1 ) .
the aerobic fitness of the tr group was enhanced significantly after training , but with detraining , this group showed a progressive impairment with decreasing vo2max ( figure 1a ) and increase in the time for the 800 m test ( figure 1b ) .
however , even after 6 weeks of detraining , the scores of the tn group remained better than at baseline and better than those of the un group ( figure 1a and b ) .
in addition , a significant high correlation was found between vo2max and the 800 m test ( figure 1c ) .
after 12weeks of training , there was significant improvement of 28% and 29% in upper and lower body strength as evaluated by the arm curl and chair stand tests , respectively .
moreover , there were improvements of 36% for the 8foot up and go test , 31% for the chair sit and reach test and 36% for static balance , showing an enhancement of the agility , flexibility and balance , respectively .
however , the neuromuscular fitness became progressively impaired when training was discontinued , and after just 4 weeks of detraining all scores were significantly lower than after 12 weeks of we training .
only lower body strength and static balance remained significantly higher than those of the un group at this time .
table 2 shows that a greater decline in the functional fitness scores occurred after 6 weeks of detraining , and all the analyzed parameters were similar in the tn and un groups .
the most pronounced effect of detraining was seen in the arm curl and sit and reach tests , which returned to their baseline level .
we training resulted in a significant general improvement in all the domains of quality of life ( figure 2 ) , but there was a progressive return to baseline levels after as early as 4 weeks of detraining , matching completely the un group and baseline levels at 6 weeks of detraining .
the major finding of this study was that after 6 weeks of detraining , the improvement of functional fitness of older healthy women , achieved with 12 weeks of we training , was lost .
not only body strength and agility , but also flexibility and static balance , and quality of life had returned to pretraining conditions .
aerobic fitness was least affected by detraining , since the vo2max values and performance on 800 m test remained higher than those of the un group and of the pretraining values .
our results show that the 12week we program improved several components of functional fitness in older women , was well tolerated , and no adverse events , such as fallrelated injuries or adverse cardiovascular effects , were seen in any subject during the exercise program , suggesting that the protocol is safe .
these indications corroborate previous studies reporting that regular participation in a structured exercise program is a safe and effective intervention for healthy older people , improving their aerobic capacity , body strength , flexibility , agility and range of motion.3,6 to evaluate whether physical exercise practice can improve social and personal wellbeing and quality of life , we used a validated questionnaire and noted a significant enhancement in the qualityoflife scores of the trained group , including physical , psychological , social and environmental domains
. these findings also agree with those of other investigators5 and are important , since studies have demonstrated a strong association between psychosocial problems such as depression , anxiety or social isolation , and chronic diseases.19 the improvement in the tn group on both the vo2max and 800 m tests owing to the 12week we program was significantly lost after 6 weeks of detraining , although their scores remained better than those of matched un subjects and the baseline values .
it is well known that improvement in aerobic capacity with training is influenced by cardiovascular and musculoskeletal adaptations.20,21 thus , the decline in cardiac output , arteriovenous difference , oxidative enzymes , hemoglobin concentration , myocytic mitochondria density , and muscle capillarization , which occur with detraining , may contribute to the decrease in aerobic fitness.22 - 24 although the lower body strength did not return to the baseline levels when exercise was stopped , our results are similar to those of others using a different exercise modality in an older people13,16,25,26 and indicate that lower body strength scores decrease significantly and are similar to un scores after detraining .
our subjects had a decrease in upper body strength as early as after 4 weeks of detraining , and returned to baseline at the sixth week of detraining .
a plausible explanation for these divergences between upper and lower body strength may be the greater adaptable response to exercise of the lower limbs .
other possible conflicting factors are the variable adjustments to different frequency , type duration and intensity of training and detraining , as well as the agerelated particular daily activity routines during the detraining period that may affect upper and lower limbs differently.25 - 28 agility / balance has an essential role in routine activities in old age .
some authors have demonstrated that the agility / balance gain is lost 6 weeks after exercise cessation,9 but other studies have found the gain to be maintained even after 313 or 12 months of detraining.8 the agility / balance improvement in the cases that use landbased exercise is probably due to a gain in strength , as described for a multicomponent exercise program.10,29,30 thus , we believe that for the we program also , the agility / balance changes should be directly correlated with improvement and impairment of both the balance and strength during training and detraining , respectively , as also proposed by ryushi et al.30 in agreement with previous multicomponent training studies in older women,9,13 flexibility was significantly affected by detraining . after only 6 weeks of detraining the improvement in flexibility achieved with the we
owing to its importance for physical autonomy and fall / injury prevention,4 the maintenance of flexibility should be evaluated with caution during detraining in older adults .
finally , a significant worsening of quality of life was associated with detraining , with the benefits of the we program being almost completely lost in just 4 weeks . moreover ,
the worsening in all domains of the quality of life ( physical , psychological , social , and environment ) matched closely reductions in aerobic capacity , body strength , agility , flexibility , and static balance , thus reinforcing scientific evidence that directly correlates regular physical exercise with psychosocial wellbeing and quality of life in older people.2,31 this is a relatively small sample and shortterm study , with no information about longterm outcomes .
nevertheless , for future public health strategies , this work reinforces the message of the importance of the frequency and regularity of this exercise modality .
in addition , there may be some inaccuracy in estimating maximum aerobic fitness ( indirect estimate of vo2max ) by measuring cardiopulmonary gas exchange .
nevertheless this drawback was common to both the un and tr groups and for all evaluations during followup , thus reducing inaccuracy . despite this significant induction of positive changes in the functional fitness of older women with the we program ,
more studies are needed to examine we practices and the effect of education , training and detraining on older men and women .
this study demonstrated that short term we training improves cardiorespiratory and neuromuscular fitness . as a result
, the ability to carry out tasks of daily living is enhanced with we training , leading to independence , psychosocial wellbeing and better quality of life in older women
. however , regularity of the exercise had a key role , as after a short period of detraining , most neuromuscular fitness parameters and qualityoflife scores returned to baseline levels .
| objective : to evaluate the effects of shortterm exercise detraining on the functional fitness of older women after a 12week waterbased exercise ( we ) program.methods:healthy older women ( trained ( tr ) group ) were submitted to 12 weeks we ( three 45 min sessions per week ) followed by a 6week detraining period .
a group of agedmatched women without any exercise training ( un group ) were evaluated during the same period .
the aerobic power , measured by vo2max and 800 meters performance , and the neuromuscular fitness and quality of life were evaluated .
all assessments were made at baseline , after 12 weeks of training and after 4 and 6 weeks of detraining.results:no changes were found for the un group during the followup study , but we induced significant improvement in aerobic capacity , neuromuscular fitness and quality of life score .
however , the upper and lower body strength , agility , flexibility , and body balance returned to un levels ( p>0.05 ) after 6 weeks of detraining.conclusions:the results confirmed that 12 weeks of we improves the functional fitness parameters and quality of life of older women .
however , after a short detraining period of 46 weeks , the neuromuscular parameters and the quality of life score returns to baseline or untrained subject levels . |
sacrococcygeal pilonidal sinus disease is a common and usually a minor disease . although wide excision has been a common practice , there are more simple alternatives , and its treatment remains controversial .
numerous surgical procedures have been described , but treatment failure and disease recurrence are frequent , leading to considerable morbidity in these , otherwise healthy patients .
the optimal surgical treatment option should be simple , inexpensive , and associated with low hospitalization periods and recurrence rates .
total excision of the involved sinus tract to the post sacral fascia is the most frequently applied surgical option but the ideal type of reconstruction is disputed .
the defect formed on the excised area might be primarily closed , partially closed as is the case in marsupialization , left open for secondary healing , or reconstructed by differing flap techniques . published literatures comparing the use of flap as off - midline procedure versus primary midline suture techniques found that primary closure would be as effective as the flap reconstruction .
midline excision techniques are associated with high morbidity and recurrence due to wound 's placement in the natal cleft .
conventional off - midline flap closures are associated with fewer serious complications , but they have less acceptable esthetic outcomes . in the present study , the author reported a novel simplified off - midline technique for closure of the defect after complete excision of the sinus tracts without flap reconstruction .
all patients completed written informed consent and received an explanation of the operative procedure from the study staff .
the study procedures were approved by the institutional ethics committee of port - fouad general hospital , represented by the hospital 's general director .
a total of two hundreds patients of both sexes with primary , non - recurrent , sacrococcygeal pilonidal sinus disease were enrolled for this prospective study from january 2002 to december 2008 regarding our modified simple closure of sacrococcygeal pilonidal sinus disease .
all procedures were performed at port - fouad general hospital , port - said , egypt by a single surgical team consisting of a consultant colorectal surgeon assisted by a general surgeon .
the surgical team treated 1 - 2 patients with pilonidal sinus disease per week in both private and public health sectors i.e. , over 500 patients , in total , during the study period .
patients were excluded from the study when they had a history of drained pilonidal abscess or recurrent disease , or declined the protocol .
patients under general anesthesia were placed in a prone , jack - knife position with buttocks widely separated using adhesive tapes , and methylene blue was injected into the sinuses .
modified d - shaped excisions were used [ figure 1a ] to include all sinuses and their ramifications to the presacral fascia and surrounding unhealthy tissues to achieve healthy , soft and supple wound margins [ figure 1b ] .
a suction drain was placed under the flap and was delivered through a separate stab incision .
the wound was closed using four or five deeply running sutures obliterating the dead space [ figure 2 ] .
the sutures were tied in a way to obliterate the dead space and permit the incision just lateral to the midline , while two sutures were left long enough to be tied over a cigar - like dressing to promote further obliteration of the dead space [ figure 3a and b ] .
the drain was removed after 48 hours and the two dressing sutures tying the dressing were cut short at the fourth day to exchange the dressing .
outpatient clinic follow - up occurred two weeks , one month , and three months after surgery , and after six months when necessary thereafter .
( a ) an operative photograph showing planning of offmidline incision ( b ) an operative photograph showing complete excision of the sinus tracks with good hemostasis an operative photograph showing planning of wound closure with the deeply running sutures in situ ( a ) an operative photograph showing complete wound closure with two sutures long enough to be tied over the dressing ( b ) an operative photograph showing final step with dressing being tightly in place the primary endpoints of the study were early failure , defined as wound disruption within 10 days of surgery and recurrence of the disease , defined as a clinically detectable sinus .
secondary endpoints were wound infection , time - off from work and painless walking and sitting time .
patients under general anesthesia were placed in a prone , jack - knife position with buttocks widely separated using adhesive tapes , and methylene blue was injected into the sinuses .
modified d - shaped excisions were used [ figure 1a ] to include all sinuses and their ramifications to the presacral fascia and surrounding unhealthy tissues to achieve healthy , soft and supple wound margins [ figure 1b ] .
a suction drain was placed under the flap and was delivered through a separate stab incision .
the wound was closed using four or five deeply running sutures obliterating the dead space [ figure 2 ] .
the sutures were tied in a way to obliterate the dead space and permit the incision just lateral to the midline , while two sutures were left long enough to be tied over a cigar - like dressing to promote further obliteration of the dead space [ figure 3a and b ] .
the drain was removed after 48 hours and the two dressing sutures tying the dressing were cut short at the fourth day to exchange the dressing .
outpatient clinic follow - up occurred two weeks , one month , and three months after surgery , and after six months when necessary thereafter .
( a ) an operative photograph showing planning of offmidline incision ( b ) an operative photograph showing complete excision of the sinus tracks with good hemostasis an operative photograph showing planning of wound closure with the deeply running sutures in situ ( a ) an operative photograph showing complete wound closure with two sutures long enough to be tied over the dressing ( b ) an operative photograph showing final step with dressing being tightly in place
the primary endpoints of the study were early failure , defined as wound disruption within 10 days of surgery and recurrence of the disease , defined as a clinically detectable sinus .
secondary endpoints were wound infection , time - off from work and painless walking and sitting time .
in the present study , there were 164 ( 82% ) male and 36 ( 18% ) female patients with age range from 15 - 33 years and male : female ratio was 4:1 .
a large majority of patients was between the ages of 15 and 25 , and the incidence or decreased thereafter [ table 1 ] .
patients with sex and subgroup distributions regarding the body mass index ( bmi ) , patients were classified as obese ( bmi > 30
kg / m ) , overweight ( bmi > 25 but < 30 kg / m ) and healthy weight ( bmi < 25 kg / m ) . by these definitions ,
64 patients were obese ( 52 males and 12 females ) , 100 were overweight ( 80 males and 20 females ) and 36 patients were of healthy weight ( 32 males and 4 females ) .
the average bmi for both sexes was 29.3 ( sd 2.8 , range 22.0 - 31.0 ) kg / m .
wound infection was observed in 24 patients ( 20 males , 4 females ) necessitating daily dressing with injection antibiotics .
the overall wound infection rate was 12% , ( 12.2% for males and 11.1% for females ) .
wound disruption was observed in 12 patients ( 10 males , 2 females ) necessitating open - wound management .
the overall wound disruption rate was 6% , ( 6.1% males , 5.5% females ) .
the overall recurrence rate was 7% , ( 6.1% males , 11.1% females ) [ tables 2 and 3 ] .
postoperative complications postoperative complications regarding the bmi the time required for pain - free walking after surgery was 20.5 3.6 days and the time required to achieve pain - free sitting on the toilet was 24 3.89 days . the time - off from work , defined as the number of days between the day of surgery and the first day a patient returned to work .
all patients were admitted as day - surgery cases ; 174 cases ( 87% ) ultimately conformed to the day - case surgery protocol with mean hospital stay 1.125 0.34 days .
after more than half a century , the best surgery for sacrococcygeal pilonidal sinus disease is still a subject of debate , and methods ranging from extensive excisions with complicated reconstructive procedures to limited debridement are being recommended with equal enthusiasm .
the main problems with the primary closure technique appear to be high - recurrence rate and high - infection rate . on the other hand ,
patients generally complain about open - packing or marsupialization methods because of painful wound management and dressing changes . in spite of the recently accepted superiority of the flap reconstructions to the non - flap techniques , morbidity related to infection and recurrence
has not been eliminated . despite the controversy about the best surgical technique for the treatment of pilonidal sinus ,
an ideal operation should minimize financial cost , allow patients to return earlier to work , be simple to perform , not require a prolonged hospital stay , inflict minimal pain , and have a low disease recurrence rate .
however , there is no surgical procedure satisfies these requirements as various operative methods utilized in the treatment of pilonidal disease are associated with a number of advantages and disadvantages .
postoperative complication rates of unroofing and marsupialization are low , but require long wound care . in addition , there were high complication rates in the primary closure and limberg flap groups .
so , the best option is to explain the advantages and disadvantages of the available surgical methods and respect the patient 's decision .
methods of simple primary closure can be broadly categorized as midline closure and off- midline techniques .
karydakis was the first to advocate off - midline asymmetric closure of pilonidal wounds to decrease recurrence by avoiding placing a wound in the midline at the depth of the natal cleft .
his off - midline procedure also flattens the cleft , reducing hair accumulation and mechanical irritation .
a clear benefit was shown in favor of off - midline rather than midline wound closure so that , the closure of pilonidal sinuses is the desired surgical option , off - midline closure is now the standard strategy . regarding the postoperative complications in the primary closure techniques , the wound infection was reported as ( 17.2% ) , and wound dehiscence as ( 13.8% ) with no hematoma formation , whereas those in the flap repair , the wound infection was ( 4.5% ) , hematoma was ( 6.8% ) , and wound dehiscence was ( 4.5% ) . according to other series with simple closure technique , it was stated that wound dehiscence was 7.7% and recurrence rate was 5% .
another study reported the postoperative complication rate in flap construction as wound infection 20% , wound dehiscence 10% and recurrence 10% .
our data came in concordance with those studies regarding the wound infection , wound dehiscence and recurrence with the simplest and less invasive technique .
it is now generally agreed that minimally invasive surgery should be used to treat pilonidal disease , whenever possible .
ghnnam and his colleagues agreed the same opinion of less invasive surgery that pilonidal sinus could be treated with limited excision and primary closure when they performed excision and primary midline closure .
the incidence of the wound infection , wound dehiscence and recurrence is directly proportional with value of bmi .
two recent studies showed that among patients with recurrent pilonidal sinus , majority had bmi 30 and 25 to 30 kg / m .
our data supported these results , where most of our patients with wound dehiscence or recurrence were of bmi 30 and bmi of 25 to 30 kg / m .
the results favoring flap closure in the treatment of sacrococcygeal pilonidal sinus are low recurrence rates , shorter hospital stay , and time off from work but the disadvantages related to unfavorable cosmetic appearance and body image are troubling
. the limitations of flap techniques are noteworthy : the unfavorable cosmetic appearance ; the surgical length and hospitalization .
the flap surgery takes longer than primary closure , requiring an additional attempt of reconstruction , which is the most complex step of the surgical treatment .
important goals in the surgical treatment of sacrococcygeal pilonidal sinus disease include minimized hospital stays , minimized time to return to work and daily activities , and esthetic satisfaction ; no single technique currently optimizes all of these criteria .
for example , patients generally complain about open packing and marsupialization methods because of prolonged and painful wound management and dressing changes .
primary closure techniques are more acceptable in those regards , but have the disadvantage of generally higher recurrence and infection rates .
flap closure has important advantages and disadvantages relative to primary closure : considerations that favor flap closure include lower recurrence rates , shortened hospital stays , and less time - off from work .
for example , a recent study compared excision with primary closure to limberg flap in terms of time required for pain - free walking ; pain - free sitting on the toilet after surgery ; and return to work .
the limberg flap technique proved superior .. importantly , however , flap surgery takes longer than primary closure because it requires additional reconstruction ; the most complex aspect of the surgery .
the simplified off - midline closure technique described in the present study may offer a favorable alternative to the flap closure : like flap closure , the simplified procedure was associated with less time required for pain - free walking , pain - free sitting on the toilet and time off from work than has been reported with other techniques .
postoperative esthetic satisfaction has been reported to be higher in patients with simplified closures , an advantage that appears to be due to the simplified technique itself , rather than due to differences in rates of wound infection or recurrence . avoiding flap reconstruction ,
the cosmetic result was better in patients with simple closure and the flap construction turned out worst while neither wound infection nor recurrence had any influence on the cosmetic result .
this means that the technique itself is responsible for the patient satisfaction of his body image after surgery .
for simple non - recurrent pilonidal sinus , less invasive surgery with limited excision and primary closure could be enough .
simple off - midline technique advocates asymmetric closure of the wound leading to decrease both wound infection and recurrence by avoiding placing a wound in the midline at the depth of the natal cleft .
the present study represented data comparable with the more aggressive used excision methods regarding the wound infection , wound dehiscence and recurrence . for simple non - recurrent pilonidal sinus disease , our simplification of off - midline limited excision with primary closure may provide a favorable alternative to conventional off - midline excisions , which involve flap closure .
our simplified procedure resembled conventional off - midline techniques in terms of favorable wound infection , wound dehiscence and recurrence rates , whereas advantages of our simplified procedure include potentially reduced surgery complexity , reduced surgery time , and improved cosmetic outcome . | background : numerous surgical procedures have been described for pilonidal sinus disease , but treatment failure and disease recurrence are frequent . conventional off - midline flap closures have relatively favorable surgical outcomes , but relatively unfavorable cosmetic outcomes.aim:the author reported outcomes of a new simplified off - midline technique for closure of the defect after complete excision of the sinus tracts.patients and methods : two hundred patients of both sexes were enrolled for modified d - shaped excisions were used to include all sinuses and their ramifications , with a simplified procedure to close the defect.results:the overall wound infection rate was 12% , ( 12.2% for males and 11.1% for females ) .
wound disruption was necessitating laying the whole wound open and management as open technique .
the overall wound disruption rate was 6% , ( 6.1% for males and 5.5% for females ) and
the overall recurrence rate was 7%.conclusion : our simplified off - midline closure without flap appeared to be comparable to conventional off - midline closure with flap , in terms of wound infection , wound dehiscence , and recurrence .
advantages of the simplified procedure include potentially reduced surgery complexity , reduced surgery time , and improved cosmetic outcome . |
isolated dislocation of the tarsal naviculum is scarcely reported in the literature.1 some few similar injuries have been described previously , whereas , they were often associated with fractures in the navicular bone or in the other midfoot bones.2 the rarity of isolated dislocation in tarsal naviculum is explained by the rigid bony support around it in the midfoot and hindfoot , moreover , the presence of sturdy ligamentous attachments .
the treatment of such entity is until now controversial.2 several options have been described based on reduction and stabilization .
a 31 year old male driver , who sustained injury in a motor vehicle accident , presented with complain of pain and deformity of his left ankle and foot .
the clinical examination revealed an obvious deformity and swelling on dorsomedial aspect of his left midfoot [ figure 1 ] .
x - ray findings showed pure isolated dorsal dislocation of the tarsal naviculum without fractures [ figure 2 ] .
hence , both open reduction and cuneio - naviculo - talar stabilization were achieved with krischner - wires ( k - wires ) [ figure 4 ] .
photograph of medial and dorsal clinical aspects of the left foot immediately after the injury showing swelling on dorsomedial aspect of foot anteroposterior and lateral x - ray of the left foot anteroposterior and lateral views showing a dislocation of the navicular bone sagittal computed tomography views of the foot revealing the isolated dorsal dislocation of the navicular bone peroperative photograph showing the dislocated navicular bone after the dorsal incision toward the talo navicular joint anatomical reduction of the naviculum was achieved [ figure 5 ] .
after surgery , the patient was made no weight bearing on the left lower extremity . at 6 weeks
postoperatively , the wires were removed , the radiographs confirmed that naviculum and midfoot are reduced in anatomical position .
the patient was given a removable , short leg boot and instructed to remain nonweight bearing on the operated foot .
partial weight bearing was started at the beginning of the 3 month postoperatively and progressed gradually total weight bearing at the end of the 3 month . at 6 months
postoperatively , the patient is ambulating in his regular shoes and has returned to his previous activities .
the eversion was 20 and inversion was 30. x - ray showed an anatomical reduction without abnormalities of the midfoot [ figure 6 ] .
the functional outcome of such entity is satisfied with normal values of motion [ figure 7 ] .
postoperative x - ray of foot anteroposterior and oblique views showing reduction and fixation of the navicular bone with k - wires . the talonaviculocuneiform fixation x - ray of the left foot anteroposterior and oblique views at last followup showing outcome without abnormalities in both talo - navicular and cuneo - navicular joints clinical photographs showing the functional outcome at the last follow up ; ( a and b ) left foot on lateral and medial views .
isolated tarsal navicular dislocations are rare.12 reviewing the literature , only 15 cases have been reported since1924.1234 usually , associated midfoot fractures , dislocations , or soft tissue damage were found simultaneously to navicular dislocation.4 the rarity of this dislocation is explained by the strong dorsal and volar ligamentous attachments and the rigid bony support surrounding the navicular.45678 several mechanisms of tarsal navicular dislocation have been reported.45 however , they are complex and not well understood.24 no specific mechanism is established for the isolated dorsal dislocation.4 the most reported one is a plantar flexion / compressive injury.45910 the position of the foot and the direction of the force at injury will determine the direction of the dislocation .
main and jowett11 had considered that longitudinal forces transmitted along the metatarsal rays would compress the navicular , produce dorsal dislocation ; this theory was based on radiographic appearances.11 whereas , this implies rupture of the dorsal ligaments , which could fail only in tension produced by plantar flexion .
knowledge of this mechanism is useful when performing.7 this makes it likely that his left foot was pressing on the brake pedal and in a plantar flexed position at impact .
associated fractures of the cuboid and calcaneus may occur when there are a hyper plantar flexion and inversion force applied to the midfoot and the location of the bifurcate ligament.46 the mechanism of a navicular dislocation has been described as being similar to a perilunate dislocation of the hand.46 some authors think that tarsal navicular dislocation can not occur without bony or ligamentous damage to the lateral column of the foot because this injury inevitably involves both columns.46 as a result , stabilization of both medial and lateral columns is recommended to have a good outcome even in cases when the dislocation seems to be isolated.45 the stability of the foot is ensured by the rigidity of both of these columns , and each supports the other.4 according to this concept , it is impossible to sustain an isolated dislocation at any level in either of the two bony columns , without disruption of the bony or ligamentous anatomy of the adjacent column ; however , internal fixation of the lateral column with k - wires was not necessary .
this allowed the injured ligaments to heal with the lateral column reduced in an anatomic position .
the appropriate treatment of this injury should consist on anatomical reduction and k - wire fixation across the naviculocuneiform and talonavicular joints , supplemented with spanning external fixation , which provides stabilization of both longitudinal columns of the foot .
percutaneous k - wire fixation of the lateral column should also be added if the injury to the lateral column is severe enough and not reducible with external fixation alone .
open reduction is recommended.461011 closed reduction alone has been shown to lead to recurrent dislocation and osteoarthritis.10 some complications may occur after complete navicular dislocation , but they are not well known .
avascular necrosis of the navicular bone is the most described complication , it is estimated at nearly ( 25% ) from all reported cases.467 the blood supply to the navicular bone is by the small branches of the dorsalis pedis and posterior tibial arteries .
after dislocation , the blood supply to the navicular is ensured by the posterior tibialis tendon.4 if the attachment of this tendon to the navicular bone is disrupted , avascular necrosis is likely to develop .
other complications have been reported like residual subluxation of the naviculum , flatfoot deformity , posttraumatic arthritis.56 at both talonavicular and naviculocuneiform joints , stiffness of the foot and soft tissue compromise.4811 concerning k - wire fixation , it is recommended to remain in place for 6 months to reduce the rate of future subluxation of the naviculum after removal of the k - wires.12 the stabilization of the medial column leads to ligamentous repair .
however ; we think that stabilization of the lateral column may reduce the midfoot pain and swelling revealed in our case .
external fixation of the lateral column seems to be an alternative to solve these problems . to conclude the present rare case report , despite the restricted followup data , adds to the limited amount of information available on these injuries and presents a viable treatment option according to the injury pattern and stability of the midfoot . | isolated dislocation of the tarsal naviculum is an unusual injury , scarcely reported in the literature .
the naviculum is surrounded by the rigid bony and ligamentous support hence fracture dislocation is more common than isolated dislocation .
the mechanism and treatment options remain unclear . in this case report
, we describe a 31 year old man who sustained an isolated dorsal dislocation of the left tarsal naviculum , without fracture , when he was involved in a motor vehicle collision .
the reported mechanism of the dislocation is a hyper plantar flexion force applied to the midfoot , resulting in a transient disruption of the ligamentous support of the naviculum bone , with dorsal displacement of the bone .
the patient was treated with open reduction and krischner - wire fixation of the navicular after the failure of closed reduction .
the wires were removed after 6 weeks postoperatively .
physiotherapy for stiffness and midfoot pain was recommended for 2 months . at 6 months
postoperatively , limping , midfoot pain and weakness were reported , no x - ray abnormalities were found .
the patient returned to his obvious activities with a normal range of motion . |
the golgi complex is the central secretory organelle of most eukaryotic cells and consists of membranous stacks called cisternae
. secreted proteins , like all other proteins , are synthesized in the endoplasmic reticulum ( er ) and are specifically packaged into vesicles that bud off from the er in a gtp - dependent process .
these lipid vesicles are coated with the copii coat protein - complex and are equipped with the atp - dependent vesicle - fusion apparatus .
they carry the secretory cargo to the cis surface of the golgi complex , with which they fuse , delivering the cargo .
a second type of vesicle , coated by the copi coat - protein complex , is part of a retrograde pathway that buds off the golgi membrane and returns proteins that are not targeted for secretion back to the endoplasm .
studies on the secretory system in crown - group eukaryotes ( plants , animals and fungi ) have uncovered a family of proteins , the p24 ( p24/gp25l / emp24/erp ) family , that have an important role in cargo selection and packaging into copii - coated vesicles .
additionally , they might also function in excluding secreted proteins from copi - coated retrograde vesicles .
members of the p24 family are type i membrane proteins , with a small carboxy - terminal cytoplasmic tail that interacts with the vesicle coat proteins and a globular lumenal region that probably interacts with the cargo .
they are abundantly distributed on the membranes of the vesicles budding off the er and the cis golgi membranes .
the p24 proteins belong to at least four distinct subfamilies and form hetero - oligomeric complexes that contain at least one member from each subfamily .
this heteromerization of the p24 proteins has been shown to require a coiled - coil stretch at the extreme carboxyl terminus of their lumenal regions .
improved understanding of the p24 family may throw light on evolution and function of the golgi apparatus in eukaryotes . with this objective
, we conducted a computational sequence analysis of the p24 proteins and show that they contain a conserved globular domain that is also present in several other golgi and lipid - traffic proteins .
we present evidence that this module is likely to serve as a common denominator in protein - protein interactions in several distinct contexts , such as in secretory vesicles and on the golgi peripheral membrane .
the proliferation of this superfamily appears to have been central to the diversification of the eukaryotic secretory apparatus .
the bona fide p24 proteins contain a short carboxy - terminal tail that interacts with the cop - complex proteins through specific short peptide motifs .
the amino - terminal region that faces the lumen is much larger and is predicted to form a compact globular unit . as this region of
the protein is likely to contain a conserved globular domain that mediates other functional interactions of these proteins , we sought to investigate its complete diversity and potential evolutionary connections .
we carried out a profile search of the non - redundant protein database ( of the national center for biotechnology information , ncbi ) using the psi - blast program , seeded with lumenal region of the caenorhabditis elegans p24 family member k08e4.6 ( the profile - inclusion threshold was set at 0.01 and the search iterated until convergence ) .
this search readily detected the classical p24 family members that are found in six to nine copies in the proteomes of most organisms belonging to the eukaryotic crown group .
in addition , this search retrieved several other proteins that do not belong to the p24 family with statistically significant expectation ( e)-values ( e < 0.001 , see figure 1 legend ) .
these proteins include yeast osh3p , a cytoplasmic oxysterol - binding protein , animal sec14-like proteins that are involved in secretion , human gcp60 ( also called pap7 , a peripheral - type benzodiazepine receptor - associated protein ) , which interacts with the golgi integral membrane protein giantin , and several other uncharacterized eukaryotic proteins with different lipid - binding domains ( figure 1 ) .
reciprocal searches initiated with this region from the newly detected proteins showed that they were more closely related to each other , but in subsequent iterations they recovered the classic p24 family members at significant e - values , suggesting that all these conserved regions define a novel superfamily of protein domains
. separate prediction of the secondary structure of this domain from the p24 family and the newly detected proteins , showed that the two groups had essentially the same core structural elements , further reinforcing their relationship .
as this conserved domain is present in at least three distinct classes of proteins related to golgi dynamics ( animal sec14 proteins , the p24 family and gcp60-like proteins ) , we name this conserved region the gold domain . the presence of the gold domain at the extreme amino or carboxyl terminus of the osh3p and animal sec14 proteins , respectively , allowed us to establish accurate boundaries for it .
the domain is typically between 90 and 150 amino acids long and , in the p24 family , it comprises almost the entire lumenal region , with the exception of an -helical extension of approximately 50 amino acids that precedes the transmembrane segment .
most of the size difference observed in the gold - domain superfamily is traceable to a single large low - complexity insert that is seen in some versions of the domain .
a secondary - structure prediction for the domain using the phd program reveals that it is likely to adopt a compact all--fold structure with six to seven strands .
most of the sequence conservation is centered on the hydrophobic cores that support these predicted strands .
the predicted secondary - structure elements and the size of the conserved core of the domain suggests that it may form a -sandwich fold with the strands arranged in two sheets stacked on each other .
experimental studies so far on diverse proteins containing gold domains point to a role for it in protein - protein interactions .
a region of the gpc60 molecule that rather precisely encompasses the gold domain has been shown to bind to the cytoplasmic region of the golgi membrane protein giantin .
cross - linking experiments have suggested that the p24 proteins interact directly with the cargo molecules that are present in the lumen of the copii - coated vesicles and that they are , accordingly , cargo receptors .
however , yeast deletion mutants lacking all the p24 proteins grow similarly to wild type , although they show delays in translocation of a subset of cargo molecules such as invertase and gas1p from the er to the golgi , and increased secretion of resident er proteins . certain members of the p24 family from vertebrates have also been shown to bind to specific ligands such as the interleukin-1 receptor - like molecule t1/st2 and might aid its proper expression on the cell surface .
these observations suggest that the p24 subset of the gold domains probably function as discriminators that selectively interact with particular proteins to influence their loading into vesicles .
the gold domains show considerable variability in some of the loops that are predicted to extrude from the core -sandwich - like structure ( figure 1 ) .
these loops might form exposed surfaces that provide the gold domains with the discriminatory capacity necessary for their interactions with diverse ligands . with the exception of the p24 proteins , which have a simple architecture with the gold domain as their only globular domain , all other gold - domain proteins contain additional conserved globular domains ( figure 2 ) . in these proteins , the gold domain co - occurs with lipid- , sterol- or fatty acid - binding domains such as ph , sec14p , fyve , oxysterol binding- and acyl coa - binding domains , suggesting that these proteins may interact with membranes .
the fyco1 protein that combines a gold domain with a fyve domain , also contains a run domain , an uncharacterized -helical domain that may have a role in the interaction of various proteins with cytoskeletal filaments .
an orthologous group of proteins typified by human sec14l1 , which is conserved in all animals , has , in addition to the carboxy - terminal fusion of the sec14p and gold domains , a previously unrecognized , conserved amino - terminal domain ( figures 2,3 ) .
this domain has so far been found only in eukaryotes , and occurs in stand - alone form in several proteins , including the human preli protein and the yeast msf1p ' protein .
the preli / msf1p ' domain is approximately 170 residues long and is predicted to assume a globular + fold with six strands and four helices ( figure 3 ) .
msf1p ' is proposed to be involved in mitochondrial protein sorting , suggesting that the preli / msf1p ' domain may also have a function associated with cellular membranes .
thus , all gold - domain proteins can be divided into two architectural categories : the p24-like category , in which the gold domains project into the lumen , anchored in the membrane by the membrane - spanning helix ( category 1 ) ; and proteins in which the gold domain occurs at the extreme amino or carboxyl terminus , with additional domains that are known to interact with lipid membranes ( category 2 ) ( figure 2 ) .
gcp60 , which is peripherally associated with the golgi membrane , is one of the proteins in the second category that has been experimentally characterized .
it has been shown that overexpression of a region of this protein encompassing the gold domain caused disassembly of the golgi structure and abrogated protein transport from the er to the golgi .
these observations can be accommodated by two ( not mutually exclusive ) hypotheses regarding the functions of these proteins .
the gold proteins belonging to the second architectural category could function as double - headed adaptors that interact with both a specific protein ( via the gold domain ) and different cellular lipid membranes .
thus , gcp60 and gold proteins with analogous architectures could help in the assembly of vesicular or golgi - membrane - associated protein complexes by tethering specific proteins to the membranes , with the gold domain binding the protein targets and the lipid - binding protein to the membrane . alternatively , at least some of the category-2 proteins could function as a previously unrecognized class of vesicular cargo - loading molecules that associate with the membrane via their lipid - binding domains and deliver their protein ligands via the gold domain .
the observation that deletion mutants lacking all the p24 proteins still show normal trafficking of certain proteins such as carboxypeptidase y , suggests that there are some protein - trafficking pathways that are unaffected by their absence .
thus , the gold - domain proteins of category 2 may have a specific role in regulating the secretion of molecules that are not affected by the p24 proteins .
the hetero - oligomerization of the p24 proteins via the coiled - coil regions carboxy - terminal to the gold domain seems to help in generating combinatorial diversity for their interactions with multiple ligands .
the presence of extensive coiled - coil segments in some of the category-2 gold - domain proteins , such as fyco1 , suggests that they might also form oligomers , like the p24 proteins .
similarity - based clustering and phylogenetic analysis divides the gold domains into two primary divisions that precisely mirror the two categories established on the basis of domain architectures ( figure 2 ) .
this division was also supported by a synapomorphic ( shared derived ) feature in the form of two conserved cysteines , which is restricted to the p24 family ( category-1 proteins ) . likewise , the presence of a specific insert between strand 1 and 2 with a characteristic conserved tryptophan serves as a synapomorphic feature for category-2 gold domains ( figure 1 ) .
an analysis of the phyletic patterns suggests that the p24 family had already differentiated into at least four distinct subfamilies in the common ancestor of plants , animals and fungi .
the detection of multiple members of the p24 family in the early branching eukaryotes such as cryptosporidium parvum and kinetoplastids suggests that some of this diversification was probably already under way early in eukaryotic evolution . within the eukaryotic crown group
, we obtained evidence of specific instances of duplications and gene losses that are restricted to particular lineages .
the most striking case is seen in arabidopsis thaliana , which appears to have proliferated the erv25 subfamily ( five to six members ) , but lacks the erp2p and erp5p subfamilies .
the second major family of gold domains ( category 2 ) is so far only attested in the crown group . in fungi , this group is typified by saccharomyces cerevisiae osh3p , which combines an amino - terminal gold domain with ph and oxysterol - binding domains .
the greatest architectural diversity of this group is seen in animals ( figure 2 ) , suggesting that there was increased proliferation and domain shuffling among these proteins concomitant with the evolutionary emergence of the animals .
the bona fide p24 proteins contain a short carboxy - terminal tail that interacts with the cop - complex proteins through specific short peptide motifs .
the amino - terminal region that faces the lumen is much larger and is predicted to form a compact globular unit . as this region of
the protein is likely to contain a conserved globular domain that mediates other functional interactions of these proteins , we sought to investigate its complete diversity and potential evolutionary connections .
we carried out a profile search of the non - redundant protein database ( of the national center for biotechnology information , ncbi ) using the psi - blast program , seeded with lumenal region of the caenorhabditis elegans p24 family member k08e4.6 ( the profile - inclusion threshold was set at 0.01 and the search iterated until convergence ) .
this search readily detected the classical p24 family members that are found in six to nine copies in the proteomes of most organisms belonging to the eukaryotic crown group .
in addition , this search retrieved several other proteins that do not belong to the p24 family with statistically significant expectation ( e)-values ( e < 0.001 , see figure 1 legend ) .
these proteins include yeast osh3p , a cytoplasmic oxysterol - binding protein , animal sec14-like proteins that are involved in secretion , human gcp60 ( also called pap7 , a peripheral - type benzodiazepine receptor - associated protein ) , which interacts with the golgi integral membrane protein giantin , and several other uncharacterized eukaryotic proteins with different lipid - binding domains ( figure 1 ) .
reciprocal searches initiated with this region from the newly detected proteins showed that they were more closely related to each other , but in subsequent iterations they recovered the classic p24 family members at significant e - values , suggesting that all these conserved regions define a novel superfamily of protein domains
. separate prediction of the secondary structure of this domain from the p24 family and the newly detected proteins , showed that the two groups had essentially the same core structural elements , further reinforcing their relationship .
as this conserved domain is present in at least three distinct classes of proteins related to golgi dynamics ( animal sec14 proteins , the p24 family and gcp60-like proteins ) , we name this conserved region the gold domain . the presence of the gold domain at the extreme amino or carboxyl terminus of the osh3p and animal sec14 proteins , respectively , allowed us to establish accurate boundaries for it .
the domain is typically between 90 and 150 amino acids long and , in the p24 family , it comprises almost the entire lumenal region , with the exception of an -helical extension of approximately 50 amino acids that precedes the transmembrane segment .
most of the size difference observed in the gold - domain superfamily is traceable to a single large low - complexity insert that is seen in some versions of the domain .
a secondary - structure prediction for the domain using the phd program reveals that it is likely to adopt a compact all--fold structure with six to seven strands .
most of the sequence conservation is centered on the hydrophobic cores that support these predicted strands .
the predicted secondary - structure elements and the size of the conserved core of the domain suggests that it may form a -sandwich fold with the strands arranged in two sheets stacked on each other .
experimental studies so far on diverse proteins containing gold domains point to a role for it in protein - protein interactions .
a region of the gpc60 molecule that rather precisely encompasses the gold domain has been shown to bind to the cytoplasmic region of the golgi membrane protein giantin .
cross - linking experiments have suggested that the p24 proteins interact directly with the cargo molecules that are present in the lumen of the copii - coated vesicles and that they are , accordingly , cargo receptors .
however , yeast deletion mutants lacking all the p24 proteins grow similarly to wild type , although they show delays in translocation of a subset of cargo molecules such as invertase and gas1p from the er to the golgi , and increased secretion of resident er proteins . certain members of the p24 family from vertebrates have also been shown to bind to specific ligands such as the interleukin-1 receptor - like molecule t1/st2 and might aid its proper expression on the cell surface .
these observations suggest that the p24 subset of the gold domains probably function as discriminators that selectively interact with particular proteins to influence their loading into vesicles .
the gold domains show considerable variability in some of the loops that are predicted to extrude from the core -sandwich - like structure ( figure 1 ) .
these loops might form exposed surfaces that provide the gold domains with the discriminatory capacity necessary for their interactions with diverse ligands .
with the exception of the p24 proteins , which have a simple architecture with the gold domain as their only globular domain , all other gold - domain proteins contain additional conserved globular domains ( figure 2 ) . in these proteins ,
the gold domain co - occurs with lipid- , sterol- or fatty acid - binding domains such as ph , sec14p , fyve , oxysterol binding- and acyl coa - binding domains , suggesting that these proteins may interact with membranes .
the fyco1 protein that combines a gold domain with a fyve domain , also contains a run domain , an uncharacterized -helical domain that may have a role in the interaction of various proteins with cytoskeletal filaments .
an orthologous group of proteins typified by human sec14l1 , which is conserved in all animals , has , in addition to the carboxy - terminal fusion of the sec14p and gold domains , a previously unrecognized , conserved amino - terminal domain ( figures 2,3 ) .
this domain has so far been found only in eukaryotes , and occurs in stand - alone form in several proteins , including the human preli protein and the yeast msf1p ' protein .
the preli / msf1p ' domain is approximately 170 residues long and is predicted to assume a globular + fold with six strands and four helices ( figure 3 ) .
msf1p ' is proposed to be involved in mitochondrial protein sorting , suggesting that the preli / msf1p ' domain may also have a function associated with cellular membranes .
thus , all gold - domain proteins can be divided into two architectural categories : the p24-like category , in which the gold domains project into the lumen , anchored in the membrane by the membrane - spanning helix ( category 1 ) ; and proteins in which the gold domain occurs at the extreme amino or carboxyl terminus , with additional domains that are known to interact with lipid membranes ( category 2 ) ( figure 2 ) .
gcp60 , which is peripherally associated with the golgi membrane , is one of the proteins in the second category that has been experimentally characterized .
it has been shown that overexpression of a region of this protein encompassing the gold domain caused disassembly of the golgi structure and abrogated protein transport from the er to the golgi .
these observations can be accommodated by two ( not mutually exclusive ) hypotheses regarding the functions of these proteins .
the gold proteins belonging to the second architectural category could function as double - headed adaptors that interact with both a specific protein ( via the gold domain ) and different cellular lipid membranes .
thus , gcp60 and gold proteins with analogous architectures could help in the assembly of vesicular or golgi - membrane - associated protein complexes by tethering specific proteins to the membranes , with the gold domain binding the protein targets and the lipid - binding protein to the membrane . alternatively
, at least some of the category-2 proteins could function as a previously unrecognized class of vesicular cargo - loading molecules that associate with the membrane via their lipid - binding domains and deliver their protein ligands via the gold domain .
the observation that deletion mutants lacking all the p24 proteins still show normal trafficking of certain proteins such as carboxypeptidase y , suggests that there are some protein - trafficking pathways that are unaffected by their absence .
thus , the gold - domain proteins of category 2 may have a specific role in regulating the secretion of molecules that are not affected by the p24 proteins .
the hetero - oligomerization of the p24 proteins via the coiled - coil regions carboxy - terminal to the gold domain seems to help in generating combinatorial diversity for their interactions with multiple ligands .
the presence of extensive coiled - coil segments in some of the category-2 gold - domain proteins , such as fyco1 , suggests that they might also form oligomers , like the p24 proteins .
similarity - based clustering and phylogenetic analysis divides the gold domains into two primary divisions that precisely mirror the two categories established on the basis of domain architectures ( figure 2 ) .
this division was also supported by a synapomorphic ( shared derived ) feature in the form of two conserved cysteines , which is restricted to the p24 family ( category-1 proteins ) .
likewise , the presence of a specific insert between strand 1 and 2 with a characteristic conserved tryptophan serves as a synapomorphic feature for category-2 gold domains ( figure 1 ) .
an analysis of the phyletic patterns suggests that the p24 family had already differentiated into at least four distinct subfamilies in the common ancestor of plants , animals and fungi .
the detection of multiple members of the p24 family in the early branching eukaryotes such as cryptosporidium parvum and kinetoplastids suggests that some of this diversification was probably already under way early in eukaryotic evolution . within the eukaryotic crown group , we obtained evidence of specific instances of duplications and gene losses that are restricted to particular lineages .
the most striking case is seen in arabidopsis thaliana , which appears to have proliferated the erv25 subfamily ( five to six members ) , but lacks the erp2p and erp5p subfamilies .
the second major family of gold domains ( category 2 ) is so far only attested in the crown group . in fungi , this group is typified by saccharomyces cerevisiae osh3p , which combines an amino - terminal gold domain with ph and oxysterol - binding domains .
the greatest architectural diversity of this group is seen in animals ( figure 2 ) , suggesting that there was increased proliferation and domain shuffling among these proteins concomitant with the evolutionary emergence of the animals .
a novel -strand - rich domain was identified in numerous eukaryotic proteins , including the p24 proteins , which appear to have a function related to the golgi complex , secretion or protein sorting .
other than the p24 proteins , gold domains are present in several proteins where they occur at the extreme termini and are combined with diverse membrane- or lipid - binding domains .
these proteins are predicted to be double - headed adaptors that may help in the assembly of protein complexes on membranes or in the packaging of specific cargo molecules in membranous vesicles .
the identification to the gold domain may help in a directed dissection of p24-family function and provide novel candidate molecules for experimental studies on secretion and sorting .
the non - redundant ( nr ) database of protein sequences ( national center for biotechnology information , nih , bethesda ) was searched using the blastp program .
profile searches were conducted using the psi - blast program with either a single sequence or an alignment used as the query , with a profile - inclusion expectation ( e)-value threshold of 0.01 , and were iterated until convergence .
previously known conserved protein domains were detected using the corresponding psi - blast - derived position - specific scoring matrices ( pssms ) .
the pssms were prepared by choosing one or more starting queries ( seeds ) for a set of most frequently encountered domains ( see reference for details ) and run against the nr database until convergence with the -c option of psi - blast to save the pssm .
this profile database can be downloaded from or used on the internet via the rps - blast program .
all globular segments of proteins that did not map to domains with previously constructed pssms were searched individually using psi - blast to detect any additional domains that may have been overlooked .
multiple alignments were constructed using the t - coffee program , followed by manual correction based on the psi - blast results .
protein secondary structure was predicted using a multiple alignment as the input for the phd program .
signal peptides were predicted using the signalp program and the transmembrane regions were predicted using the topred program .
phylogenetic analysis was carried out using the maximum likelihood , neighbor - joining and least - squares methods .
briefly , this process involved the construction of a least - squares tree using the fitch program or a neighbor - joining tree using the neighbor program ( both from the phylip package ) , followed by local rearrangement using the protml program of the molphy package to arrive at the maximum likelihood ( ml ) tree .
the statistical significance of various nodes of this ml tree was assessed using the relative estimate of logarithmic likelihood bootstrap ( protml rell - bp ) with 10,000 replicates .
a multiple alignment of the gold domain was constructed using t - coffee and realigning the sequences by parsing high - scoring pairs from psi - blast search results .
the phd - secondary structure is shown above the alignment , with e representing a strand ( upper - case letters indicate predictions with > 82% accuracy , and lower - case letters denote predictions with > 72% accuracy ) .
a search with the lumenal region of caenorhabditis elegans p24-family member k08e4.6 ( region 20 - 191 ) recovers ralbp from todarodes pacificus , the squid ortholog of sec14l2 ( e = 7 10 , iteration 1 ) . a reciprocal search with ralbp_todarodes pacificus ( region 189 - 343 ) recovers gcp60 ( e = 2 10 , iteration 1 ) , fyco1 ( 8 10 , iteration 1 ) , spac23h4.01c ( 10 , iteration 2 ) , kiaa0420 ( 10 , iteration 2 ) and k08e4.6 ( 7 10 , iteration 2 ) .
the 80% consensus shown below the alignment was derived using the following amino - acid classes : h , hydrophobic ( alicvmyfw , yellow shading ) ; l , the aliphatic subset of the hydrophobic class ( alivmc , yellow shading ) ; a , aromatic ( fhwy , yellow shading ) ; s , small ( acdgnpstv , green letters ) ; u and p are the tiny subsets of the small class ( u , gas , green shading ) and polar ( p , cdehknqrst , blue letters ) .
the different families are shown on the right . a , p24 family ; b , osh3p family ; c , cg9528 family ( sec14-like proteins with an amino - terminal preli / msf1p ' domain )
; d , sec14-like proteins ; e , gcp60 family ; and f , fyco1 .
the sequences are denoted by their gene name followed by the species abbreviation and genbank identifier .
ce , caenorhabditis elegans ; dm , drosophila melanogaster ; hs , homo sapiens ; sc , saccharomyces cerevisiae ; sp , schizosaccharomyces pombe ; top , todarodes pacificus .
a phylogenetic tree of the gold - domain - containing proteins is shown along with the various architectures , drawn approximately to scale , and the phyletic distributions of individual architectural classes .
the thickness of a given branch is approximately proportional to the number of proteins contained within it .
ph , pleckstrin - homology domain ; oxb , oxysterol - binding domain ; aco , acyl - coa - binding domain ; sec14 , domain found in sec14 proteins ; run ( for rpip8 , unc-14 and nesca ) and fyve ( for fab1p , yotb , vac1p and eea1 ) .
a multiple alignment of the preli / msf1p ' domain was constructed using t - coffee and realigning the sequences by parsing high - scoring pairs from psi - blast search results .
the phd - secondary structure is shown above the alignment with e representing a strand ( upper - case is for predictions with > 82% accuracy ; lower - case denotes predictions with > 72% accuracy ) .
the 100% consensus shown below the alignment was derived using the following amino - acid classes : h , hydrophobic ( alicvmyfw , yellow shading ) ; l , the aliphatic subset of the hydrophobic class ( alivmc , yellow shading ) ; a , aromatic ( fhwy , yellow shading ) ; c , charged ( dehkr , pink letters ) ; s , small ( acdgnpstv , green letters ) and p , polar ( cdehknqrst , blue letters ) .
the limits of the domains are indicated by the residue positions on each side ( except for the unfinished genome of cryptosporidium parvum ) .
the preli and kisir subgroups contain stand - alone versions of the domain , whereas the cg9528 family comprises sec14-like proteins with an amino - terminal preli and a carboxy - terminal gold domain .
the sequences are denoted by their gene name followed by the species abbreviation and genbank identifier . at , arabidopsis thaliana ; ce , caenorhabditis elegans ; cpar , cryptosporidium parvum ; dm , drosophila melanogaster ; hs , homo sapiens ; mm , mus musculus ; sc , saccharomyces cerevisiae ; sp , schizosaccharomyces pombe . | backgroundmembers of the p24 ( p24/gp25l / emp24/erp ) family of proteins have been shown to be critical components of the coated vesicles that are involved in the transportation of cargo molecules from the endoplasmic reticulum to the golgi complex .
the p24 proteins form hetero - oligomeric complexes and are believed to function as receptors for specific secretory cargo.resultsusing sensitive sequence - profile analysis methods , we identified a novel -strand - rich domain , the gold ( golgi dynamics ) domain , in the p24 proteins and several other proteins with roles in golgi dynamics and secretion .
this domain is predicted to mediate diverse protein - protein interactions .
other than in the p24 proteins , the gold domain is always found combined with lipid- or membrane - association domains such as the pleckstrin homology ( ph ) , sec14p and fyve domains.conclusionsthe identification of the gold domain could aid in directed investigation of the role of the p24 proteins in the secretion process . the newly detected group of gold - domain proteins , which might simultaneously bind membranes and other proteins , point to the existence of a novel class of adaptors that could have a role in the assembly of membrane - associated complexes or in regulating assembly of cargo into membranous vesicles
. |
steptoe et al . defined health behavior as the activities undertaken by people in order to protect , promote or maintain health and to prevent disease.1 the personal health behavior is important in individual for the maintenance of general health or total health of the body and this depends to some or more extent on the oral health behavior .
many systemic diseases are related to oral conditions and thus general health requires efforts of both medical and dental health professionals.2 - 4 the dental health professionals can play an important role in the oral health education of their patients , families , and friends ; and also at the community levels .
however before dental health professionals play a role as oral health educators , it is important to know the level of their own knowledge , attitude , and behavior toward oral health.2,5 it is of primary need that , as the dental students are specialists in conditions related to oral regions , they have good knowledge and expertise in oral health behaviors according to professional criteria.3 the attitude and behavior toward oral health maintenance of the dental professionals reflect their understanding of the preventive oral health measures , and this is very important for the improvement of their patient s oral health.5,6 hence , the present study was carried out to assess the oral health knowledge , attitude and behavior among the dental students .
the dental students in india spend their first 2 years in preclinics and next 2 years in clinics and last year of rotatory internship .
this study was formulated to study and comparatively evaluate between preclinical and clinical students about oral health knowledge , attitude and behavior .
this cross - sectional study was conducted on total 147 voluntarily participated 1 to 4 year students of malabar dental college , kerala .
it included 48 of 1 year , 34 of 2 year , 37 of 3 year and 28 of final year students . out of these 147 students ,
81 students were female and 66 were male ( table 1 and graph 1 ) .
first pilot study was carried out using a self - administered structured questionnaire written in english and which was later applied to the study group .
no other academic records except year of study were obtained of the participant study group students .
a total of 30 questions were designed to evaluate the oral health knowledge , attitude and behavior of students between male and female students ; and between preclinical and clinical students .
the questionnaire was in the format multiple choice questions and yes / no type questions .
the students were allowed to interact with the study committee for the meaning of any word or question .
the questionnaire included 30 items for the evaluation of oral health knowledge , attitude and behavior of the students :
oral health knowledge :
it included 10 questions on the basic knowledge of the oral health practices and purpose of maintaining oral healththe questions were in the form of multiple choice questions and student told to select the correct response .
attitude towards oral health :
it includes seven questions based on the attitude of the students for the maintenance of the oral healthin this section , yes / no answer type questions were selected .
except for the questions with marked as ( n ) , for which score one was given for the answer no ( appendix ii ) .
behavior or practices of the student towards oral health maintenance :
13 questions were included in this section , which cover the basic behavior or measures taken by the students towards maintenance of the oral health.out of 13 questions selected , some were of multiple choice questions and each correct answer was given score one .
for the scoring , yes answer given score one , except for the questions with marked as ( n ) were given score
oral health knowledge :
it included 10 questions on the basic knowledge of the oral health practices and purpose of maintaining oral healththe questions were in the form of multiple choice questions and student told to select the correct response . each correct answer was given
it included 10 questions on the basic knowledge of the oral health practices and purpose of maintaining oral health the questions were in the form of multiple choice questions and student told to select the correct response .
attitude towards oral health :
it includes seven questions based on the attitude of the students for the maintenance of the oral healthin this section , yes / no answer type questions were selected .
for the scoring , yes answer given score one , except for the questions with marked as ( n ) , for which score one was given for the answer no ( appendix ii ) .
it includes seven questions based on the attitude of the students for the maintenance of the oral health in this section , yes / no answer type questions were selected . for the scoring ,
one , except for the questions with marked as ( n ) , for which score one was given for the answer no ( appendix ii ) . behavior or practices of the student towards oral health maintenance :
13 questions were included in this section , which cover the basic behavior or measures taken by the students towards maintenance of the oral health.out of 13 questions selected , some were of multiple choice questions and each correct answer was given score one .
for the scoring , yes answer given score one , except for the questions with marked as ( n ) were given score
13 questions were included in this section , which cover the basic behavior or measures taken by the students towards maintenance of the oral health . out of 13 questions selected , some were of multiple choice questions and each correct answer was given score
in this section also some yes / no answer type questions were selected . for the scoring ,
one , except for the questions with marked as ( n ) were given score
descriptive statistics was calculated , and mean scores , standard deviation , and frequency distribution were obtained .
the difference of the oral health knowledge , attitude and behavior between preclinical and clinical students was assessed by student s t - test .
the variation of the scores from 1 to 4th year students of each of knowledge , attitude , and behavior was analyzed using one - way anova test .
the difference between males and female students of preclinical and clinical students was assessed by using student s t - test .
the assessment of the total score of the oral health knowledge , attitude and behavior between preclinical and clinical students were also done by using student s t - test .
the questionnaire included 30 items for the evaluation of oral health knowledge , attitude and behavior of the students :
oral health knowledge :
it included 10 questions on the basic knowledge of the oral health practices and purpose of maintaining oral healththe questions were in the form of multiple choice questions and student told to select the correct response .
attitude towards oral health :
it includes seven questions based on the attitude of the students for the maintenance of the oral healthin this section , yes / no answer type questions were selected .
for the scoring , yes answer given score one , except for the questions with marked as ( n ) , for which score one was given for the answer no ( appendix ii ) .
behavior or practices of the student towards oral health maintenance :
13 questions were included in this section , which cover the basic behavior or measures taken by the students towards maintenance of the oral health.out of 13 questions selected , some were of multiple choice questions and each correct answer was given score
for the scoring , yes answer given score one , except for the questions with marked as ( n ) were given score
oral health knowledge :
it included 10 questions on the basic knowledge of the oral health practices and purpose of maintaining oral healththe questions were in the form of multiple choice questions and student told to select the correct response . each correct answer was given
one score while wrong answer and do nt know the answer given zero score ( appendix i ) .
it included 10 questions on the basic knowledge of the oral health practices and purpose of maintaining oral health the questions were in the form of multiple choice questions and student told to select the correct response .
attitude towards oral health :
it includes seven questions based on the attitude of the students for the maintenance of the oral healthin this section , yes / no answer type questions were selected .
for the scoring , yes answer given score one , except for the questions with marked as ( n ) , for which score one was given for the answer no ( appendix ii ) .
it includes seven questions based on the attitude of the students for the maintenance of the oral health in this section , yes / no answer type questions were selected .
for the scoring , yes answer given score one , except for the questions with marked as ( n ) , for which score one was given for the answer no ( appendix ii ) . behavior or practices of the student towards oral health maintenance :
13 questions were included in this section , which cover the basic behavior or measures taken by the students towards maintenance of the oral health.out of 13 questions selected , some were of multiple choice questions and each correct answer was given score
for the scoring , yes answer given score one , except for the questions with marked as ( n ) were given score
13 questions were included in this section , which cover the basic behavior or measures taken by the students towards maintenance of the oral health . out of 13 questions selected ,
some were of multiple choice questions and each correct answer was given score one .
one , except for the questions with marked as ( n ) were given score
descriptive statistics was calculated , and mean scores , standard deviation , and frequency distribution were obtained .
the difference of the oral health knowledge , attitude and behavior between preclinical and clinical students was assessed by student s t - test .
the variation of the scores from 1 to 4th year students of each of knowledge , attitude , and behavior was analyzed using one - way anova test .
the difference between males and female students of preclinical and clinical students was assessed by using student s t - test .
the assessment of the total score of the oral health knowledge , attitude and behavior between preclinical and clinical students were also done by using student s t - test .
totally 147 students were participated in the study , of which 66 ( 44.89% ) were males and 81 ( 55.11% ) were females .
the male to female ratio was 1:1.25 and most of the study group students belong to the age between 18 and 25 years .
the distribution of the students according to the academic year was 48 , 34 , 37 and 28 , respectively ( table 1 and graph 1 ) .
table 2 shows the mean scores ; and a score of males and females , of the knowledge , attitude and behavior of the 1 - 4 year students .
the difference between each of knowledge , attitude and behavior scores between preclinical and clinical students was analyzed , which had shown statistically highly significant difference in all cases ( student s t - test ; p < 0.001 ) ( table 3 ) .
student s t - test for comparison of knowledge , attitude and behavior scores of preclinical and clinical students .
total score ( of knowledge , attitude and behavior together ) differences between preclinical and clinical students were analyzed by student s t - test , which also shown statistically highly significant difference ( p < 0.001 ) ( table 4 ) .
student s t - test for comparison of total score of knowledge , attitude and behavior of preclinical and clinical students .
the difference of total scores of male and female students according to academic years ; of knowledge , attitude and behavior was analyzed .
these shown females were better than males , but this was found to be statistically insignificant in all three categories i.e. , knowledge ( student s t - test , p > 0.01 ) , attitude ( student s t - test , p > 0.01 ) and behavior ( student s t - test , p > 0.01 ) ( table 5 and graph 2 ) .
student s t - test for comparison of scores of knowledge , attitude and behavior of male and female students .
distribution of knowledge , attitude and behavior scores according to year of study and gender .
the analysis of variation in the scores from 1 to 4 year students in each of the knowledge , attitude and behavior questionnaires was done by one - way anova test , which shown highly significant increase with the increasing academic year of study ( table 6 and graph 3 ) .
one - way anova test for the comparison of scores of knowledge , attitude and behavior of 1 year to 4 year students .
variations in the scores of knowledge , attitude and behavior scores according to year of study .
three models of professionalism have been described by the researchers in the literature , which have an impact on the quality of health care provided by the practitioner .
this ultimately depends on the knowledge and attitude of the health practitioner about their own profession.2
commercial model : in this , the services are based on patient s ability to buy rather than their needguild model : in this model , the practitioner is ethically trusted through their knowledge and wisdom by patient to provide best quality careinteractive model : in this model , both the practitioner and the patient decide the care provided through combination of experience and patients values .
commercial model : in this , the services are based on patient s ability to buy rather than their need guild model : in this model , the practitioner is ethically trusted through their knowledge and wisdom by patient to provide best quality care interactive model : in this model , both the practitioner and the patient decide the care provided through combination of experience and patients values .
education of oral health of the individual and community has a major impact from the knowledge of the dentists , which ultimately depends on the knowledge , attitude and behavior of the students of the dental colleges .
increased awareness of the oral health care among the dental students through academic learning can motivate their patients for the maintenance of the oral health and prevention of the development of the different oral diseases.7 as different health professionals combine work constitute the health team for the provision of the complete medical care including oral health , the variation in the knowledge , attitude and behavior of oral health during the years of university academic study will reflect the variation in the educational training experience of the dental students.1,2 in the present study , the scores of the knowledge , attitude and behavior of clinical students of 3 and 4 year was compared with that of the preclinical students of 1 and 2 year , which shown that the scores of the clinical students was higher than the preclinical students ( p < 0.001 ) .
this reflects the awareness of the oral health knowledge ; attitude and behavior during the clinical years of university study .
the variation of the scores with the years of study i.e. , from 1 year to 4 year also showed statistically highly significant increase , which reflects the variation in the educational training experience of the dental students according to years of the study ( p < 0.001 ) .
this variation of the scores is in accordance to the study done by neeraja et al.,8 sharda and shetty1 and mani et al.,9 but this was in contrast to the study done by dagli et al.,10 which showed non - significant increase in the scores of clinical students when compared with preclinical students .
the questions of knowledge like meaning of gum bleeding , effect of fluorides on teeth and reasons for oral cancer had shown much higher frequency ( 72% , 81% and 78% respectively ) of correct answers of clinical students than preclinical .
this may be due to the year wise education of the clinical knowledge among dental students . in the present study ,
the scores in general , of the knowledge , attitude , and behavior between females was higher than males , but the difference was not significant statistically ( p > 0.01 ) , which indicates that both males and females were equally aware about their oral health .
this was also reported in the study done by sharda and shetty1 and khami et al.11 and in contrast with the results of the study by al - omari and hamasha,12 ostberg et al.13 fukai et al.,14 schwarz and lo15 and kawamura et al.,16 which found that female students take better care of their teeth and had better health attitudes than their male colleagues .
when analysis of the individual questions was done , it was found that females ( 90% ) more likely to use dental floss and mouth wash than males ( 40% ) .
the present result was same as it was in kawas et al.17 and in contrast with the results of al - omari and hamasha12 and khami et al.,11 in which male students more frequently use dental floss than female .
similarly , female students ( 82% ) showed significantly positive attitude to the visit dentist regularly than male students .
this result was similar to study done by baseer et al.7 and strm and masalu18 the difference between males and females may be explained by habitual personal neglect by males and females caring more about their body and appearance .
the present study thus showed the improvement of knowledge , attitude and behavior with the academic years and there was no bias of gender with reference to oral health .
oral health knowledge , attitude and behavior was expected to be better among the clinical dental students than preclinical students in the present study , since it is a primary content in dental professional education , as they require it to educate their patients and also community , when they start working in health care system .
various previous researchers showed conflicting results of the studies on oral health knowledge , attitude and behavior among health professionals , but the results of these studies can be helpful in the planning of different oral health education programs , preventive measures and other activities related to the improvement of the community level oral health .
these studies can be taken as a guide to improving the oral health among the dental students , which ultimately helpful at the community level .
one of the limitations in these types of studies , as the scores depends on self - reported data ; the results may be biased though over- and underreporting due to social desirability , though the confidentiality is maintained .
thus we recommend that oral health awareness should be increased among dental students , which are the future health professionals .
for this purpose , we suggest that oral health and preventive dentistry topics should be stressed in the curriculum of students and also continuing dental education programs on these topics can be helpful . | background : dentists play very important role in the oral health education of the community . thus it is important to know the status of knowledge , attitude , and behavior toward the maintenance of oral health at the student level .
the aim of the present study was to evaluate the oral health knowledge , attitude , and behavior among preclinical and clinical dental students.materials and methods : a cross - sectional study was conducted on 147 dental students from 1st to 4th year of malabar dental college , kerala , in the year of march - june 2010 and was carried out with the help of 30 questionnaires .
age , gender and academic year data were recorded .
statistical analysis was performed with the help of spss version 20 using the student s t - test and one - way anova tests.results:the difference in scores of oral health knowledge , attitude and behavior between preclinical and clinical dental students was found statistically highly significant ( student s t - test , p < 0.001 ) .
the variation of scores of knowledge , attitude , and behavior also showed highly significant increase with the year of study ( one - way anova , p < 0.001 ) . while the difference in scores between males and females showed that females have better oral health knowledge than males , but the difference was not statistically significant ( student s t - test , p > 0.01 ) .
the present study thus showed improvement of knowledge , attitude and behavior with the academic years and there was no bias of gender with reference to oral health.conclusions:although oral health knowledge , attitude and behavior showed improved results from 1st to 4th year dental students , it should be improved in order to serve better for the community in the future . |
anatomical parameters of intracranial aneurysms such as aneurysm shape , size , and location clearly correlate with their natural progressions [ 14 ] . in general
past clinical studies also raised a possibility that there was a difference in the aneurysm shape between ruptured and unruptured intracranial aneurysms [ 13 ] .
the international study on unruptured intracranial aneurysms and many other clinical reports have indicated that the risk of aneurysm rupture varies in anterior and posterior circulations [ 4 , 5 ] .
intra - aneurysmal hemodynamics is believed to be another important factor related to the etiology and natural history of intracranial aneurysms .
consequently , a variety of intra - aneurysmal hemodynamic studies have been published in the last decade [ 69 ] .
recent hemodynamic studies of intracranial aneurysms use patient - specific radiographic data , since the geometry of a parent artery and aneurysm itself significantly affect the hemodynamic result . a few studies with the use of patient - specific data
have successfully demonstrated statistically significant differences in intra - aneurysmal hemodynamic patterns between ruptured and unruptured intracranial aneurysms [ 6 , 7 ] .
however , many past hemodynamic studies included intracranial aneurysms with different anatomical parameters ( size , shape , and location ) , which might have obscured the true hemodynamic differences between ruptured and unruptured aneurysms or between stable and unstable aneurysms .
various anatomical parameters affect on the intra - aneurysmal hemodynamics . nevertheless , a basic question that how the shapes of real patient aneurysms affect on their intra - aneurysmal hemodynamics remains unanswered . such basic knowledge would greatly help to deeply understand the effect of intra - aneurysmal hemodynamics on the natural history , considering the fact that different size aneurysms carry different rupture risks . in the present study
, we examined the hemodynamic characteristics of intracranial aneurysms from a single anatomical location and evaluated the effect of aspect ratio , aneurysm dome size , and dome neck ratio on the intra - aneurysmal hemodynamics .
internal carotid artery ophthalmic artery ( ica oph ) aneurysms , which are categorized as side - wall aneurysm , are selected for this hemodynamics simulation study .
oph aneurysm cases from the three - dimensional ( 3d ) digital subtraction angiography database at the division of interventional neuroradiology , ronald reagan ucla medical center were reviewed by interventional neuroradiologists ( fv and st ) . only good - quality angiogram ( sufficient contrast opacification of all major branches and no patient motions ) was considered for the selection of this study .
aneurysms with intraluminal thrombus were excluded from the selection . ruptured aneurysms with an irregularly shaped sac with contrast stagnation in the sac
were also excluded from the selection , since such an aneurysm may have a pseudoaneurysm component in its dome .
if a parent artery vasospasm was observed , the case was also excluded from the selection . moreover ,
the case with a fetal - type posterior cerebral artery and posterior communicating artery was excluded due to the complexity of outlet flow condition .
oph aneurysms were selected from the database . there were three ruptured aneurysms and five unruptured aneurysms .
three dimensions of the aneurysm dome ( anterior - to - posterior , width , and craniocaudad directions ) with the largest size of the aneurysm neck were measured by the interventional neuroradiologists ( fv and st ) using the philips integris workstation ( philips medical systems , best , the netherlands ) . the mean size of the eight ica
the 3d rotational cerebral angiograms were obtained using a philips integris unit ( philips medical systems , best , the netherlands ) .
data from those images were transferred to the philips integris workstation for 3d voxel generation and image restoration .
the 3d voxel data obtained by rotational angiography were then transferred to a dell 490 hemodynamic analysis workstation in our division .
image - based computational fluid dynamics ( cfd ) software developed by the department of computational sciences , george mason university , was utilized for aneurysm flow simulation [ 6 , 7 ] .
the 3d computational model was constructed semiautomatically through segmentation , surface generation , and 3d grid generation for each ica oph aneurysm .
the internal carotid artery was reconstructed from the level of the bony carotid canal to the bifurcation of the anterior and middle cerebral arteries .
cases with a large posterior communicating artery were excluded from this study , posterior communicating artery and anterior choroidal artery were not reconstructed .
normal pulsatile flow conditions acquired from a healthy subject using magnetic resonance phase - contrast measurement ( general electric 1.5 t scanner , waukesha , wi ) were imposed on the cfd simulation . the unsteady incompressible navier
blood was assumed to have uniform viscosity of 0.004 pa s. since the accurate information on the pulsatile aneurysm wall motion was not available in 3d rotational angiography , the rigid and no - slip boundary condition was assumed for the aneurysm wall in this cfd study [ 68 ] . in order to perform a quantitative hemodynamic analysis , intra - aneurysmal blood flow velocity and wall shear stress ( wss ) were collected from three measurement planes in each aneurysm : planes crossing the aneurysm dome at the level of the aneurysm neck , middle , and top ( fig . 1 ) .
the value of intra - aneurysmal flow velocity and wss in each aneurysm was defined by averaging the flow velocity and wss data from all of the three measurement planes .
the correlation coefficients ( r ) were obtained by a biostatistician ( js ) between the hemodynamic values and the following anatomical parameters : averaged dimension of aneurysm dome , the largest aneurysm dome dimension , aneurysm dome aspect ratio , and dome neck ratio .
three planes in the aneurysm sac were leveled at the neck ( a1 ) , the middle ( a2 ) , and the top ( a3 ) of the aneurysm to divide the aneurysm sac into three equal portions schematic representation of planes for the quantitative analyses .
three planes in the aneurysm sac were leveled at the neck ( a1 ) , the middle ( a2 ) , and the top ( a3 ) of the aneurysm to divide the aneurysm sac into three equal portions the averaged dimension of the aneurysm dome was defined as an averaged value of anterior - to - posterior , transverse , and craniocaudad dimensions .
the aneurysm aspect ratio was defined as aneurysm craniocaudad dimension divided by transverse or anterior - to - posterior dimension , whichever larger .
the dome neck ratio was given as aneurysm width or anterior - to - posterior dimension , whichever larger , divided by the largest aneurysm neck dimension .
internal carotid artery ophthalmic artery ( ica oph ) aneurysms , which are categorized as side - wall aneurysm , are selected for this hemodynamics simulation study .
oph aneurysm cases from the three - dimensional ( 3d ) digital subtraction angiography database at the division of interventional neuroradiology , ronald reagan ucla medical center were reviewed by interventional neuroradiologists ( fv and st ) . only good - quality angiogram ( sufficient contrast opacification of all major branches and no patient motions ) was considered for the selection of this study .
aneurysms with intraluminal thrombus were excluded from the selection . ruptured aneurysms with an irregularly shaped sac with contrast stagnation in the sac
were also excluded from the selection , since such an aneurysm may have a pseudoaneurysm component in its dome .
if a parent artery vasospasm was observed , the case was also excluded from the selection . moreover ,
the case with a fetal - type posterior cerebral artery and posterior communicating artery was excluded due to the complexity of outlet flow condition .
oph aneurysms were selected from the database . there were three ruptured aneurysms and five unruptured aneurysms .
three dimensions of the aneurysm dome ( anterior - to - posterior , width , and craniocaudad directions ) with the largest size of the aneurysm neck were measured by the interventional neuroradiologists ( fv and st ) using the philips integris workstation ( philips medical systems , best , the netherlands ) . the mean size of the eight ica
the 3d rotational cerebral angiograms were obtained using a philips integris unit ( philips medical systems , best , the netherlands ) .
data from those images were transferred to the philips integris workstation for 3d voxel generation and image restoration .
the 3d voxel data obtained by rotational angiography were then transferred to a dell 490 hemodynamic analysis workstation in our division .
image - based computational fluid dynamics ( cfd ) software developed by the department of computational sciences , george mason university , was utilized for aneurysm flow simulation [ 6 , 7 ] .
the 3d computational model was constructed semiautomatically through segmentation , surface generation , and 3d grid generation for each ica oph aneurysm .
the internal carotid artery was reconstructed from the level of the bony carotid canal to the bifurcation of the anterior and middle cerebral arteries .
cases with a large posterior communicating artery were excluded from this study , posterior communicating artery and anterior choroidal artery were not reconstructed .
normal pulsatile flow conditions acquired from a healthy subject using magnetic resonance phase - contrast measurement ( general electric 1.5 t scanner , waukesha , wi ) were imposed on the cfd simulation . the unsteady incompressible navier
blood was assumed to have uniform viscosity of 0.004 pa s. since the accurate information on the pulsatile aneurysm wall motion was not available in 3d rotational angiography , the rigid and no - slip boundary condition was assumed for the aneurysm wall in this cfd study [ 68 ] .
in order to perform a quantitative hemodynamic analysis , intra - aneurysmal blood flow velocity and wall shear stress ( wss ) were collected from three measurement planes in each aneurysm : planes crossing the aneurysm dome at the level of the aneurysm neck , middle , and top ( fig . 1 ) .
the value of intra - aneurysmal flow velocity and wss in each aneurysm was defined by averaging the flow velocity and wss data from all of the three measurement planes .
the correlation coefficients ( r ) were obtained by a biostatistician ( js ) between the hemodynamic values and the following anatomical parameters : averaged dimension of aneurysm dome , the largest aneurysm dome dimension , aneurysm dome aspect ratio , and dome neck ratio .
fig .
three planes in the aneurysm sac were leveled at the neck ( a1 ) , the middle ( a2 ) , and the top ( a3 ) of the aneurysm to divide the aneurysm sac into three equal portions schematic representation of planes for the quantitative analyses .
three planes in the aneurysm sac were leveled at the neck ( a1 ) , the middle ( a2 ) , and the top ( a3 ) of the aneurysm to divide the aneurysm sac into three equal portions the averaged dimension of the aneurysm dome was defined as an averaged value of anterior - to - posterior , transverse , and craniocaudad dimensions .
the aneurysm aspect ratio was defined as aneurysm craniocaudad dimension divided by transverse or anterior - to - posterior dimension , whichever larger . the dome neck ratio was given as aneurysm width or anterior - to - posterior dimension , whichever larger , divided by the largest aneurysm neck dimension .
the general characteristics of the hemodynamics patterns in the eight ica oph aneurysms are shown in fig .
2 . this cfd study of patients aneurysm at a single location shows some correlation between the intra - aneurysmal hemodynamics and such factors as aneurysm diameter , aspect ratio , and dome neck ratio .
particularly , the averaged values of intra - aneurysmal flow velocity and wss tend to become smaller as the aneurysm size becomes larger ( fig . 2 ) .
in fact , there are negative linear correlations between intra - aneurysmal flow velocity and the averaged dimension of aneurysm dome ( correlation coefficient : r = 0.735 ) and also wss ( r = 0.736 ; fig . 3a , b ) .
there is also a negative linear correlation seen between the intra - aneurysmal flow velocity and the largest aneurysm dimension ( r = 0.731 ; fig .
there is a weak negative correlation between the intra - aneurysmal wss and the largest aneurysm dimension with correlation coefficient of 0.496 ( fig .
2the general characteristics of the hemodynamics patterns in eight ica oph aneurysms . blood flow stream line and wss during peak systolic phase are presented in each ica oph aneurysm . in the stream line pictures ,
a red color line indicates faster flow , and a blue one means slower flow . in the wss distribution pictures , area in red color indicates higher wss , and area in blue means lower wssfig .
3a h correlation between the aneurysm shape ( dome diameter , aspect ratio , and dome neck ratio ) and flow parameters ( velocity and wss ) .
the number in the right upper corner indicates the correlation coefficients ( r ) . a correlation between
averaged aneurysm dome dimension and velocity , b between averaged aneurysm dome dimension and wss , c between the largest dome diameter and velocity , d between the largest dome diameter and wss , e between aspect ratio and velocity , f between aspect ratio and wss , g between dome neck ratio and velocity , and h between dome neck ratio and wss the general characteristics of the hemodynamics patterns in eight ica oph aneurysms .
blood flow stream line and wss during peak systolic phase are presented in each ica oph aneurysm . in the stream line pictures , a red color line indicates faster flow , and a blue one means slower flow . in the wss distribution pictures , area in red color indicates higher wss , and area in blue means lower wss a h correlation between the aneurysm shape ( dome diameter , aspect ratio , and dome neck ratio ) and flow parameters ( velocity and wss ) .
the number in the right upper corner indicates the correlation coefficients ( r ) . a correlation between averaged aneurysm dome dimension and velocity , b between averaged aneurysm dome dimension and wss , c between the largest dome diameter and velocity , d between the largest dome diameter and wss , e between aspect ratio and velocity , f between aspect ratio and wss , g between dome neck ratio and velocity , and h between dome
neck ratio and wss the aspect ratio of the aneurysm dome does not show a strong correlation with the intra - aneurysmal flow condition .
some weak negative correlations are observed between the aneurysm aspect ratio and intra - aneurysmal flow velocity ( r = 0.381 ) and wss ( r = 0.501 ; fig .
there is a clear negative linear correlation between the intra - aneurysmal flow velocity and the dome neck ratio ( r = 0.708 ; fig .
in other words , there is a positive linear correlation between the relative size of aneurysm neck and intra - aneurysmal flow velocity .
although wide - necked aneurysms ( smaller dome neck ratio ) show a faster intra - aneurysmal flow condition , there is only a weak negative correlation observed between the intra - aneurysmal wss and the dome neck ratio ( r = 0.392 ; fig .
we studied the relationship between the shape of intracranial aneurysms and the intra - aneurysmal hemodynamic parameters in a single anatomical location using actual patients computed tomography angiograms .
the result of this study showed that the intra - aneurysmal hemodynamics was significantly affected by such anatomical factors as aneurysm diameter , aspect ratio , and dome neck ratio . by eliminating other anatomical factors such as aneurysm location
, we could demonstrate a clear trend that the intra - aneurysmal blood flow was slower , and the averaged value of intra - aneurysmal wss was lower in the larger aneurysms .
in addition , our study showed that there was a weak correlation between the aspect ratio of the aneurysm dome and the intra - aneurysmal hemodynamic values .
some clinical studies found that the aspect ratio of the aneurysm dome was higher in the ruptured aneurysms than that in the unruptured aneurysms [ 1 , 3 ] .
therefore , when a researcher conducts a hemodynamic comparison study in rupture and unruptured aneurysms in order to investigate the hemodynamic risks of aneurysm rupturing , it is critical to match the size of the aneurysms in both groups as well as aspect ratio and dome neck ratio .
otherwise , such a pre - existing condition , which is the difference in the shape and size of intracranial aneurysms between ruptured and unruptured aneurysm groups , may create a false hemodynamics difference and leads to a false conclusion .
it is not clear whether high shear stress , low shear stress , or high shear stress gradient is associated with the aneurysm wall degeneration [ 79 , 11 , 12 ] .
furthermore , it is still controversial whether wss per se plays a role in the natural history of intracranial aneurysms or not .
the result of this study indicates that the larger aneurysms , which is known to carry a higher risk of rupture , showed lower intra - aneurysmal wss values . although it does not directly denote that the low wss induces the aneurysm rupturing , the low shear condition may be associated with the process of the degeneration of aneurysm walls .
a hemodynamic and histopathological comparison study of a large number of ruptured and unruptured aneurysm cases with similar size ranges and shapes in both ruptured and unruptured groups will reveal the true contribution of wss to the natural progression of intracranial aneurysm .
an immunocytochemical / histopathological study of intracranial aneurysms demonstrates that larger aneurysms develop more advanced atherosclerotic lesions on the aneurysm walls than smaller aneurysms .
given that a lower value of wss is a well - known predisposing factor of atherosclerosis plaque in such arteries as carotid bulb , coronary artery , and aorta , lower wss in larger intracranial aneurysms may explain the reason why lager aneurysms tend to have more advanced atherosclerotic lesions on them [ 1315 ] .
our study showed an interesting correlation that the intra - aneurysmal blood flow was faster as the aneurysm neck was wider .
a larger blood flow inlet relative to the volume of the aneurysm dome in a wide - necked aneurysm may be associated with this finding .
although a major factor for the higher recanalization rate after coil embolization of small / wide - necked aneurysm is an insufficient packing of embolic materials , faster intra - aneurysmal blood flow may also contribute to it .
the long - term durability of coil embolization in wide - necked aneurysms seems to be better with the use of self - expandable intracranial aneurysm stents such as neuroform and enterprise [ 1619 ] .
again , the primary reason for that must be due to an enhanced packing density with stent assists . however , flow velocity reduction after the placement of aneurysm stents in wide - necked aneurysms may also contribute to the better long - term results .
the relationship between aneurysm dome aspect ratio and intra - aneurysmal hemodynamics has been studied in the past .
the current study failed to reveal a strong correlation between aspect ratio and such hemodynamic parameters as averaged intra - aneurysmal flow velocity and wss .
since a higher aspect ratio is known to be associated with a secondary circulation zone in the deeper part of the aneurysm dome , it may correlate with blood flow structure rather than quantitative intra - aneurysmal hemodynamic values .
one of the limitations of this study is the fact that it is conducted in a single location .
since aneurysms at a different location may have different hemodynamic characteristics , similar hemodynamic studies in different anatomical locations are mandatory .
creation of a research consortium among centers of excellence in intracranial aneurysm is necessary for a larger hemodynamics study with a large number of aneurysms in each intracranial aneurysm location .
a quantitative cfd analysis was conducted in eight ica oph aneurysms with various geometries .
the intra - aneurysmal hemodynamics was significantly affected by such factors as aneurysm diameter , aspect ratio , and dome neck ratio .
the size of the aneurysm dome showed a negative linear correlation with intra - aneurysmal flow velocity and wss . | introductionvarious anatomical parameters affect on intra - aneurysmal hemodynamics . nevertheless
, how the shapes of real patient aneurysms affect on their intra - aneurysmal hemodynamics remains unanswered.methodsquantitative computational fluid dynamics simulation was conducted using eight patients angiograms of internal carotid artery
ophthalmic artery aneurysms .
the mean size of the intracranial aneurysms was 11.5 mm ( range 5.8 to 19.9 mm ) .
intra - aneurysmal blood flow velocity and wall shear stress ( wss ) were collected from three measurement planes in each aneurysm dome .
the correlation coefficients ( r ) were obtained between hemodynamic values ( flow velocity and wss ) and the following anatomical parameters : averaged dimension of aneurysm dome , the largest aneurysm dome dimension , aspect ratio , and dome neck ratio.resultsnegative linear correlations were observed between the averaged dimension of aneurysm dome and intra - aneurysmal flow velocity ( r = 0.735 ) and also wss ( r = 0.736 ) .
the largest dome diameter showed a negative correlation with intra - aneurysmal flow velocity ( r = 0.731 ) and wss ( r = 0.496 ) .
the aspect ratio demonstrated a weak negative correlation with the intra - aneurysmal flow velocity ( r = 0.381 ) and wss ( r = 0.501 ) . a clear negative correlation was seen between the intra - aneurysmal flow velocity and the dome neck ratio ( r = 0.708 ) . a weak negative correlation is observed between the intra - aneurysmal wss and the dome neck ratio ( r = 0.392).conclusionthe aneurysm dome size showed a negative linear correlation with intra - aneurysmal flow velocity and wss .
wide - necked aneurysm geometry was associated with faster intra - aneurysmal flow velocity . |
the term reprocessing is defined in 3 no . 14 mpg ( medizinproduktegesetz ) as follows : the reprocessing of medical devices intended to be applied semi - sterile or sterile , is the cleaning , disinfection and sterilisation , including the processes connected therewith , as well as the testing and restoration of technical - functional safety , following their use for the purpose of renewed use .
it clearly follows from this definition that the term reprocessing refers not only to the cleaning , disinfection and sterilisation of medical devices .
rather , the technical - functional safety must also be tested and , if necessary , restored .
reprocessed medical devices must offer the same standard of safety as a new product in terms of their material characteristics , their functions ( e.g. recording , cutting , coagulating ) etc . at the same time
, in addition to servicing , inspection and repair , reprocessing constitutes a fourth sub - category of maintenance , and is thus recognised as another form of operation of medical devices .
a large number of these articles can following appropriate professional reprocessing be re - used in medical treatment . without a doubt , it is particularly health economics arguments that influence the decision as to whether to reprocess medical devices for re - use .
the basic rule here is that reprocessing regardless of whether a medical device is declared for single use or for repeated use is even more worthwhile the greater the input of materials and resources required in the manufacture of the medical device in question .
this economic interest is , however , limited by the need to protect patients against functional , immunological , toxicological or hygienic impairment through reprocessed medical devices . in healthcare practice
, this economic / legal conflict initially triggers questions in particular with regard to the regulatory aspects of medical device law .
the regulatory requirements for reprocessing medical devices are set forth in the law on medical devices .
the basic norm here is the german act on medical devices ( medizinproduktegesetz , hereinafter mpg ) , which transposed directive 90/385/eec ( active implantable medical devices ) , 98/79/ec ( in - vitro - diagnostics ) and 93/42/eec ( other medical devices ) into german law .
the general provisions of the mpg are governed in detail in a number of ordinances , e.g. the medical device operators ordinance ( medizinproduktebetreiberverordnung , hereinafter mpbetreibv ) .
the requisite reprocessing quality is defined in 4 para . 2 sentence 1 mpbetreibv .
the reprocessing of medical devices intended to be applied semi - sterile or sterile is to be carried out taking into account the manufacturer s specifications using suitable validated procedures , such that the success of these procedures can be verifiably ensured and the safety and health of patients , users or third parties is not put at risk .
. 2 sentence 1 mpbetreibv , is given : proper reprocessing pursuant to sentence 1 is assumed if the joint recommendation of the commission for hospital hygiene and the prevention of infection at the robert koch institute and the federal institute for drugs and medical devices concerning hygiene requirements when reprocessing medical devices is observed . in terms of the law , a rule of assumption , which can be refuted in individual cases , was chosen ( vol .
if an operator or , as the case may be , an external processor ( service provider ) , carries out the reprocessing in accordance with the joint recommendation of the commission for hospital hygiene and the prevention of infection at the robert koch institute ( rki ) and the federal institute for drugs and medical products ( bfarm ) ( hereinafter : joint rki and bfarm recommendation ) , then it shall initially be assumed that the reprocessing was carried out properly .
the joint rki and bfarm recommendation governs the details concerning the hygiene requirements when reprocessing medical devices .
medical devices are divided into various categories of risk on the basis of the type of application , and the risk involved : non - critical medical devices ( e.g. ecg electrodes),semi - critical medical devices ( e.g. endoscope),critical medical devices .
non - critical medical devices ( e.g. ecg electrodes ) , semi - critical medical devices ( e.g. endoscope ) , critical medical devices . pursuant to the joint rki and bfarm recommendation , critical medical devices are devices used for the application of blood , blood products and other sterile medicinal products and medical devices that penetrate the skin or mucous membrane and thereby come into contact with blood , internal tissues or organs , including wounds . in turn , the critical medical devices are divided into sub - categories : critical a ( without particular reprocessing requirements , e.g. hook retractors),critical b ( with more stringent reprocessing requirements , e.g. mic trocars),critical c ( with particularly stringent reprocessing requirements , e.g. heart catheters ) .
critical a ( without particular reprocessing requirements , e.g. hook retractors ) , critical b ( with more stringent reprocessing requirements , e.g. mic trocars ) , critical c ( with particularly stringent reprocessing requirements , e.g. heart catheters ) .
the joint rki and bfarm recommendation provides with regard to medical devices belonging to the category
critical c , that the reprocessor s quality management system must be certified by a body accredited by the central authority of the lnder for health protection with regard to medicinal products and medical devices ( zentralstelle der lnder fr gesundheitsschutz bei arzneimitteln und medizinprodukte , zlg ; named body pursuant to 20 para . 1 mpg ) .
this certification must be carried out in accordance with din en iso 13485:2003+ac:2007 , i.e. in accordance with the only din norm currently applicable in the area of quality management systems of medical device reprocessing .
pursuant to the joint rki and bfarm recommendation , not every body can certify the quality management systems for the reprocessing of medical devices belonging to the category
critical c . instead , a designated body ( explanation : named bodies are independent certifying offices organised under private law .
they are , however , subject to state supervision and must be accredited by the zlg , for example .
the suitability as a designated body is examined and ascertained in the accreditation proceedings ) must be specifically accredited by the zlg for this purpose .
in contrast to the zlg , the central authority of the lnder for safety technology ( zentralstelle der lnder fr sicherheitstechnik , zls ) does not certify any appointed offices for the reprocessing of medical devices . as per
may 1 , 2008 the following designated bodies have been accredited by the zlg ( http://www.zlg.de ) for this purpose : dekra certification gmbh stuttgart , lga intercert nrnberg , tv rheinland product safety gmbh kln , medcert hamburg .
dekra certification gmbh stuttgart , lga intercert nrnberg , tv rheinland product safety gmbh kln , finally , it must be pointed out that the certificates issued by the accredited designated body have a limited applicability
reprocessors must , as explained under point 2 , prove their ability to properly reprocess medical devices belonging to category
critical c as defined in 4 mpbetreibv , by way of a certificate issued by a designated body accredited for this purpose by the zlg . the quality management system of the company for the reprocessing of medical devices belonging to the category
this certification relates both to the introduction and to the maintaining and application of the quality management systems .
the operator of medical devices is thus able , with the help of the certificate , to examine in each case whether the contractor is able to properly reprocess the medical devices submitted by the operator for reprocessing .
neither the mpg nor the ordinances enacted on the basis thereof expressly prohibit the reprocessing of single - use products .
this is a result of the structure of the law : pursuant to 2 para .
1 mpbetreibv , medical devices may only be constructed , operated , used and maintained in accordance with their specifications .
the central issue in this context is whether the manufacturer s designation as single - use medical device belongs to the specification or whether this is merely a restriction on the use , or a definition of a term on the part of the manufacturer .
according to one view represented in the literature , the single - use designation is part of the specification , since the specification does not depend only on the relevant characteristics in the medical device definition set forth in 3 no
instead , the manufacturer would have to define additional purposes , e.g. with regard to the classification of the medical device
. this view would correspond with an interpretation in line with the directive , taking into account the safety concept on which medical device law is based .
were one to follow this understanding , the reprocessing of single - use products would be contrary to their purpose , and , thus , prohibited . according to the opposing view
, however , only the characteristics of a medical device specified in 3 no . 1 mpg belong to the specifications .
this would result from the definition of specifications in 3 no . 10 mpg , which refers to the use of the product and therefore constitutes a reference exclusively to the definitions in 3 no . 1 mpg .
it is argued that with the specifications the manufacturer could only pursue goals that are designated in the definition of medical devices contained in 3 no . 1 mpg . all further declarations are solely for the purpose of labelling ( , 9 , margin note 56 ) .
the latter opinion should be confirmed , since the express emphasis of the intended use in the definition set forth in 3 no .
10 mpg constitutes a pretty clear reference to the statements in the medical device definition contained in 3 no .
this does not , however , include the designation single - use product .
the safety concept of medical device law does not demand the prohibition of reprocessing of single - use products either .
what is important is ensuring an appropriately high standard of quality when reprocessing , which is secured by way of the provisions of the mpbetreibv .
ultimately the purpose of the norm , which can be derived from the legislative intent and historic interpretation , supports the permissibility of reprocessing single - use products .
the proposed resolution of the health committee on the 2 act to amend the mpg states that the amendment shall serve to remove the legal uncertainties in the area of the reprocessing of single - use products .
it states that , in the interests of consumer protection , the federal ministry for health is called upon to introduce appropriate supervision of the reprocessing of single - use products .
thus , the legislator is obviously proceeding on the basis that the reprocessing of single - use products is permissible , and wanted to ensure in the course of the 2 act to amend the mpg that this reprocessing attains a high qualitative standard by way of appropriate supervision .
the federal government s answer to a parliamentary question regarding the reprocessing of single - use products leads to the same outcome . according to the unequivocal answer , the reprocessing of single - use products is not prohibited .
the reprocessing requirements do not differentiate between single - use and multi - use products .
so , the reprocessing of single - use products is legally permissible . citing the legislative purpose , the higher courts seconded this outcome in a decision .
as explained under point 6 , the reprocessing of medical devices that are intended to be applied semi - sterile or sterile is , although it entails more stringent requirements , permissible under european and german federal law .
however , this legislative system of values does not release the manufacturers , nor the professional operators of medical devices , from their liability under liability law for damage resulting from the use of incorrectly processed medical devices .
the civil and criminal law responsibility of a manufacturer of medical devices , or that of his authorised agents in the course of the first placing on the market of a medical device for faults in the construction , the manufacture and the instructions , as well as for product supervision measures that were disregarded , was expressly clarified with the entry into force of the 2 act to amend the mpg on january 1 , 2002 through 6 para .
4 together with 5 mpg : pursuant to this , the operators of medical devices are obligated to monitor compliance and correct implementation of the provisions of the mpg and the mpbetreibv by way of organisational and technical measures .
2 sentence 1 mpbetreibv , the reprocessing of medical devices intended to be applied semi - sterile or sterile is to be carried out taking into account the manufacturer s specifications using suitable procedures such that the success of this procedure can be verifiably ensured and the safety and health of patients , users and third parties is not put at risk ( in distinction the reprocessing of medical devices that are not applied sterile usually amounts to nothing more than the measures to maintain the normal condition ( servicing ) , i.e. simple cleaning ) . according to a decision of the administrative court arnsberg it is therefore not sufficient if a clinic assumes that the medical devices treated in a steam steriliser are sterile , without there being a validated procedure . in order to avoid any claim against the operator under liability law
, the medical devices must be reprocessed in accordance with a validated and documented procedure . only through the validation of the reprocessing procedure
are the correct parameters defined , which are necessary in order to prove that the process in question was carried out in a form that guarantees effective reprocessing ( chapter ii 3 ,
the subject of a validation procedure must , therefore , refer to a documented procedure for the performance , recording and interpretation of the results .
this is the only way that proof can be provided that products of consistently equal quality can be achieved in a reprocessing procedure .
the success of the proper reprocessing process is linked to a decisive degree with the knowledge of the condition of a medical device .
it is above all the manufacturers who have well - founded expert knowledge of this . on grounds of patient - protection and for liability law protection ,
the operators should therefore request details of the appropriate validated reprocessing procedures from the manufacturers .
when a claim is made against an operator of medical devices under liability law ,
2 sentence 3 mpbetreibv plays a central role . as explained under clause 2b , it is assumed such assumption being refutable that an operator or an external processor has carried out the reprocessing correctly if the reprocessing procedure complies with the joint rki and bfarm recommendation . complying with the recommendation
does not constitute a direct statutory obligation , i.e. failure to comply with it does not lead to any direct punishment of the operator .
however , in the event that a claim is made under liability law , failure to comply with the recommendation can lead to an alleviation of the burden of proof on the claimant s side .
the allocation of the burden of proof in the patient / operator relationship basically follows the general rules , pursuant to which the claimant carries the burden of proof for all conditions establishing his claim . from the point of view of the law of evidence , this initially results in a comfortable position for the operator of medical devices .
if , however , the aforementioned recommendations are not observed when reprocessing , then in the event of an incident the onus of presentation and the burden of proof of proper reprocessing would be incumbent upon the operator of the medical device .
the reprocessing of medical devices belonging to the highest risk category ( critical c ) , or of medical devices for which the manufacturer has restricted re - use ( single - use products ) , constitute a special case . as explained above
, the reprocessing of the aforementioned articles is not directly or indirectly prohibited at any point in the mpg or the mpbetreibv . on the contrary the federal government follows the strategy that it is possible to safely reprocess these medical devices . with regard to the reprocessing too , the quality management system set up by the reprocessor must have been certified by a body accredited by the competent authority . in the case of single - use products , for which , accordingly , the manufacturer has not provided a validated procedure , it is of the utmost importance for ensuring safety that the reprocessor is able to understand the function and condition of the medical device to be reprocessed . only well - founded expert knowledge with regard to the material characteristics and the technical construction of a product ensure that an adequate validated reprocessing procedure can be developed , implemented and certified ( chapter ii 3 , 4 mpbetreibv , margin note 4 ) .
if the operator outsources the task of reprocessing and has it carried out by an external third party , the selection of the reprocessor is placed in his responsibility . from the point of view of an operator of medical devices ,
it is therefore essential that a validated and verified procedure be provided when outsourcing the reprocessing . in this connection
, the certification of the processing company constitutes a milestone in the course of liability law risk management , because this enables the operator to ensure that the external reprocessor carries out the reprocessing properly and professionally .
when deciding whether to reprocess medical devices for the purpose of re - use , the benefits and risks must be carefully weighed up against each other in the interests of the efficient development of our healthcare system . according to the legislator s intention ,
when discussing how to finance the services required pursuant to social security law , in addition to many economic issues , sustainability must also be taken into account , i.e. in addition to economic factors , environmental aspects are increasing in importance in the course of the selection of a medical device . here
, it is the case that reprocessing , regardless of whether the medical device is declared for single useor for repeated use , appears even more worthwhile the greater the input of material and resources required in the manufacture of the medical device in question . from the legal point of view
, however , this discretion in the selection is , pursuant to these parameters , limited by an incontrovertible liability law factor : patient protection .
consequently , with regard to the reprocessing of single - use products in particular , it must be examined for each individual product with the greatest degree of care whether the material characteristics and the construction of the medical device allow it to be reprocessed , without this posing a risk to life or limb of the patient . | the processing of single - use products is permissible pursuant to medical device law .
this is apparent both from the wording of the german law on medical devices and from the purpose and the objectives underpinning the legislative materials .
the prerequisite for processing is , however , compliance with the the joint recommendation of the commission for hospital hygiene and the prevention of infection at the robert koch institute ( rki ) and the federal institute for drugs and medical products ( bfarm).for medical devices in the category critical c , the rki / bfarm - recommendation provides that the processor s quality management system must be certified by a body accredited by the central authority of the federal states for health protection with regard to medicinal products and medical devices ( zentralstelle der lnder fr gesundheitsschutz bei arzneimitteln und medizinprodukten , zlg ) .
the certification must be carried out in accordance with en iso 13485:2003+ac:2007.on april 4 , 2008 the federal health ministry ( bundesministerium fr gesundheit , bmg ) presented a progress report on the processing of medical devices .
the bmg concludes that the legal framework for the processing of medical devices is sufficient , and that a prohibition on the processing of single - use products is inappropriate . |
csip was initiated to address a perceived decline in the scientific workforce by attracting more students to majors in science , technology , engineering , and mathematics ( stem ) .
csip was originally targeted to primarily undergraduate institutions ( puis ) , which , as a result of the 1985 oberlin report ( davis - van atta , 1985 ) , were ( and still are ) largely viewed as one of the nation s important incubators for phds in the sciences .
these grants , aimed at improving undergraduate laboratory instruction , enabled awardees to purchase modern , up - to - date equipment that they could not otherwise afford and that would enable them to enrich both the content of student laboratories and the way in which they were taught .
csip s early annual budget of about $ 7 million supported small awards of $ 5,000 to $ 50,000 , each requiring dollar - for - dollar matching from the submitting institution .
in addition , nsf required that csip awards and matching funds be used solely for instructional equipment ; no overhead or administrative costs could be included in proposal budgets or taken from grant funds .
the combination of nsf funding and institutional matching went a long way toward modernizing the equipment used in teaching laboratories in puis . in 1988 ,
the range of csip - eligible institutions was expanded to include both two - year and doctoral - granting institutions , and the name of the program was changed to the instrumentation and laboratory improvement ( ili ) program .
the objectives of ili were similar to those of csip , namely to support improved laboratory experiences and thereby attract more students to majors in the sciences .
however , ili also emphasized the need to educate nonscience majors and preservice teachers , as well .
in addition , the program put increased emphasis on projects that introduced laboratories that went beyond the traditional cookbook approach . with the advent of ili , nsf was beginning to realize the potential for grants targeted to undergraduate education to influence the culture of more research - focused institutions .
it was hoped that faculty members who were successful at landing educational grants could reap some of the same recognition from their institutions as those who regularly brought in research funds . as with csip ,
ili required awardee institutions to match nsf funds dollar - for - dollar for equipment purchases ; institutional overhead was prohibited . despite these limitations ,
ili consistently received about 2000 proposals and funded about 28 percent , or an average of 550 proposals , each year . by 1994 ,
nearly 100 percent of doctoral granting institutions and 75 percent of four - year colleges had submitted at least one ili proposal , and 87 percent of doctoral - granting and 55 percent of four - year schools had received one or more awards .
the course and curriculum development ( ccd ) program was initiated in the same year that csip morphed into ili and was limited in the first three years to curricular reform projects in engineering , calculus , and precalculus . but as well - equipped student laboratories in the bench sciences ( funded with ili grants ) became more commonplace , creative faculty in all science disciplines began seeking support for curricular reform .
the focus in undergraduate laboratory teaching was shifting from passive experiments with well - defined outcomes to more research - like exercises , in which students designed experiments , collected data , interpreted results , and communicated their findings in a variety of ways . in 1991 , ccd was opened to all disciplines .
ccd supported revisions in courses or entire curricula , implementation of nontraditional pedagogies , and development of materials to support new methods in both the classroom and the laboratory , with the more ambitious goal of catalyzing major changes in undergraduate science education at a national level .
an evaluation of ccd and ili undertaken in 1996 ( see www.nsf.gov/pubs/1998/nsf9833/ili-1.htm ) concluded that these programs were succeeding in revitalizing education and re - energizing faculty at the local level in the classrooms and departments of awardees but fell short of the national impact the programs sought to realize .
in addition , research in science education how students learn and how we measure it was maturing .
concerned faculty could now choose from tested methods to improve their classes , thus approaching science education as a science in and of itself .
in 1999 , ccd and ili were folded into a new , four - track funding instrument , the course , curriculum , and laboratory improvement ( ccli ) program .
ccli incorporated most of the features of both ccd and ili , including the option to submit instrumentation or equipment proposals , but gave increased priority to testing the effectiveness of materials and practices in terms of gains in student learning . adapting and implementing proven materials and classroom practices
was incorporated into ccli as a separate track , and what had been ili became , in 1999 , the adaptation and implementation ( a & i ) track of ccli .
a & i projects enabled science faculty to apply proven innovations or develop their own , as well as purchase the equipment required to implement them .
ccd was folded into either the a & i track or the educational materials development ( emd ) track of the new ccli , which supported innovative textbooks , software , course modules , laboratory exercises , and web - based teaching materials . reflecting the need for better assessment of student learning and the desire to train the nation s faculty in tested educational approaches , two new initiatives were introduced , the assessment of student achievement and national dissemination tracks , respectively .
ccli budgets could include institutional overhead , although the a & i track still required that awardees provide matching funds to purchase instrumentation . in 2000 , the first year ccli grants were awarded , four projects with neuroscience themes were funded .
three institutions , the university of wisconsin - la crosse , regis university , and gustavus adolphus college , used funds from the a&i track to equip open - ended teaching laboratories that employ research - like pedagogies . in these revised courses , students ask research questions and propose the appropriate experimental methods to answer them .
students at regis university have been authors on four abstracts submitted to professional research conferences , and the principle investigators report substantial gains in cognitive skills as a result of these laboratory reforms .
the fourth project , developed at rice university and funded from the emd track , supported a web - based multimedia textbook in behavioral science ( see psych.rice.edu/mmtbn/ ) , which is freely available to all .
throughout its history and in all its various forms , the trend in ccli has been toward a more holistic approach to stem undergraduate education .
the scope of the program has steadily expanded to include increased emphasis on evaluation of the effectiveness of educational reform and better dissemination of tested innovations , so as to broaden the impact of successful projects .
scientists and science educators are converging into a community of scholars sharing similar goals , methods , and expectations , so that projects concerned with undergraduate science education more closely resemble traditional science research projects in their attention to documenting results . in 2005
ccli was substantially revised to reflect these changes . using the model of disciplinary research proposals
, the revised ccli requires that the rationale for undertaking a project be built on prior work . to foster projects that are effective agents of change at the national level , proposers are encouraged not only to cite prior work by themselves and others , but also to describe how their project will be assessed and how the results of that assessment will contribute to the improvement of undergraduate stem education . in the words of the program s solicitation ( nsf 06 - 536 ; www.nsf.gov/publications/pub_summ.jsp?ods_key=nsf06536 ) , proposals must describe how the project will contribute to building a community of scholars .
the 2005 revisions to ccli were , themselves , informed by the literature in education .
the revised program employs a model for educational reform originally published to describe how mathematics education progresses ( ball , 2003 ) .
analysis of existing reform efforts ( stage 1 , research on teaching and learning ) leads to creation of new learning materials and strategies ( stage 2 ) .
new approaches require that faculty gain expertise ( stage 3 ) , so that they can implement tested educational innovations ( stage 4 ) .
finally , widely disseminated innovations must be assessed ( stage 5 , determination of resulting learning gains ) , which leads to information about teaching and learning that can be used to inform the next cycle of innovation ( stage 1 ) .
proposals to ccli can address one or more elements of the cycle , depending on the size and scope of the project , but all must pay appropriate attention to assessment and dissemination . in place of separate tracks reflecting different kinds of program activities , ( a&i , emd , etc . ) , the revised ccli is divided into three phases reflecting the size , scope , and maturity of the project being proposed .
indirect costs are permitted and no institutional matching is required for any of the phases .
this new emphasis in all phases of ccli on evaluation and dissemination means that it is no longer adequate to assume ( or hope that reviewers will assume ) that simply implementing a reform in the classroom or laboratory will result in gains in student learning .
instead , proposers should state explicitly what they expect students to learn as a result of the project , how those learning gains will be measured and quantified , what outcomes constitute success , and how they will inform others of these outcomes . for many successful ccli projects ,
this has meant engaging the aid of educational researchers ( defined broadly , see below ) to help define and assess outcomes .
inclusion of educational researchers also helps to determine how readily generalized successful teaching strategies are and how easily reforms can be implemented in different settings .
although it is not a requirement of the revised ccli solicitation that proposals include outside evaluators , many include colleagues in education departments , sociologists or anthropologists interested in cultural change , or consultants external to the university .
these collaborators can help conduct a thorough and unbiased evaluation of project outcomes , both during the course of the project and at its conclusion .
proposals for larger , more expensive projects ( submitted as phase two or three proposals , for example ) are more likely to include the services of an outside evaluator .
the pis ( carol ann paul , bruce johnson , and julio ramirez ) , who are from three different types of institutions , are developing pedagogically sound , inexpensive , on - line laboratories for undergraduate neuroscience courses , called the neuron connection .
these digital - format laboratories include simulations , animations of classical experiments , and referrals to the primary literature in neuroscience .
the digital labs are being assessed for the appropriateness and effectiveness of delivery in a wide range of college and university environments .
preliminary results were presented at the society for neuroscience annual meeting in san diego in 2004 and in washington , dc in 2005 . a software package for teaching neurobiology through interactive laboratory simulations .
building on a previous nsf - funded project , the pi ( william j. moody ) and collaborators have developed a neuron simulation software package that allows students to create a virtual electrophysiology recording set - up by placing amplifiers , stimulators , and other equipment into a virtual rack and wiring them together .
students can manipulate electrodes into virtual neurons and record changes in membrane voltage under various experimental conditions .
volunteer users were videotaped to identify areas of confusion , and appropriate changes were made to improve the product .
post - testing at intervals after using the software indicated that students inquiry skills improved .
beta versions of the software were sent to more than 100 educators in over 20 countries , including many minority - serving institutions in the usa . comments from beta testers
were used to finalize the software . while it is too soon to provide examples of neuroscience projects funded under the 2005 revised program solicitation ( in 2005 ,
only one neuroscience proposal was submitted under the new program ) , listed below are some highlights of ongoing projects that we believe illustrate current funding priorities .
in addition due maintains a list of abstracts from recently funded projects on its project information resource system ( pirs ) website ( www.ehr.nsf.gov/pirs_prs_web/search ) .
most principle investigators ( pis ) are happy to share information about their projects when asked .
pis , david deerfield , eric jakobsson , barbara kucera and gregory hood , are using the access grid group - to - group communication technology at the pittsburgh supercomputer center to deliver a course in computational neuroscience , particularly aimed at students in epscor states .
the project s objective is to test whether courses originating at universities with supercomputing facilities can be effectively disseminated using synchronous interactions .
results indicated that students were comfortable with the access grid technology and would consider taking another access grid course if the topic were one not offered at their home institution .
the pis did identify one important hurdle to be overcome before cross - institutional courses can gain wide acceptance : administrative issues concerning student credit and instructor compensation will need to be addressed at each participating institution .
( this project was jointly funded by due and the computer and information science and engineering directorate at nsf . )
as expertise in the cellular and molecular interfaces between biological and artificial systems grows , a new generation of neural prostheses , devices that can interact with or emulate living nervous systems , is being developed . in response to these developments ,
the uic departments of bioengineering and biological sciences have created a two - year , cross - college undergraduate curriculum in neural engineering , one of the first such programs in the country .
pis , john hetling and christopher comer , used ccli funds to equip a neural engineering laboratory course that serves as the capstone experience in the new undergraduate curriculum . the novel curriculum , including the capstone laboratory course , was described in presentations to the asee ( american society for engineering education ) annual meeting and the whitaker foundation biomedical engineering summit .
some of the instrumentation was also used in a bioengineering summer camp program for high school teachers . | for over 20 years , the division of undergraduate education ( due ) at the national science foundation ( nsf ) has been supporting undergraduate curricula in the sciences , including neuroscience .
nsf s priorities in undergraduate education , however , have evolved during that period , and the competition for grants has increased .
this history and overview of the current course , curriculum and laboratory improvement program ( ccli ) illustrates the changing philosophy of due with regard to its curricular programs .
it is hoped that understanding the current emphasis on assessing the outcomes of curricular changes and disseminating their results will help interested science faculty write better proposals and compete more effectively for funds . |
neuroleptic malignant syndrome ( nms ) rare but life - threatening side effect of antipsychotic medications .
it is suggested that the ability to create nms by neuroleptics is associated with their ability to block dopamine in the nigrostriatal pathway , mesocortical pathway , and hypothalamic nucleus .
its incidence has been reported from 0.69 to 3.23 percent . according to various studies ,
the growing use of atypical antipsychotic has reduced the incidence of nms and it is estimated that 0.01 to 0.02% of patients treated with antipsychotics will suffer from its fatal side effect . although nms is less common with atypical antipsychotic , but it seems that its incidence is increasing with the administration of these medications .
perhaps such increase is due to rising atypical forms of the disease caused by different therapeutic mechanisms of modern medicine .
there are several reports about nms following atypical antipsychotic drugs such as risperidone , clozapine , olanzapine , and quetiapine .
2004 ) carried out a review study on nms associated with atypical antipsychotic drugs . in medline database , they identified reviews on 68 patients ( 21 women and 47 men ) suffering from nms with atypical antipsychotic drugs , namely clozapine ( n=21 ) , risperidone ( n=23 ) , olanzacine ( n=19 ) , and quetiapinen=5 ) .
( 2013 ) reported a patient with bipolar disorder that showed nms symptoms following the administration of intramuscular haloperidol .
the medication was replaced with olanzapine , but nms appeared again . herein , we report a case of nms related to olanzapine therapy in a young male patient .
the significance of this case stems from the scarcity of reports on nms associated with olanzapine as well as similar presentations with traditional antipsychotic after one year of treatment .
we present a 27-year - old man with a history of paranoid schizophrenia who showed signs consistent with nms that occurred after one year of treatment with olanzapine ( 20 mg / day ) .
symptoms included fever , altered level of consciousness , changes in mental status , drooling , diaphoresis , urinary incontinence , sweating , development of tremors , lead pipe rigidity , tachycardia , and increased level of creatine phosphokinase .
his level of consciousness according to the glasgow coma score ( gcs ) was 11 .
the recorded vital signs were temperature of 38.2 c , blood pressure 100/60 mmhg , heart rate 100 beats / min , and respiratory rate 11/min . on the second day ,
neurologic examination showed an increase of muscle tone in four limbs and bilateral tremor of upper limbs .
the level of creatine kinase was 30,445 iu / l , creatine phosphokinase 4,145.5 mb , and lactate dehydrogenase ( ldh ) 2,599 .
the hemoglobin level was 15.9 g / dl , leukocyte count of 14,300/mm3 , and platelet count of 43,000/mm3 .
sgpt and sgpt were 123 u / l and 138 u / l , respectively .
the kidney function , analysis of cerebrospinal fluid , and serum electrolyte levels were normal .
early differential diagnoses were seizure , aspiration , stroke , and infection . these diagnoses were ruled out and nms was considered after which olanzapine was discontinued .
the naranjo probability scale indicated a possible relationship between the development of nms and olanzapine .
bromocriptine began ( dose of 2.5 mg per 8 hours ) through gastric feeding tube and was increased to 5 mg every 8 hours .
the patient received traditional treatment for nms , such as intravenous fluids and supportive care .
creatine kinase ( ck ) level fell and dropped to 403 iu / l on day 13 ( normal level : 15 - 300 ) and by day 15 it was within the normal range .
briefly , the patient had decreased level of consciousness , intense muscle rigidity , fever , drooling , diaphoresis , urinary incontinence , sweating , and increased blood pressure .
clinical features of this syndrome include high fever , extrapyramidal symptoms , and dysfunction in autonomic system such as unstable blood pressure , cardiac arrhythmia , dyspnea , sweating and urinary incontinence . however , fever and muscle rigidity are the most recognized signs in various studies .
our patient showed these signs and his other criteria were high too .. as mentioned , patients with nms may present different symptoms .
several factors such as male gender , agitation , physical exhaustion , dehydration , and neurological defects are associated with an increased risk of nms .
the majority of patients are in the age range of 20 - 50 years . in our case , there were anomalies in clinical findings and laboratory tests ( such as cpk : 40000 iu / l ) after exposure to the causative agent .
these symptoms could not be clarified by other diseases or medications and discontinuation of the drug resulted in significant clinical improvement .
we assessed the causality of this side effect with who - umc causality assessment system and naranjo assessment scale . according to typical clinical features and laboratory abnormal findings in nms , there was no need to rechallenge with the drug . according to the who - umc causality assessment system , side effects were probably related to olanzapine . in terms of naranjo assessment scale ,
symptoms following atypical antipsychotics include fewer extrapyramidal symptoms , reduced creatine kinase elevations , reduced muscle rigidity , and frequent mild fever .
occasionally , for instance , rigidity can be seen in the extremities instead of general muscular rigidity .
the study by hassan and buckley showed that clinical findings following typical and atypical antipsychotic drugs are the same , but increased cpk has been observed more by typical antipsychotic .
our report indicates that even with olanzapine treatment in the therapeutic range , a high - risk nms is curable even after one year .
therefore , it is essential that adequate and safe dose of medication is chosen and the patient is monitored by the signs and symptoms of this lethal syndrome .
in this report , we described a young male patient with nms caused by olanzapine treatment . this case underlines the need for vigilance regarding the side effects of taking atypical antipsychotic .
neuroleptic malignant syndrome can occur with atypical antipsychotic drugs such as olanzapine , particularly when risk factors are present .
we should pay attention to this rare but life - threatening event associated with fatal complications .
it is important to realize that an early detection and treatment of this disorder is vital .
clinicians should be aware that nms with eps could occur with olanzapine at steady state doses without recent dosage adjustments or titration . | neuroleptic malignant syndrome ( nms ) is a rare but life - threatening idiosyncratic side effect resulting from neuroleptic drugs .
nms mainly occurs in patients treated with high - potency typical antipsychotics , but rarely caused by atypical antipsychotics .
although nms is less common with atypical antipsychotic , but it seems that its incidence is rising due to increased administration of such drugs .
we present the case of a 27-year - old man with a history of paranoid schizophrenia that showed signs consistent with nms that occurred after treatment with olanzapine .
the patient was adherent to treatment .
he had decreased level of consciousness , muscle rigidity , diaphoresis , fever , drooling , urinary incontinence , and high blood pressure .
this patient illustrates that nms can occur due to treatment with atypical antipsychotic drugs like olanzapine , particularly in the presence of risk factors .
this phenomenon is often unrecognized , underdiagnosed , or not treated properly .
physicians should be aware that nms with extrapyramidal syndrome could occur with olanzapine at steady state doses without recent dosage adjustments or titration .
it is essential that adequate and safe dose of medication is chosen and the patient is monitored by the signs and symptoms of this lethal syndrome . |
resilience is defined as an active and adaptive biological , psychological , and social response to an event that may otherwise impair one 's normal function ( mcewen , 2007 , dudley et al . , 2011 , russo et al . , 2012 ) .
resilience typically implies the presence of insult - related pathologies that are overcome by molecular , cellular , synaptic , and finally behavioral changes that enable coping and normal function .
much has been written about the origins of resilience ( barker , 1989 , yehuda et al . , 2006 , gluckman et al . , 2007 , feder et al . , 2009 ,
there is clear evidence that resilience and vulnerability are influenced by genetic factors ( caspi et al . , 2003 , binder et al . , 2008 ) and gene - environment interactions ( caspi et al . , 2003 , bale et al . , 2010 , dincheva et al . , 2014 )
in addition , a large body of work has supported strong correlations of early - life experience / environment and resilience to cognitive and emotional illnesses later in life ( schmidt et al . , 2011 , baram et al . , 2012 , lucassen et al . , 2013 , huang , 2014 , insel , 2014 , santarelli et al . , 2014 ) .
several theories have been put forth that strongly suggest a causal and adaptive relationship between early - life experience and lifetime vulnerability or resilience to disease ( barker , 1989 , mcewen , 2000 , gluckman et al .
2012 ) . whereas human studies produce associations which can strongly suggest a causal relationship between early - life experience and vulnerability or resilience to disease , direct manipulations of early - life experience in animal models
have been shown to lead
to persistent changes in aspects of brain function , including resilience to subsequent insults such as stress .
indeed , a large number of primate and rodent models have been created to directly manipulate early - life experience , in order to generate resilience or vulnerability ( see maras and baram , 2012 , huang , 2014 for recent reviews ) .
broadly categorized , these paradigms aim to model early - life adversity such as chronic stress ( schmidt et al . , 2011 ,
2014 ) , or to create a nurturing early - life environment , typically based on optimized maternal care or novelty ( see akers et al . , 2008 , champagne et al . , 2008 ,
indeed , rodents raised in these distinct environments generally develop vulnerability ( huot et al . , 2002 , romeo et al . , 2004 , brunson et al . , 2005 , champagne et al . , 2008 ,
2012 ) or resilience ( liu et al . , 1997 , fenoglio et al . , 2005 ,
2012 ) to future stress and to cognitive and/or emotional deficits . although the influence of early - life experience on life - time resilience and vulnerability are well established , the underlying mechanisms are not fully understood .
it is now generally agreed that enduring changes in the expression of important genes might be involved , and that these changes might persist via epigenetic mechanisms including histone and dna modifications ( meaney and szyf , 2005 , borrelli et al . , 2008 , roth et al . , 2009 , mcclelland et al . , 2011 , sun et al . , 2013 , morrison et al . , 2014 )
what is the essence of the experience or environmental - signal that is perceived by the developing brain ?
how does the signal reach important neurons that change in response to the early - life experience ?
what are these neurons that are re - programmed to enable the structural and functional plasticity that underlies resilience ? how do these neurons know to modulate their epigenetic machinery ?
as mentioned above , direct manipulation of maternal care patterns has yielded long - lasting resilience or vulnerability to cognitive and emotional deficits .
we briefly describe the frameworks for bi - directional manipulation of maternal signals to young rodents that have been employed by our group , because the robust outcomes enable examination of the underlying mechanisms . the handling paradigm ( levine , 1957 , plotsky and meaney , 1993 , avishai - eliner et al .
, 2001a ) , which involves brief ( 15 min ) daily separation of rat pups from the mother during the first weeks of life , was used as a model of enhanced maternal care .
these brief separations promoted increased maternal - derived sensory input upon reunion with their mothers ( fig . 1 ) ( liu et al . , 1997 , fenoglio et al . ,
this paradigm led to increased resilience to depressive - like behavior ( meaney et al . , 1991 ) and improved learning and memory ( liu et al .
commonly , early - life stress is generated by maternal separation ( ms ) , a manipulation believed to be stressful .
extended absence of the mother provokes hypothermia and starvation , so many models use intermittent maternal deprivation and hence intermittent stress . in the human condition ,
when infants and children grow up in famine , war , or in the presence of drug - abusing mothers , the stress is typically chronic rather than intermittent , and the mother is typically present .
maternal care behaviors during these conditions might be the source of stress in the infant ( whipple and webster - stratton , 1991 , koenen et al .
, 2003 , kendall - tackett , 2007 , baram et al . , 2012 ) , as is particularly well documented in neglect / abuse situations , where maternal care is unpredictable and fragmented ( whipple and webster - stratton , 1991 , gaudin et al . ,
aiming to recapitulate the human condition , we generated a model of chronic early - life stress ( ces ) where the mother is continuously present .
the paradigm involves limiting the bedding and nesting material in the cage ( for a detailed review , see molet et al . , 2014 ) .
this impoverished cage environment resulted in abnormal maternal care , i.e. , fragmented maternal - derived sensory input to the pups .
the latter , as reported in humans , provoked chronic uncontrollable early - life emotional stress ( gilles et al . , 1996 ,
, 2001b , ivy et al . , 2008 , baram et al . , 2012 ) .
there was minimal change in the overall duration of maternal care or of specific aspects of care ( licking and grooming , nursing , etc ) ( ivy et al . , 2008 ) .
however , in both mice and rats , maternal care was fragmented and unpredictable : each bout of behavior is shorter and the sequence of nurturing behaviors is unpredictable ( rice et al . , 2008 , baram et al . , 2012 ) . in some cases ,
especially when cage environment was altered later in the development of the pups ( postnatal days 38 and 812 rather than 29 ) , rough handling of the pups by the mother was noted ( moriceau et al .
the ces model of aberrant maternal care and early - life experience led to emotional and cognitive vulnerabilities , and eventually overt pathology , including early cognitive aging ( for a detailed review , see molet et al .
, found depressive - like symptoms measured as increased immobility time in the forced swim test ( fst ) in adolescent rats that experienced ces . when tested during adolescence and young adulthood using paradigms such as novelty induced hypophagia , open - field , and elevated plus maze ,
rodents stressed early in life showed anxiety - like behaviors ( wang et al .
finally , memory deficits were unveiled by the morris water maze and novel object recognition tasks in adult rats and mice that were exposed to fragmented maternal care using the ces model ( brunson et al .
the ability to manipulate early - life experience in both adverse and salubrious directions provides powerful frameworks for examining the mechanisms for the resulting vulnerability and resilience .
a significant body of work has established a molecular signature of the resilience or vulnerability phenotypes generated by early - life experience in rodents . in adult rats experiencing augmented maternal care , an enduring upregulation of glucocorticoid receptor ( gr ) expression in hippocampus , and a repression of corticotropin releasing hormone ( crh ) expression in hypothalamic paraventricular ( pvn ) neurons was reported ( plotsky and meaney , 1993 , avishai - eliner et al . , 2001a ) .
the epigenetic basis of the enduring enhancement of hippocampal gr expression was uncovered by pioneering studies by the meaney group ( weaver et al . , 2004 ) .
examination of the temporal evolution of the molecular signature of rats experiencing augmented maternal care revealed that repression of crh expression in hypothalamus preceded the increased gr expression in hippocampus , and was directly dependent on recurrent predictable barrages of maternal care ( avishai - eliner et al .
these data suggested that the crh neuron in the hypothalamus may be an early locus of maternal care - induced brain programming .
notably , it is unlikely that changes in crh or gr expression in themselves explain the remarkable resilient phenotype of rats experiencing augmented maternal care early in life .
whereas the gr and crh are likely important mediators of long - lasting effects of maternal care , they may also serve as marker genes , a tool to study mechanisms of broad , enduring gene expression changes .
in addition , determining the locations of the changes in gene and protein expression helps to identify specific
target neurons that are re - programmed to enable the structural and functional plasticity that underlies resilience .
as mentioned above , the repression of gene expression in crh neurons occurred early and was already present after a week of
handling , i.e. , on postnatal day 9 in the pups ( avishai - eliner et al . ,
in addition , the crh - expressing neurons in the hypothalamus were identified as a component of a neuronal network activated by maternal care ( fenoglio et al . ,
the latter finding emerged from fos - labeling and mapping studies that queried which neurons were activated at several time points after returning of pups to their mothers following brief ( 15 min ) separations .
the fos mapping studies demonstrated that the maternal signal traveled via the central nucleus of the amygdala ( ace ) and bed nucleus of the stria terminalis ( bnst ) to the hypothalamic pvn ( fenoglio et al . , 2006 ) .
interestingly , while single augmented maternal care activated these structures and did not result in repressed crh expression , a week of recurrent daily barrages of maternal care activated also the stress - memory center of the brain , the paraventricular nucleus of the thalamus ( pvt ) ( bhatnagar and dallman , 1998 , fenoglio et al . , 2006 ) . the combinatorial output of the signal to the hypothalamic crh cells emerging from activation of pvt , ace , and bnst of recurrently handled pups differed from that of single - handled pups , and resulted in robust and enduring suppression of crh gene expression in these neurons ( fig .
2 ) ( fenoglio et al . , 2006 , karsten and baram , 2013 ) .
this reduction in crh expression in hypothalamic pvn , together with the apparent network changes involving this neuronal population , led us to focus on the crh - expressing cells in the pvn as important mediators of molecular changes associated with resilience .
neurons receive information mainly by synaptic contact , so that altered excitatory and/or inhibitory synaptic input onto crh neurons as a result of maternal care might be a plausible mechanism for the alteration of molecular machinery in these neurons that enduringly reduces crh expression .
synaptic innervation of neurons is now known to be dynamic and modulated by experience ( brunson et al .
, 2001 , verkuyl et al . , 2004 , horvath , 2005 ) . for crh neurons ,
the majority of input is mediated by gabaergic and glutamatergic synapses ( aubry et al . , 1996 ,
cullinan , 2000 , miklos and kovacs , 2002 , ziegler et al . , 2012 ) , via gabaa ( cullinan , 2000 ) and glutamate receptors ( aubry et al . , 1996 , kiss et al .
, 1996 , cullinan , 2000 , di et al . , 2003 , ulrich - lai et al . , 2011 , ziegler et al . , 2012 ) .
combining electrophysiology , quantitative analyses of vesicular transporters and quantitative confocal and electron microscopy , korosi et al . , studied if enhanced early - life experience reduced excitation to crh neurons or augmented their inhibition ( korosi et al . , 2010 ) . using similar methodologies , gunn et al .
, examined the excitatory and inhibitory input onto crh - expressing hypothalamic neurons of mice experiencing aberrant , fragmented maternal care in cages with limited bedding and nesting material ( gunn et al . , 2013 ) .
using several different methods , korosi et al . , discovered reduced number and function of excitatory synapse that abut onto crh - expressing neurons in pups experiencing a week of recurrent augmented maternal care ( korosi et al . , 2010 ) . while enhanced maternal care resulted in reduced levels of the glutamatergic transporter vglut2 via western blot , no change in the levels of the gaba - a transporter vgat was detected .
dual - label confocal microscopy revealed a reduced number of vglut2-positive puncta ( presynaptic terminals ) abutting identified crh neurons ( fig . 3 ) .
quantitative electron microscopy revealed reduced number of asymmetric ( excitatory ) synapses onto crh neurons in pups experiencing augmented maternal care .
finally , the frequency of miniature post synaptic excitatory currents ( epscs ) , a functional measure of excitatory synapses , was reduced in presumed crh cells of rats experiencing recurrent increased maternal care ( korosi et al . , 2010 ) . obviously , if optimal early - life experience and specifically maternal signals reduce excitatory synapses , then aberrant maternal care should increase excitatory synapses onto crh neurons . indeed , a recent study by gunn et al .
( 2013 ) found that mice experiencing the limited bedding and nesting cage environment , which provokes fragmented maternal care and chronic stress , had increased levels of crh expression in the pvn ( gunn et al . , 2013 ) .
remarkably , immunohistochemical and electrophysiological approaches demonstrated a robust increase in excitatory input onto the stress - sensitive crh - expressing neurons , in direct contrast to the observation following enhanced early - life experience ( fig . 4 ) .
together , these findings support the idea that early - life experience influences resilience via tuning of the level of excitatory input into stress - sensitive neuronal populations , which in turn affects intracellular programs .
notably , at least in the case of optimal early - life experience , the synaptic changes were transient .
tweak gene expression regulatory pathways and epigenetic mechanisms that maintain the expression changes enduringly . whereas we do not understand how the transient synaptic changes modulate downstream intracellular signaling , we propose that the decrease in the excitatory drive onto the crh neurons following augmented maternal care leads to reduced calcium influx into the crh cells , which can potentially initiate transcriptional programs , resulting in decreased crh expression . once initiated ,
the transcriptional changes may then be stably maintained via epigenetic mechanisms ( mcclelland et al .
early - life experience interacts with genetic factors to shape cognitive and emotional outcomes . specifically , early - life experiences influence resilience or vulnerability to emotional and cognitive illnesses . salient signals by which early - life experiences program the brain include recurrent sensory inputs from the mother .
fragmentation and unpredictability of maternal - derived signals might promote vulnerability to mental illness , whereas consistency and predictability might promote resilience .
the salient signal from the early - life environment is transported to stress - sensitive neurons via neuronal networks , and it modulates the numbers and function of synapses impinging on these neurons .
optimal early - life experience seems to reduce excitation to crh - expressing hypothalamic neurons whereas chronic early - life stress and fragmented maternal care increases excitation onto these same neurons .
the plasticity of synaptic wiring of crucial stress - responsive neurons likely initiate gene expression programs within these neurons , and the resulting changes of gene expression may persist - perhaps to subsequent generations - via epigenetic mechanisms . | genes and environment interact to influence cognitive and emotional functions throughout life .
early - life experiences in particular contribute to vulnerability or resilience to a number of emotional and cognitive illnesses in humans . in rodents , early - life experiences
directly lead to resilience or vulnerability to stress later in life , and influence the development of cognitive and emotional deficits .
the mechanisms for the enduring effects of early - life experiences on cognitive and emotional outcomes are not completely understood . here , we present emerging information supporting experience - dependent modulation of the number and efficacy of synaptic inputs onto stress - sensitive neurons .
this synaptic
rewiring , in turn , may influence the expression of crucial neuronal genes .
the persistent changes in gene expression in resilient versus vulnerable rodent models are likely maintained via epigenetic mechanisms .
thus , early - life experience may generate resilience by altering synaptic input to neurons , which informs them to modulate their epigenetic machinery . |
rett syndrome ( rtt ) is a genetically determined , neurodevelopmental disorder with autistic features [ 1 , 2 ] .
although relatively rare , rtt represents the second most common cause of severe intellective disability in the female gender . to date , the disease has been classified into a typical form and three main atypical variants , that is , preserved speech , early seizure , and congenital . in up to 95% of cases , rtt is caused by de novo mutation in the x - linked gene encoding mecp2 , a protein known to either activate or repress several transcriptional genes [ 3 , 4 ] .
cumulating evidence indicates that rtt , for a long time considered exclusively a disease of the brain , is actually a systemic disease with involvement of several organs besides the brain , including autonomic nervous system , lung , bone , and heart [ 58 ] .
girls affected by typical rtt show a 300-fold increased risk for sudden cardiac death as compared to general population ( about 26% of all deaths are sudden and of unknown cause ) , although a satisfactory explanation for the association is still missing . in the lack of evidence for an increased prevalence of congenital heart defects , the attention of several authors has been focused on the presence of cardiac dysautonomia and rhythm abnormalities . in particular , a prolonged qt interval , an indicator of a repolarization abnormality and a well - established risk factor for sudden cardiac death , is reported in nearly 20% of patients with rett syndrome [ 9 , 1113 ] .
abnormally persistent sodium currents have been reported in cardiomyocytes from mecp2 mice and found to be normalized by the sodium channel - blocking antiepileptic drug phenytoin , which strongly suggests a brain - heart link as a possible explanation for the increased risk of sudden death in rtt .
however , our recent observation of a subclinical myocardial biventricular dysfunction in a large series of typical and atypical rtt patients may add new perspectives to the heart involvement in this neurodevelopmental disease .
evidence of enhanced oxidative stress ( os ) and , in particular , lipid peroxidation has been well established by our group in blood samples from patients with rtt and recently confirmed in primary skin fibroblasts cultures [ 1521 ] .
however , the molecular pathways linking the mecp2 gene mutation to the os derangement remain to be explored and , in particular , whether the nature of the relationship between mecp2 gene mutation and abnormal redox homeostasis is causal or correlational remains currently unclear . at the same time
, experimental models have shown that os is detrimental for myocardial function [ 23 , 24 ] .
therefore , we speculate that os may play a role in the myocardial dysfunction of rtt patients . omega-3 polyunsaturated fatty acids ( -3 pufas ) are natural molecules with a wide range of physiological functions on multiple tissues including the heart .
in particular , -3 pufas are able to affect a myriad of molecular pathways , including alteration of physical and chemical properties of cellular membranes , direct interaction with and modulation of membrane channels and proteins , regulation of gene expression via nuclear receptors and transcription factors , changes in eicosanoid profiles , and conversion of -3 pufas to bioactive metabolites .
-3 pufas have gained increasing attention in the prevention of cardiovascular disease , although their biological effects and molecular mechanisms are highly debated . in previous studies , we have demonstrated that supplementation of -3 pufas moderately reduces clinical severity and significantly reduces the levels of several os biomarkers in the blood of rtt patients [ 17 , 19 , 26 ] .
the aim of the present study was to assess the effects of 12 months of dietary supplementation with high - dose -3 pufas on the rtt - related subclinical myocardial dysfunction .
in this study , a total of 66 rtt patients ( mean age 12.7 9.1 years ) with typical presentation and demonstrated mecp2 mutation were enrolled ( table 1 ) .
rtt diagnosis and inclusion / exclusion criteria were based on the recently revised rtt nomenclature consensus [ 28 , 29 ] .
rtt clinical severity was assessed using the clinical severity score ( css ) , a validated clinical rating specifically designed for rtt , based on 13 individual ordinal categories measuring clinical features common in rtt .
all the patients were admitted to child neuropsychiatric unit , university hospital azienda ospedaliera universitaria senese ( head dr .
blood samplings in the patients ' group were performed during the routine follow - up study at hospital admission .
sampling from the control group was carried out during routine health checks , sports , or blood donations obtained during the periodic clinical checks .
the study was conducted with the approval of the institutional review board and all informed consents were obtained from either the parents or the legal tutors of the enrolled patients .
patients were randomized at admission ( n = 33 treated , mean age at supplementation time zero : 13.0 8.6 years ; n = 33 untreated , mean age at time zero : 12.4 9.3 years ) to either oral supplementation with -3 pufas oil for twelve months or no treatment .
administered -3 pufas were in the form of fish oil ( norwegian fish oil as , trondheim , norway , product number ho320 - 6 ; italian importer : transforma as italia , forl italy ; italian ministry registration code : 10 43863-y ) at a dose of 5 ml twice daily , corresponding to docosahexaenoic acid ( dha ) 71.9 13.9 mg / kg b.w./day and eicosapentaenoic acid ( epa ) 115.5 22.4 mg / kg b.w./day , with a total -3 pufas 242.4 47.1 mg / kg b.w./day .
all the subjects , included patients , examined in this study were following a standard mediterranean diet .
the study was performed using a commercially available echocardiography equipment ( philips ie 33 vision 2009 , qlab 7.0 software ; 5 and 8 mhz transducers ) as previously reported .
briefly , two - dimensional right and left chambers quantification ( areas and volumes ) , left ventricle ejection fraction ( simpson 's method ) , and pulmonary arterial systolic pressure ( pasp ) were estimated .
mitral flow velocities ( e wave , a wave , and e / a ratio ) were recorded using pulsed wave ( pw ) doppler on the mitral valve .
the evaluation of left and right ventricular longitudinal systolic function was performed by ( a ) mitral annular plane systolic excursion ( mapse ) and tricuspid annular plane systolic excursion ( tapse ) , using m - mode , and ( b ) systolic ( s ) and early diastolic ( e ) peak velocities , using pw tissue doppler imaging ( tdi ) of the lateral ( lat ) and septal ( sep ) mitral annulus for left ventricle ( lv ) and of tricuspidal annulus for right ventricle ( rv ) in four - chamber apical view .
the e / elat ratios were determined as surrogate of lv filling pressures . in order to reduce operator - dependent bias ,
all measures were performed by two operators , blinded for clinical and therapeutical data of rtt group .
blood sampling was carried out in all subjects at around 8.00 am after the overnight fast . for the -3 pufas
treated group , blood sampling was performed the day before starting the supplementation and the day after the end of the selected 12-month period .
blood was collected in heparinized tubes , and all manipulations were carried out within 2 h after collection .
blood samples were centrifuged at 2,400 g for 15 min at room temperature .
the platelet poor plasma was saved , and the buffy coat was removed by aspiration .
the erythrocytes were washed twice with physiological solution , resuspended in ringer solution ( 125 mm nacl , 5 mm kcl , 1 mm mgso4 , 32 mm hepes , 5 mm glucose , 1 mm cacl2 ) , ph 7.4 as a 50% ( vol / vol ) suspension , and then used for the determination of erythrocyte non protein - bound iron ( npbi ) .
plasma was used for free isoprostanes ( f2-isoprostanes , f2-isops , and f4-neuroprostanes , f4-neurops ) , 4-hydroxynonenal protein adducts ( 4-hne pas ) , and npbi determinations .
for all isoprostane determinations , butylated hydroxytoluene ( bht ) ( 90 m ) was added to plasma as an antioxidant and stored under nitrogen at 70c until analysis .
npbi is a pro - oxidant factor , associated with hypoxia , hemoglobin oxidation , and subsequent heme iron release .
intraerythrocyte and plasma npbi were determined as a desferrioxamine ( dfo)-iron complex by high - performance liquid chromatography , as previously reported .
isoprostanes are considered the gold standard for the os in vivo evaluation [ 31 , 32 ] .
specifically , f2-isops are the end products of arachidonic acid oxidation , a polyunsaturated fatty acid which is abundant in both brain grey and white matter .
plasma f2-isops and f4-neurops were determined by a gas chromatography / negative ion chemical ionization tandem mass spectrometry ( gc / nici - ms / ms ) analysis after solid - phase extraction and derivatization steps [ 33 , 34 ] .
for f2-isops , the measured ions were the product ions at m / z 299 and m / z 303 derived from the [ m-181 ] precursor ions ( m / z 569 and m / z 573 ) produced from 15-f2t - isops and pgf2-d4 , respectively .
for f4-neurops , the measured ions were the product ions at m / z 323 and m / z 303 derived from the [ m-181]precursor ions ( m / z 593 and m / z 573 ) produced from oxidized dha and the pgf2-d4 , respectively . plasma 4-hydroxynonenal protein adducts ( 4-hne pas ) are markers of protein oxidation due to aldehyde binding from lipid peroxidation sources .
plasma proteins ( 30 g protein ) were resolved on 420% sds - page gels ( lonza group ltd . , switzerland ) and transferred onto a hybond ecl nitrocellulose membrane ( ge healthcare europe gmbh , milan , italy ) .
after blocking in 3% nonfat milk ( biorad , hercules , ca , usa ) , the membranes were incubated overnight at 4c with goat polyclonal anti-4-hne adduct antibody ( code ab5605 ; millipore corporation , billerica , ma , usa ) .
following washes in tbs tween and incubation with specific secondary antibody ( mouse anti - goat horseradish peroxidase - conjugated , santa cruz biotechnology inc . ,
ca , usa ) for 1 h at rt , the membranes were incubated with ecl reagents ( biorad , hercules , ca , usa ) for 1 min .
quantification of the significant bands was performed by digitally scanning the amersham hyperfilm ecl ( ge healthcare europe gmbh , milan , italy ) and measuring immunoblotting image densities with imagej software .
differences between groups were evaluated using independent - sample t - test ( continuous normally distributed data ) , mann - whitney rank sum test ( continuous nonnormally distributed data ) , and kruskal - wallis test .
the medcalc version 12.1.4 statistical software package ( medcalc software , mariakerke , belgium ) was used .
all patients of the -3 pufas arm of the study completed the 12-month supplementation and no side effects were observed .
phenotypical severity , biometric data , and bone densitometry estimates as well as serum 25-oh vitamin d levels were found to be comparable between the -3 pufas - supplemented and unsupplemented rtt subpopulations ( table 1 ) . following -3 pufas ( epa plus dha )
supplementation , significant improvements in systolic longitudinal parameters of both ventricles were observed ( figure 1 ) , along with increased pasp . on the other hand ,
following 12 months of -3 pufas supplementation , npbi , plasma f2-isops , and f4-neurops were significantly reduced as compared to time 0 values ( figures 2(a)2(d ) ) .
significant differences were already observed in the treated group at time of 6 months for plasma npbi , intraerythrocyte npbi , and plasma f2-isops .
the correlation matrix for os markers and myocardial function variables in rett syndrome following -3 pufas supplementation is reported in table 2 .
plasma f2-isops and f4-neurops and 4-hne pas were found to be inversely related to the left ventricular systolic function parameters . on the other hand , no significant changes in os markers were detectable in the untreated rtt patients ( data not shown ) . in the -3 pufas - supplemented group ,
clinical severity decreased to 25.5 and 30.1% at 6 and 12 months , respectively ( css at time zero : 26.2 11.2 ; css at time 6 months : 19.52 8.7 ; css at time 12 months : 18.3 7.8 ; anova p < 0.005 ; pairwise comparisons time 0 > time 6 months = time 12 months ) .
conversely , no significant differences in clinical severity were observed in the unsupplemented group of patients ( css at time zero : 26.0 11.1 ; css at time 12 months : 26.5 10.9 ; p = 0.911 ) .
significant improvements were observed in the areas of attention , breathing abnormalities , muscular tone , ambulation , autonomic dysfunction , and somatic growth .
our findings indicate , for the first time , that the subclinical myocardial dysfunction observed in typical rtt can be , at least partially , rescued by 1-year high - dose -3 pufas dietary supplementation .
specifically , -3 pufas appear to reverse all the examined longitudinal systolic parameters ( mapse , slat , ssep and tapse , srv ) of the left and right ventricles .
moreover , the improvement in the systolic myocardial function was found to be associated with a marked decrease of os markers as determined in plasma , whereas no significant changes in the diastolic function were detectable in -3 pufas - treated patients .
taken as a whole , these findings suggest that os may play a key role in the systolic performance of the rtt myocardium and that it can be at least partially rescued by -3 pufas dietary supplementation . to date , among the molecular mechanisms potentially underlying the -3 pufas action there are changes in membrane structures and gene expression , direct interactions with ion channels , and alterations in eicosanoid biosynthesis .
in particular , epa and dha have been reported to compete with arachidonic acid for the conversion by cytochrome p450 enzymes , thus resulting in the formation of alternative , physiologically active , metabolites which could likely mediate some of their beneficial effects .
our current working hypothesis on the beneficial effects of -3 pufas in rtt is that the increased isoprostanes levels in rtt are not simply the effect of the peroxidation of the pufas precursors following the attack by radical oxygen species ( ros ) , but rather the effect of a potential dysregulation of the molecular targets of -3 pufas .
contrary to expectations , the assumed fatty acids are not further oxidized , while the actual endogenous isops production is reduced ( the fatty acid paradox ) together with amelioration of the clinical disease severity .
conceivably , an excess of peroxidation end products from -6 and -3 pufas would actually imply an excessive consumption of these pufas in the cell membranes , thus paving the way for a new perspective on the nutritional horizons in rtt .
as rtt girls appear to chronically suffer from oxidation of pufas , either -3 ( i.e. , dha / epa ) or -6 ( i.e. , aa , ada ) , but , at the same time , benefit from -3 pufas supplementation , it can be inferred that these patients would need -3 pufas replacement as a consequence of a persistent pufas oxidation within the chronic os context . on the other hand , it is also possible that , in rtt , the endogenous pufas are , for their own nature , more susceptible to the os as compared to the exogenous ones .
further research is needed to explore this point , although a very interesting recent report indicates that -3 pufas supplementation , as fish oil , in mice with nonalcoholic fatty liver disease is able to prevent hepatic lipid accumulation and improve lipid metabolism without causing oxidative stress .
this report lends further support to our fatty acids paradox theory by generalizing it to different abnormal lipid metabolism conditions , either genetic or environmental .
a further critical new piece of research indicates that cholesterol synthesis is impaired in a mutant mouse model in rtt .
this latter work strongly indicates that a congenital lipid metabolism error may play a role in the rtt pathogenesis and suggests the use of statins as a potentially valuable alternative treatment for the human disease . in line with this hypothesis ,
we have previously described an unrecognized hypercholesterolemia in girls affected by the syndrome and pointed out the possibility of an abnormal cholesterol synthesis with a likely partial block in the squalene catabolism due to coexistence of heterozygous mutations in cyp24a1 ( omim126065 ) or tm7sf2 ( omim603414 ) , which encode the proteins cp24a and erg24 , respectively . moreover
, a promising recent line of research suggests that the beneficial actions of -3 pufas ( or their secondary metabolites ) could be related to the modulation of an unrecognized subclinical inflammatory status in rtt , a point certainly in need of further exploration , but well fitting with the known anti - inflammatory properties of -3 pufas [ 26 , 42 ] . on the other hand , a possible explanation for the incomplete rescue of the myocardial dysfunction in rtt could reside in the fact that mecp2 appears to be involved in myocytes differentiation and maturation .
therefore , the relationship between the mecp2 protein and heart needs to be further evaluated in the experimental models of the disease .
our data indicate that -3 pufas are able to improve the subclinical biventricular myocardial systolic function observed in typical rtt and that this functional gain is partially mediated through a regulation of the redox balance . | rett syndrome ( rtt ) is a devastating neurodevelopmental disorder with a 300-fold increased risk rate for sudden cardiac death .
a subclinical myocardial biventricular dysfunction has been recently reported in rtt by our group and found to be associated with an enhanced oxidative stress ( os ) status . here , we tested the effects of the naturally occurring antioxidants -3 polyunsaturated fatty acids ( -3 pufas ) on echocardiographic parameters and systemic os markers in a population of rtt patients with the typical clinical form .
a total of 66 rtt girls were evaluated , half of whom being treated for 12 months with a dietary supplementation of -3 pufas at high dosage ( docosahexaenoic acid ~71.9 13.9 mg / kg b.w./day plus eicosapentaenoic acid ~115.5 22.4 mg / kg b.w./day ) versus the remaining half untreated population .
echocardiographic systolic longitudinal parameters of both ventricles , but not biventricular diastolic measures , improved following -3 pufas supplementation , with a parallel decrease in the os markers levels .
no significant changes in the examined echocardiographic parameters nor in the os markers were detectable in the untreated rtt population .
our data indicate that -3 pufas are able to improve the biventricular myocardial systolic function in rtt and that this functional gain is partially mediated through a regulation of the redox balance . |
the first reported hiv cases in china were found among foreign travellers in 1985 and the first indigenous infections were identified in 1989 among injection drug users ( idus ) in yunnan province .
subsequently , hiv infections spread to henan province through contaminated plasma collection and to xinjing uygur autonomous region by idus . recently
, hiv transmission through sexual contact has increased for both women and men who have sex with men . in 2009 ,
the national hiv prevalence was 0.057% , and the number of hiv - infected persons was estimated to be 740 000 .
since the late 1980s , a series of national strategic , managerial and technical plans have been set up to build a tiered laboratory system to accurately identify infected persons .
free hiv antibody screening has been provided to the general public since 2004 to increase testing coverage .
free cd4 cell enumeration , viral load ( vl ) determination and antibody confirmatory testing were provided in 2004 , 2006 and 2008 , respectively . by the end of 2009 ,
8273 serologic screening laboratories were in place and more than 220 million cumulative screening tests had been performed nationwide since 2002 . to monitor trends in incidence
, the efficacy of anti - retroviral treatments ( arts ) and vertical transmission , laboratory capacity was further expanded to include more sophisticated molecular methodologies . in this report
, we describe the hierarchical structure , function , measures and challenges of the laboratory expansion process .
the establishment of the chinese hiv laboratory network can be divided into three developmental stages .
the initial stage , from 1985 to 1988 , was a spontaneous reaction to the hiv epidemic outside of china .
the laboratory capacity was inadequate to carry out timely testing and there was a general lack of scientific knowledge , technical skills and specialized equipment . recognizing the need for an organized national response , china formulated a national hiv / aids prevention plan and additional hiv / aids surveillance regulations in 1987 , focusing on serologic surveillance in most - at - risk populations and of blood products .
initially , the role of the national reference laboratory was played by the institute of virology at the chinese academy of preventive medicine . in 1998 ,
the national aids reference laboratory ( narl ) was established in the national center for aids prevention and control of chinese center for disease control and prevention ( china cdc ) . by 2002 , 44 confirmatory laboratories and 1870 screening laboratories were operational , which conducted more than 22 000 confirmatory and 12.3 million screening tests ( figure 1 ) .
figure 1the yearly increase of screening ( a ) and confirmatory and confirmatory central laboratories ( b ) and the respective tests performed in china between 1992 and 2009 .
diamonds : numbers of laboratories ; squares : numbers of tests performed the yearly increase of screening ( a ) and confirmatory and confirmatory central laboratories ( b ) and the respective tests performed in china between 1992 and 2009 .
diamonds : numbers of laboratories ; squares : numbers of tests performed the third stage of laboratory development began in 2003 when china equipped its hiv network laboratories with contemporary instruments , and used standardized operating procedures to homogenize operations nationwide .
sophisticated molecular methodologies , optimized quality assurance programmes and an electronic data reporting system were developed . in 2004 , china issued national hiv / aids management guidelines to delineate the overall configuration and managerial structure of a tiered screening and confirmatory laboratory network ( table 1 ) .
the base of the network was comprised of screening laboratories located mostly in county - level public health facilities and hospitals .
they primarily performed serologic tests such as enzyme - linked immunosorbent assays ( elisa ) and rapid tests ( rts ) .
all reactive specimens were transported to confirmatory laboratories where western blot ( wb ) assays were used to confirm results before a positive report was issued .
table 1distribution and major functions of each tier of the hiv testing facilities in chinafacilitiesnumberlocationmajor functionsnarl1china cdc in beijingformulation of national testing guidelines , implementation and coordination of national testing projects , provision of quality assurance programmes , supervision , evaluation of novel technologies and reagent kitsconfirmatory central laboratories35one each in 31 provincial cdcs and four other special organizationsformulation of provincial policies and conducting in - province staff training , reagent kit evaluations , supervisory visits of lower tier laboratories and provincial serologic pt programmesconfirmatory laboratories254prefecture cdcs , large hospitals , border health quarantine and other facilitiesconductance of confirmatory tests to verify all specimens screened reactive or indeterminate from the screening laboratories , provision of qa , training and supervision to screening laboratoriesscreening laboratories8273county cdcs , hospitals , blood stations , mch clinics , border health quarantine and other facilitiesconductance of screening tests including elisa and rtsxinjiang production and construction corps , border health quarantine , armed forces and military police bureau .
mch : maternal and child health ; pt : proficiency testing ; rts : rapid tests . distribution and major functions of each tier of the hiv testing facilities in china xinjiang production and construction corps , border health quarantine , armed forces and military police bureau .
mch : maternal and child health ; pt : proficiency testing ; rts : rapid tests .
the number of screening laboratories tripled from 2382 in 2003 to 8273 in 2009 ( figure 1 ) .
screening laboratories used control specimens provided by reagent kits and external quality controls provided by commercial sources to perform run - to - run levey
the total number of confirmatory central and confirmatory laboratories increased from 48 in 2003 to 289 in 2009 .
the number of screening and confirmatory tests performed in 2009 were 52 million and 88 498 , respectively ( figure 1 ) .
the screening laboratories were widely distributed in county cdcs , hospitals , blood banks , maternal and child health ( mch ) clinics and border quarantine health - care facilities . originally , these screening laboratories were located in cdc facilities ; but by the end of 2009 , 57% were in the hospital system .
rts were introduced into china in 1999 and their use followed the who recommendations of using a serial testing algorithm .
initially , rts were used primarily in prevention of mother - to - child transmission ( pmtct ) programmes to quickly identify hiv - infected mothers .
it is now widely used in voluntary counselling and testing ( vct ) facilities , hospital surgery and out - patient departments and for hard - to - reach populations such as men who have sex with men ( msm ) or female sex workers in prefecture and lower health - care facilities .
beginning in 2006 , the narl conducted a proficiency testing ( pt ) evaluation on confirmatory laboratories , and , by 2009 , 269 of the 289 ( 93% ) laboratories participated in the programme .
provincial and confirmatory laboratories performed cd4 enumeration , hiv-1 incidence tests and molecular assays . in 2009 , 84 of these laboratories conducted vl tests , 12 dried blood spot ( dbs)-based polymerase chain reaction ( pcr ) for early infant diagnosis ( eid ) , 6 drug resistances ( drs ) and 15 bed - based incidences ( table 2 ) . because of the expansion of art , the annual numbers of cd4 and vl tests performed increased by 52 and 90% , respectively , from 2008 to 2009 . in 2009 , nationally , 238 385 cd4 enumeration , 63 408 vl and 3946 dr tests were completed .
table 2the establishment and participants of various pt programmes organized and offered by the narlcategoriestestsprogramme initiation yearno .
( coverage ) of participating laboratories in 2009 ( % ) frequency ( no . per year)serologic testselisa2004289 ( 100)3wb2004289 ( 100)3rt2006269 ( 93)3incidence ( bed)200615 ( 100)2immunologic testcd42004234 ( 74)2virologic testsvl200579 ( 94)2dbs for eid200612 ( na)2dr20066 ( na)1subtyping200612 ( na)2serologic pt programmes were only offered to confirmatory central and confirmatory laboratories .
provincial confirmatory central laboratories were responsible for the serologic qa of their respective screening laboratories .
the numbers of laboratories performing the tests of high complexity ( i.e. cd4 , vl , bed , dr and subtyping ) are small .
some of these laboratories were sub - confirmatory laboratories in high - hiv - prevalent areas .
na : data not available in 2009 . the establishment and participants of various pt programmes organized and offered by the narl serologic pt programmes were only offered to confirmatory central and confirmatory laboratories .
provincial confirmatory central laboratories were responsible for the serologic qa of their respective screening laboratories . the numbers of laboratories performing the tests of high complexity ( i.e. cd4 , vl , bed , dr and subtyping ) are small .
some of these laboratories were sub - confirmatory laboratories in high - hiv - prevalent areas .
clean , semi - contaminated and contaminated. pcr - based tests ( i.e. vl , eid , dr and subtyping ) have to be conducted in a facility with three separate rooms for reagent preparation , blood sample processing and amplification .
laboratory staff are required to take specific biosafety and test - specific training and biannual refresher training in order to perform clinical testing .
provincial health authorities and technical hiv experts perform annual laboratory inspections and issue operational permits .
laboratories without operational permits or those that fail inspections are ordered to close until corrective remedies are instituted .
if a laboratory is closed , the narl and the provincial cdc provide onsite supervisory visits and training to raise the operational level of both staff and laboratory procedures . with the input from domestic and international experts , the narl formulated and established hiv - related technical guidelines .
the national hiv / aids testing technical guidelines were published in 2004 and revised in 2009 to incorporate recent methodological changes and international recommendations .
the narl is responsible for the dissemination of the policies to laboratory staff through training workshops , electronic notifications and the semi - annual laboratory network newsletter distributed to all screening facilities .
these trainees would in turn provide training to the staff at the lower level laboratories .
the narl is responsible for national training courses for sophisticated tests including bed , eid , dr and subtyping .
provincial laboratories are responsible for sub - provincial training on wb , rts , elisa and cd4 . in 2009 ,
the 229 technical staff from the 35 provincial laboratories received an average of 2.7 trainings per person .
the training used to prepare the laboratory staff to obtain operational permits was conducted in laboratories with repetitious hands - on practice .
in addition to the technical staff , 10% of the trainees were non - laboratory staff responsible for general quality assurance and procurement service with classroom training .
the inclusion of these staff in the training enhances co - ordination of operations so that reagent procurement and equipment maintenance would be carried out appropriately .
post - exposure prophylactic treatment for hiv was available . in 2009 , 920 accidental exposures were reported by personnel working in hiv control and prevention programmes and laboratories .
six hundred and eighty - four persons received art prophylaxis , counselling and follow - up .
to build and strengthen its own capacity , the narl first participated in several international pt programmes .
narl staff were trained in hiv laboratories in western countries to learn to prepare pt materials .
it also participated in the following programmes : ( i ) college of american pathologists hiv wb and p24 antigen tests ; ( ii ) u.s .
cdc s incidence assay , and dbs - based eid detection ; ( iii ) rush - presbyterian - st luke s medical center on vl and dr qa programmes ; ( iv ) australia national serology reference laboratory on antigen and antibody tests ; and ( v ) cd4 pt provided by united kingdom s national external quality assessment site programme and canadian national immunology programme for the quality assessment and standardization of immunological measures relevant to hiv / aids .
the narl began offering its own elisa and wb pt programmes to domestic laboratories in 2004 ( table 2 ) . in 2009 , these programmes were expanded to cover rts , bed , cd4 , vl , dbs - based eid , dr and subtyping .
the laboratory coverage in elisa , wb , rts , cd4 and viral load was 100% ( 289/289 ) , 100% ( 289/289 ) , 93% ( 269/289 ) , 74% ( 234/317 ) and 94% ( 79/84 ) , respectively .
provincial confirmatory central laboratories also offered pt programmes on serologic tests ( elisa and rts ) . in 2009 , 97% ( 34/35 ) of confirmatory central laboratories provided this service to screening laboratories .
the coverage of 29 provinces was 100% , but one province had a low coverage of 25% because of the lack of human resources and experience .
however , narl personnel provided feedback and discussed findings with laboratory staff . if a laboratory exhibited low performance , the narl conducted on - site visits to resolve problems .
problems identified included transcriptional error or reporting specimens not pertaining to the pt programme ( 65% ) and delay of reporting ( 35% ) .
technical managers of provincial laboratories participated in the quality monitoring process . with the exception of one confirmatory central laboratory in 2008 and another one in 2009 ,
all others provided elisa pt for their constituent screening laboratory , similar to the process used by the narl .
on - site laboratory evaluations are conducted annually , as stipulated by the national management regulation of hiv / aids detection and the national technical guidelines for detection of hiv / aids .
the evaluation covers staff training , biosafety , documentation of standard operating procedures , record keeping equipment maintenance and laboratory performance .
the narl inspected 2963 wb - positive specimens nationwide and found the average turnaround time ( tat ) for reporting was 5 working days ( range : 121 days ) .
the one with the longest tat was 21 working days and occurred in a laboratory located in a region of low hiv prevalence where the number of specimens required to be confirmed was small and thus the test was not done until a larger number of specimens was collected .
this laboratory was instructed to forward the specimens to other laboratory to complete the tests in a timely manner .
in 2002 , the narl and provincial cdcs adopted a multi - province evaluation to determine the sensitivity and specificity of commercial hiv antibody diagnostic kits in china ( table 3 ) . in 2008 ,
the narl and five cdcs in high - hiv - prevalence areas ( anhui , sichuan , guangxi , guangdong and guizhou ) jointly evaluated 16 elisa and 12 rt kits after they had received approval from china s state food and drug administration ( table 3 ) .
kits were purchased randomly from the market and tested using standard panels previously confirmed by wb .
the foreign rt kits evaluated had an average sensitivity of 100% and a specificity of 99.51% ( range : 99.02100% ) .
the domestic rt kits had an average sensitivity of 98.09% ( range : 87.5100% ) and specificity of 99.51% ( range : 98.04100% ) .
one domestic kit performed especially poorly with a sensitivity of 87.5% and specificity of 100% .
the finding of an rt kit with suboptimal performance strengthens the notion that frequent inspections of domestic products are important .
the kit performance data were disclosed in the china cdc web site and published in the china aids laboratory network newsletter .
evaluations of this kind convey a message to domestic manufacturers to continue to improve their manufacturing technologies and enhance product quality .
table 3evaluation of the performance of commercial hiv antibody diagnostic elisa and rt kits ( 200208)elisartyearnumber of kitssensitivity ( % ) average ( range)specificity ( % ) average ( range)number of kitssensitivity ( % ) average ( range)specificity ( % ) average ( range)2002domestic 1099.10 ( 97.88100)98.41 ( 96.4999.58)foreign 199.6599.402003domestic 1199.75 ( 98.98100)96.72 ( 94.8299.10)494.37 ( 91.4896.77)97.72 ( 94.6899.77)foreign 299.61 ( 99.21100)98.41 ( 98.0998.73)398.31 ( 96.7199.15)98.10 ( 97.7798.61)2004domestic 1599.47 ( 82.35100)91.87 ( 83.0099.50)689.48 ( 83.3395.24)98.33 ( 97.6199.52)foreign 399.61 ( 98.82100)97.50 ( 96.5098.00)492.26 ( 80.9597.62)97.85 ( 96.6599.52)2005domestic 1399.29 ( 96.64100)94.16 ( 90.7597.24)797.81 ( 95.7399.15)97.82 ( 95.7699.58)foreign 399.44 ( 99.16100)98.68 ( 97.8099.56)598.44 ( 96.5899.15)98.45 ( 95.3499.58)2006domestic 1499.65 ( 98.04100)97.91 ( 94.8699.21)899.23 ( 96.91100)97.99 ( 94.6499.23)foreign 399.67 ( 99.02100)98.41 ( 96.8499.21)598.76 ( 95.88100)98.71 ( 97.70100)2007domestic 1199.91 ( 99.04100)99.38 ( 97.9799.75)1194.83 ( 84.21100)99.09 ( 94.62100)foreign 310098.90 ( 97.4699.75)398.31 ( 96.7199.15)98.10 ( 97.7798.61)2008domestic 1399.53 ( 97.69100)98.72 ( 94.61100)998.09 ( 87.50100)99.51 ( 98.04100)foreign 310098.20 ( 94.61100)310099.51 ( 99.02100 ) evaluation of the performance of commercial hiv antibody diagnostic elisa and rt kits ( 200208 )
china has established a well - defined hiv laboratory network along with national and provincial qa systems to attain a high quality of testing .
the laboratory network follows a top - down hierarchical management approach . because of the high testing volume and low number of staff
, there is often insufficient human resources . with rapid technological advancements and guideline modifications ,
technical guidelines from the narl are broad and thus may not be understood by staff at lower levels .
most instruments use english as the operating language , which presents problems for the technical staff at the confirmatory laboratory level . with the expected expansion of rt use , maintaining rt quality will become a challenge . the narl and provincial cdcs will increase rt testing training and standardized record keeping in testing facilities as recently recommended by parekh et al .
more than one brand of molecular testing instrument may be present in the same facility .
these challenges will need to be resolved to guarantee sound development of the chinese hiv / aids testing laboratory network and its qa system .
the developmental history of the chinese hiv / aids testing laboratory network and qa system is reliant on strong financial support from central and local governments .
the role of the narl in laboratory network development , qa and new technology research is important and the effectiveness well documented . technical research and method
evaluations carried out by the narl , such as hiv diagnosis technologies and strategies , hiv-1 vl , eid , incidence assay and dr testing , have offered key scientific basis for the establishment of national technical guidelines and the application of new technologies . provincial laboratories
these laboratories are pivotal in the training and dissemination of new technology , qa and site - directed technical support at the provincial level .
in addition , frequent international cooperation and communications have produced constructive suggestions and ideas for the laboratory network and qa / qc development .
the majority of infected persons are still unaware of their infection status , and testing cd4 or vl in patients who reside in hard - to - reach rural areas prior to and post - art treatment is difficult .
the low - cost point - of - care technologies such as cd4 measurement , with easy instrument maintenance and simple qa procedures , may provide one of the solutions needed in many resource - constrained areas in china in the future .
chinese ministry of health national aids program ( 131 - 08 - 105 - 02 ) ; national science and technology major project of the ministry of science and technology of china ( grant no . | background in 2009 , there were 8273 local screening laboratories , 254 confirmatory laboratories , 35 provincial confirmatory central laboratories and 1 national aids reference laboratory ( narl ) in china .
these laboratories were located in center for disease control and prevention ( cdc ) facilities , hospitals , blood donation clinics , maternal and child health ( mch ) hospitals and border health quarantine health - care facilities.methods the narl and provincial laboratories provide quality assurance through technical , bio - safety and managerial training ; periodic proficiency testing ; on - site supervisory inspections ; and commercial serologic kit evaluations.results from 2002 to 2009 , more than 220 million hiv antibody tests were performed at screening laboratories , and all reactive and indeterminate samples were confirmed at confirmatory laboratories .
the use of highly technically complex tests , including cd4 cell enumeration , viral load , dried blood spot ( dbs)-based early infant diagnosis ( eid ) , drug resistance ( dr ) genotyping , hiv-1 subtyping and incidence assays , have increased in recent years and their performance quality is closely monitored.conclusion china has made significant progress in establishing a well - coordinated hiv laboratory network and qa systems .
however , the coverage and intensity of hiv testing and quality assurance programmes need to be strengthened so as to ensure that more infected persons are diagnosed and that they receive timely prevention and treatment services . |
chronic low back pain ( clbp ) is one of the common health problems in modern society , with
7080% of the population having experienced back pain at least once in their lifetime1 .
people with clbp show more muscle atrophy , especially of the lumbar deep muscles3 , 4 .
movement limited by low back pain causes morphological changes in the lumbar muscles that
induce decreased muscle strength and endurance5 .
decreased muscle strength and endurance negatively affect lumbar
stability and eventually cause functional limitations6 . back
pain in the lumbar area weakens the lumbar flexors and extensors6 . specifically , aside from the erector spinae and other
abdominal muscles , the transverse abdominis of low back pain patients is weaker than that of
healthy people7 .
back pain diminishes
activity levels and results in muscle atrophy , with decreased muscle strength and a
decreased sectional area of the muscles around the spine . as endurance and flexibility
decrease due to these reasons ,
recent studies on and treatments for clbp have been focused on increasing trunk stability
to minimize recurrent episodes and maximize prevention .
the main topics of these studies are
trunk stability and the muscles involved in spine alignment and posture9 .
therefore , the co - activation of the trunk muscles is necessary to achieve trunk stability
and to prevent and treat back pain10,11,12 .
many studies have suggested trunk stability exercises in different positions with or without
an unstable platform device to strengthen the co - activation of trunk muscles13 , 14 .
bridge exercises are the most commonly used among the trunk
stability exercises with different positions .
back pain patients are comfortable in assuming
the bridge position and pain is reduced .
moreover , bridge exercises activate the superficial
and deep trunk muscles at an appropriate ratio and strengthen the gluteus and lower leg
muscles15 .
in previous studies , prone
bridge exercises activated the rectus abdominis , external oblique , and internal oblique more
than supine bridge exercises16 , and prone
bridge exercises on a swiss ball activated the rectus abdominis and external oblique more
than supine bridge exercises17 .
this study aimed to compare the effects of conventional supine bridge exercises and prone
bridge exercises on trunk muscle thickness to determine which type of exercise is more
efficient .
a total of 37 patients ( 13 males and 24 females ) with clbp participated in this study . for
patients with chronic lumbar pain , basic physical therapy ( h / p , ict , and u / s ) was performed
in 40-minute sessions , three times a week , along with a bridge exercise .
all the patients
met the following inclusion criteria : older than 20 years old ; more than 6 months since
diagnosis of clbp by x - ray , ct or mri ; no history of vestibular disease , neurologic disease ,
or spine surgery ; no medicine for balance - related impairments ; and able to do exercises .
their mean age , weight , and height were 41.2 8.3 years , 61.5 7.6 kg , and 163.8 5.9 cm ,
respectively ( table 1table 1.general characteristics of the subjects ( n=37)characteristicstotal(n = 37)exercise 1(n = 11)exercise 2(n = 13)exercise 3(n = 13)age ( yr)41.28.339.47.741.210.242.97.1height ( cm)163.85.9163.55.2163.26.5164.76.2weight ( kg)61.57.660.27.659.46.7 64.87.8gendermale13 ( 35.1)4 ( 36.4)4 ( 30.8)5 ( 38.5)female24 ( 64.9)7 ( 63.6)9 ( 69.2)8 ( 61.5)meanstandard deviation ) .
the subjects understood the experimental purpose and methods and agreed
voluntarily to participate in the study .
all the participants read and signed an informed
consent form approved by the local ethics committee of the catholic university of daegu .
meanstandard deviation all participants were randomly assigned to one of the three exercise groups ( supine bridge
exercise , supine bridge on a swiss ball exercise , and prone bridge exercise ) .
first , the
participants in the supine bridge exercise group ( exercise 1 ) performed the bridge exercise
in the supine position with their knee joints at 90 degrees , both arms spread at about 30
degrees , and both hands on the ground .
they kept their head and neck in a straight position ,
with their eyes looking at the ceiling .
second , the participants in the supine bridge on a
swiss ball exercise group ( exercise 2 ) performed the same exercise as the exercise 1 group ,
but they placed their legs on a swiss ball .
lastly , the prone bridge exercise group
( exercise 3 ) performed the bridge exercise in a prone position with their elbows at 90
degrees , their bodies supported by their forearms and toes , their neck slightly extended ,
and their eyes looking toward the front .
the subjects performed three sets of five reps for
each exercise , which consisted of 30 s work and 30 s rest periods , on three days each week .
the exercise programs were conducted for eight weeks at a designated training facility under
the supervision of a trained physical therapist who ensured that each subject performed the
exercises correctly . to measure trunk muscle thickness ,
ultrasonography ( sonoace 6000 , medison , republic of
korea ) with a frequency of 68.5 mhz and a gain range of 2080 was conducted .
ultrasound
images of the tra , eo , and io were obtained in b - mode with a 7.5 mhz linear transducer . to
minimize the difference between testers , one trained physical therapist participated as an
examiner . during measurement ,
the ultrasound transducer was positioned at the intersection point between a
line from the anterior superior iliac crest to the midaxillary area and a horizontal line
passing through the belly button18,19,20 .
all images were collected at the end of normal exhalation to control for the influence of
respiration on the transverse abdominis21 .
image acquisition was performed three times , and the mean values
were used for statistical analysis .
the shapiro - wilk test was used
to verify the normal distribution of the variables .
a paired t - test was used to determine
the changes in the trunk muscle thicknesses before and after the exercises , and an analysis
of covariance of muscle thickness before the exercise was used as the covariate .
the
bonferroni post hoc test was also performed to identify the differences in the thicknesses
of the trunk muscles among the various exercises .
the three exercise groups showed significant changes in tra , eo , and io thicknesses after
exercise compared with the thicknesses before the exercises ( p<0.05 ) .
the thicknesses of
the tra and io showed significant differences among the groups ( p<0.05 ) . among the
groups , the prone bridge exercise group showed the highest increase in muscles
thicknesses
of the tra , io , and eo , followed by the supine bridge exercise on a swiss ball group and the
supine bridge exercise group ( table 2table 2.comparison of thickness of trunk muscles among groups ( unit : mm)groupvariableexercise 1exercise 2exercise 3meansdmeansdmeansdiopre 4.971.01 5.141.60 5.931.56post 6.010.90 6.281.53 7.451.82change1.030.351.140.491.520.70trapre 2.620.48 2.530.67 3.300.57post 2.930.48 3.070.72 4.200.75change0.300.120.540.180.890.39eopre 3.771.08 3.571.06 4.040.66post 4.711.02 4.571.00 5.220.72change0.940.860.990.391.170.65*p<0.05 , * * p<0.001 .
io : internal oblique , tra : transverse abdominis , eo :
external oblique . significantly different compared with exercise 1 .
significantly different compared with exercise 2 .
significantly different compared with exercise 3 ) .
io : internal oblique , tra : transverse abdominis , eo :
external oblique . significantly different compared with exercise 1 .
significantly different compared with exercise 2 .
a bridge exercise was selected to improve trunk muscle stability among patients with clbp .
to investigate an effective method of bridge exercise
, the existing traditional bridge
exercise was performed along with a prone bridge exercise , and the trunk muscles
thicknesses were subsequently measured .
the purpose of trunk stability exercises is to
protect the spine from recurrent micro - damage of muscles , pain due to spine instability , and
degenerative changes in the spine12 .
muscle strengthening exercises are performed for the prevention and treatment of
musculoskeletal diseases and for the improvement of exercise capacity14 .
generally , patients with low back pain show decreased
muscle strength of the spine extensors and abdominals . specifically , the strength of the tra
is weaker in individuals with low back pain than in those without22 .
the strength of the deep muscles around the lumbar spine in patients with low back pain is
also unbalanced .
patients with low back pain demonstrate decreased proprioception , which
causes position sense impairments and eventually spine instability . as a result ,
for this reason , many studies have been conducted to improve deep
muscle strength . however , to date , minimal research has been conducted on direct measurement
of deep muscles .
ultrasound can be used to show deep muscle thickness even when muscles
contract for certain movements24 , 25 .
this study used ultrasound to measure the effect of eight weeks of bridge exercise on
muscle thicknesses .
the results show that the thicknesses of the tra , io , and eo increased
significantly after prone bridge exercise , followed by supine bridge exercise on a swiss
ball and then supine bridge exercise .
interestingly , the muscle thicknesses of the tra , io ,
and eo after prone bridge exercise increased significantly more than after conventional
bridge exercise . with prone bridge exercise ,
the deep muscles , tra , and io are activated
more to stabilize the trunk and make it thicker .
subjects with low back pain are known to have postural control
impairments ; therefore , a postural control program should be considered part of their
rehabilitation plan27 .
trunk stability
plays an important role in postural control , and the functions of the tra , io , and eo are
crucial for trunk stability28 . in this study ,
prone bridge exercise significantly increased the thicknesses of these
muscles compared with other bridge exercises .
therefore , prone bridge exercise could be
suggested as a method of improving postural control for patients with back pain .
the reasons
for the significant improvement of muscle thicknesses with prone bridge exercise compared
with the other bridge exercises can be suggested biomechanically and physiologically .
biomechanically , the prone position has a smaller base of support compared with the other
bridge exercises .
moreover , the prone bridge
exercise raises the individual to resist gravity during the process of the spinal flexion
pattern .
that is , as the elbows and toes touch the ground , the trunk muscles are required to
counteract gravity through co - contraction .
physiologically , the prone bridge position , with
the upper and lower extremities acting as a bridge , activates the muscles placed
transversely first ( e.g. , the tra and then the io and eo ) for trunk stability .
the subjects in this study reported difficulty in lifting their pelvis during supine bridge
exercises when pain worsened .
however , they reported easiness in assuming the prone bridge
posture compared with the supine bridge postures .
accordingly , patients with acute back
pain , not only those with clbp , can also perform the prone bridge exercise safely . in
conclusion ,
prone bridge exercise is the more efficient for improving trunk stability and
increasing trunk muscle thicknesses compared with the supine bridge exercises in patients
with clbp .
a limitation of this study is that we only measured the thicknesses of the io ,
eo , and tra muscles .
as the study measured only muscle thickness , the study results are hard
to generalize .
moreover , bridge exercises mobilize many muscles in the trunk and
extremities , and other factors can affect muscle thickness . | [ purpose ] this study aimed to investigate the effects of prone bridge exercise on trunk
muscle thickness .
[ subjects and methods ] thirty - seven chronic low back pain patients
participated in this study .
each participant was randomly assigned to one of three
exercise groups , namely , a prone bridge exercise group , supine bridge exercise on a swiss
ball group , and supine bridge exercise group .
the thicknesses of the transverse abdominis
( tra ) , internal oblique ( io ) , and external oblique ( eo ) were measured using ultrasound .
[ results ] after eight weeks of training , the three groups showed significant increases in
the thicknesses of the tra , io , and eo . among
the groups , tra and io showed significantly
different muscle thicknesses .
[ conclusion ] the prone bridge exercise significantly
affected the thicknesses of the tra , io , and eo unlike the supine bridge exercises . based
on the results of this study ,
the prone bridge exercise is a more effective method to
improve trunk stability than conventional supine bridge exercises . |
the eye mutant resource ( emr ) and the translational vision research models ( tvrms ) programs currently housed at the jackson laboratory are tailored to provide genetically defined models of vision - associated diseases to the research community .
the emr screens for spontaneous mutations in the large production and repository colonies , while the tvrm program screens for chemically induced mutations in third - generation ( g3 ) offspring of mutagenized mice .
both programs are motivated by the need for well - characterized models for studying the function of particular molecules in the eye , for examining disease pathology , and for providing a resource to test therapeutic regimens . in the early phases of the emr program ,
the tools for examining mice for ocular abnormalities were adapted for the small size of the mouse eye [ 1 , 2 ] .
these tools included indirect ophthalmoscopy , slit lamp biomicroscopy , fundus photography , and electroretinography ( erg ) .
initially , mice from various stocks and inbred strains were screened to identify spontaneous ocular mutants using the first two methodologies .
currently , erg screening is done as well to identify and characterize new retinal mutants . as secondary screens ,
fluorescein angiography is used to detect vascular changes , and noninvasive tonometry is used to assess changes in intraocular pressure .
screening has also been expanded to include genetically engineered strains from the jackson laboratory 's genetic resource sciences ( grs ) repository that are systematically examined as they are removed from the shelf or are retired from breeding .
also , in addition to the initial phenotypic characterization , the emr strives to identify the mutations underlying the disorders .
systematic chemical mutagenesis screens have been successfully carried out in several model organisms , including drosophila , c. elegans , and zebrafish [ 6 , 7 ] .
the zebrafish screens have provided valuable eye models , especially those pertaining to eye development .
in addition to our efforts , other mutagenesis screens for eye phenotypes in mice have been reported in which a number of mutants have been described [ 911 ] .
although different methods for mutagenizing mice are available , the alkylating agent , n - ethyl - n - nitrosourea ( enu ) , is the mutagen most commonly used .
enu mainly induces point mutations resulting in a range of consequences including total or partial loss - of - function , dominant - negative , or gain - of - function alleles [ 1316 ] .
its effectiveness as a mutagen is dependent on dosage , frequency of administration , and mouse strain .
effectiveness , in terms of identifying mutants , depends upon the type of screen ( e.g. , dominant versus recessive ) and the reproducibility of the phenotypic assay utilized .
mutant recovery has ranged from a rate of 1/175 , to ~1/666 in specific locus tests ( slts ) , and to an average of 1/1470 based on recessive screening in a defined chromosomal region .
the mutation rates for individual loci can vary by almost tenfold [ 12 , 17 , 18 ] . the majority of large - scale mutagenesis screens have been dominant screens .
this is probably due to the relative ease of creating mutagenized mice for dominant screens compared to recessive ones .
screening for dominants on a genome - wide basis can be done in one generation ( g1 ) , while recessives generally require three .
the neuherburg cataract mutant collection of ~170 dominant mutants was assembled through screening over 500,000 first - generation mice exposed to various mutagens .
the gsf - munich and mrc - harwell [ 13 , 20 ] programs were established using a phenotype - based approach to screen thousands of mice for dominant mutations affecting a variety of biological processes . a major drawback to dominant screens , however , is that not all mutations have dominant effects .
estimates suggest that the frequency of diseases caused by recessive mutations is 410-fold higher than for dominant ones .
in fact , of 218 eye mutants surveyed in the mouse genome informatics database , 80% were recessive mutations and only 20% were dominant or semidominant .
therefore , the tvrm program screened a g3 population of mutagenized mice for recessive mutations .
screening for spontaneous and chemically induced mutants provides an important source of models to study the effects of single - gene mutations found in human patients .
additionally , new mutations within the same gene provide allelic series in which splice variants or domain - specific effects can be queried .
finally , mutations in novel genes that lead to retinal disorders can be discovered using a forward genetic approach .
the ages at which the visual system is affected by disease can vary considerably . for the emr program , an initial screen of jax mice & services ( jmss ) production colonies and mice removed from the grs repository
is routinely performed at ~2 months of age and if necessary , additional screening is done at an older age , usually at 6 months of age . also ,
as with other neuronal diseases , diseases of the visual system are not reversible , so ocular diseases can be captured in retired breeders .
therefore , when available , retired breeders that are older than 1 year of age are screened .
c57bl/6j ( b6 ) g3 enu mutagenized mice were screened by the tvrm program . for the chemically induced mutations , enu was administered to male b6 mice in three weekly injections of 80 mg / kg .
g3 offspring were generated using a three - generation backcross mating scheme ( figure 1 ) .
g3 mice were screened at 24 weeks of age in order to enhance our ability to identify late onset diseases . to determine
if the disease phenotype was inheritable , mutant mice were outcrossed to wild - type ( wt ) mice to generate f1 progeny with subsequent intercrossing of the resultant f1 mice to generate f2 progeny .
if f1 mice were affected , the pedigree was designated as segregating a dominant mutation . if f1 mice were not affected but ~25% of f2 mice were affected , the pedigree was designated as segregating a recessive mutation .
once the observed ocular phenotype was determined to be genetically heritable , mutants were bred and maintained in the research animal facility at jax .
mice were provided with nih 6% fat chow diet and acidified water , with 12:12 hour dark : light cycle in pressurized individual ventilation caging which are monitored regularly to maintain a pathogen - free environment .
mice , dark adapted for a minimum of 1 hour , were treated with atropine prior to examination by indirect ophthalmoscopy with a 60 or 78 diopter aspheric lens .
fundus photographs were taken with a kowa small animal fundus camera using a volk superfield lens held 2 inches from the eye as previously described . for electroretinographic evaluation of mutants , following a 2-hour dark adaptation ,
mice were anesthetized with an intraperitoneal injection of xylazine ( 80 mg / kg ) and ketamine ( 16 mg / kg ) in normal saline .
briefly , dark - adapted , rod - mediated ergs were recorded with the responses to short - wavelength flashes over 4.0-log unit to the maximum intensity by the photopic stimulator .
cone - mediated ergs were recorded with white flashes after 10 min of complete light adaptation .
genomic dna was isolated from tail tips using a pbnd ( pcr buffer with nonionic detergents ) preparation , which was adapted from a protocol from perkin elmer cetus .
samples were heated to 95c for 10 minutes , and 1 l of the dna preparation was used in a 12 l pcr reaction .
amplicons were visualized with ethidium bromide after electrophoretic separation on a 4% agarose gel . for mapping purposes , phenotypically affected mice , presumed to be homozygous for the mutations , were mated with dba/2j mice .
the resulting f1 offspring were intercrossed to generate f2 offspring if recessive and backcrossed ( bc ) to wt parental if dominant . resulting progeny were phenotyped by indirect ophthalmoscopy .
dna isolated from tail tips from a minimum of 10 affected and 10 unaffected mice was pooled and subjected to a genome - wide scan using 4880 simple sequence length polymorphic markers distributed throughout the genome .
total rna was isolated from whole eyes and brains of affected mutants and b6 mice using trizol reagent ( life technologies ) per manufacturer 's protocol .
total rna was treated with rnase - free dnasei ( ambion ) and quantity was determined using a nanodrop spectrophotometer ( thermo scientific ) .
primers to sequence the coding region of the candidate genes were designed from exon sequences obtained from the ensembl database .
rt - pcr was done using eye cdna in a 24 l pcr reaction containing 1xpcr buffer ( 10 mm tris - hcl ph 8.3 , 50 mm kcl ) , 250 m of each datp , dctp , dgtp , dttp , 0.2 m of each forward and reverse primer , 1.5 mm mgcl2 , and 0.6 u taq polymerase .
the following pcr program was used : 94c for 1 minute 30 sec followed by 35 cycles of 94c for 30 sec , 55c for 45 sec , and 72c for 45 sec , and a final extension of 72c for 2 minutes .
pcr products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining .
dna fragments were sequenced on an applied biosystems 3730xl ( using a 50 cm array and pop7 polymer ) .
mice were asphyxiated by carbon dioxide inhalation , and enucleated eyes were fixed overnight in cold methanol / acetic acid solution ( 3 : 1 , v / v ) .
the paraffin - embedded eyes were cut into 6 m sections , stained by hematoxylin and eosin ( h and e ) , and examined by light microscopy .
since its inception in the 1980s , the emr program has identified and/or imported more than 100 mouse models with ocular abnormalities for research .
table 1 lists some of the retinal degeneration mouse models of human disease developed and/or currently maintained in the emr that are available to the research community .
the tvrm program was built upon the success of the neuromutagenesis facility ( nmf ) at the jackson laboratory , and 15 of the 60 mutant lines ( tables 2 and 3 ) in which a disease phenotype has been subsequently fixed as a coisogenic inbred strain by the tvrm program were first identified in screens conducted by the nmf .
the remaining 45 tvrm lines were established by screening ~14,000 g3 mice for anterior and posterior segment abnormalities by indirect ophthalmoscopy and/or slit lamp biomicroscopy .
six of the 60 mutations ( 10% ) are inherited in a dominant or codominant manner , and the remaining are recessive mutations .
forty six of the mutants have retinal phenotypes ranging between pan - retinal spots or patches , pigmentation defects , and/or attenuation of blood vessels with or without morphological changes that were detectable by light microscopy .
six of the mutant lines have reduced or absent erg responses for either rod and/or cone cells without photoreceptor loss .
forty six of the mutations ( 23 reported in table 3 ) have been localized to a chromosome , and the molecular basis has been identified for 23 of them ( table 2 ) .
fourteen lines are still in the process of being mapped ( data not shown ) .
nineteen of the 23 mutations in table 2 were novel alleles in genes in which mutations had previously been reported .
it should be noted that the current bias for reoccurrences of mutations , herein referred to as remutations , versus identification of novel genes in table 2 is probably due to the fact that once a mutation is mapped , candidate genes previously associated with an eye disease can be quickly sequenced .
regions containing no obvious candidate genes need to be narrowed further and/or all genes within the region may need to be sequenced to identify the disease - causing mutation .
interestingly , new phenotypes were observed in 8 of the remutations that have been examined ( see ; [ 5155 ] , personal communication pmn ) .
for example , outer segments ( oss ) were either formed abnormally or did not initiate in retinas from homozygous rpgrip1 mice .
this was in contrast to the rpgrip1 targeted null mutant , hereafter , rpgrip1 in which os discs were formed and stacked vertically rather than horizontally .
targeted alleles of lama1 were reported to be embryonic lethal [ 57 , 58 ] .
the enu - induced allele , lama1 , provides a viable , hypomorphic allele in which abnormalities in the adult animal could be examined .
histologically , persistent hyaloid vessels and fibrous tissue were found in the vitreal space , and the inner limiting membrane was disrupted . in an allelic series of mutations within the rhodopsin gene ,
heterozygous rho and rho mice raised in standard vivarium lighting did not exhibit any morphological changes until exposed to bright light .
previously rho alleles showed spontaneous and pan - retinal degeneration , even when mice were reared from birth in darkness .
tvrm65 segregates as a recessive mutation that is characterized by a pan - retinal , grainy fundus appearance that eventually progresses with age to patches of depigmentation within the central retina ( data not shown ) .
the mutation was mapped to chromosome ( chr . ) 7 between flanking markers d7mit75 and d7mit190 . a single nucleotide polymorphic ( snp ) marker ( snp i d : rs13479126 ) served to narrow the interval .
crx , a reasonable biological candidate gene , contained within the minimal interval , was examined for a mutation .
mice and humans share a 97% sequence similarity . to date , two crx transcripts have been reported .
the long isoform ( genbank nm_001113330 ) has 25 additional amino acids ( aa ) in its n terminus when compared to the shorter isoform ( genbank nm_007770 ) .
a t > a nonsense mutation identified in crx is located in the last exon and is expected to affect both isoforms .
the tvrm65 mutation is predicted to cause an early termination at leu277 ( ttg ) of the 323 aa from the longer isoform or at leu253 of a 299 aa product from the shorter isoform ( figure 2(a ) ) .
phenotypically , crx mutants resemble the null mouse model in which the single homeodomain containing region of crx was targeted .
crx mutants show a rapid photoreceptor degeneration ( figure 2(b ) ) . at postnatal day
( p ) 14 and p21 , oss were absent and inner segments ( iss ) were rarely observed ( figure 2(b ) ) . by p21 , photoreceptor cell bodies were reduced to ~60% of controls .
the outer plexiform layer ( opl ) was also thinner , approximately 40% of controls . by 3 months of age ,
the oss and iss were absent and only 2~3 layers of outer nuclear layer ( onl ) were remained .
the photoreceptor degeneration observed in the crx mutants occurs more rapid than reported for the null allele .
this may , in part , be due to the difference in genetic background of the two alleles as crx was generated on a b6 background , whereas the previous null allele was described on a segregating b6 and 129sb genetic background .
tvrm64 segregates as a recessive mutation that is characterized by a grainy fundus appearance and attenuated retinal vessels ( data not shown ) .
the mutation mapped to chr.1 between the centromere and d1mit427 , an interval in which rp1 resides .
rp1 encodes a large protein of 2095 aa in mouse and 2156 aa in humans .
rp1 localizes in the connecting cilia and appears to play a structural and/or functional role in molecular transport through the connecting cilia [ 61 , 62 ] .
structurally , it has two ubiquitin homolog ( ubq ) domains in its amino terminus .
rp1 was tested for a mutation , as the phenotype of homozygous tvrm64 mutants was similar to that of mice carrying either of two targeted rp1 alleles , involving homologous recombination in which exons 2 and 3 were targeted ( rp1 ) or a truncation after codon 662 , rp1 , analogous to the r667ter mutation in humans .
direct sequencing of homozygous rp1 retinal cdna revealed an a > t transversion at nucleotide 1769 ( genbank nm_011283 ) , creating a nonsense mutation in which arg522 ( aga ) is changed to a termination codon ( tga ; figure 3(a ) ) .
the os length of rp1 mutant retina was approximately 50% shorter than wt controls at 1 month of age ( figure 3(b ) ) .
the difference in is length between mutant and controls , however , was barely discernable at 1 month of age but was obviously shorter in rp1 mutants at 3 months of age .
the photoreceptor degeneration was progressive with little difference in cell body number in the onl at 1 month of age but by 3 months , cell nuclei were reduced to ~50% in mutants in comparison to controls . in contrast , the photoreceptor morphology of rp1 mice appeared normal by light microscopy at p30 with comparable length of os in mutant and controls .
also , rp1 mice at p30 showed shorter os lengths and a 12-layer reduction in onl .
therefore , the disease progression in rp1 at similar age appears to be more severe than observed in rp1 mice but less severe than rp1 mice .
this difference between the models was also discernable functionally . at 1 month of age , dark - adapted ergs of rp1 mice were comparable to wt ( figures 3(c ) and 3(d ) ) .
the recessive tvrm148 mutation is characterized by late onset retinal spotting and by patches of depigmentation that is readily discernable by indirect ophthalmoscopy at 5 months of age ( data not shown ) .
rpe65 was screened by direct sequencing for a mutation as it fell within the minimal interval identified , and the disease phenotype was similar to that reported for the rpe65 targeted knockout animal ( herein referred to as rpe65 ) and rpe65 alleles .
a t > c point mutation was found by direct sequencing of retinal cdna from rpe65 mice and is expected to generate a mutant protein with an f229s point mutation ( figure 4(a ) ) .
f229 is evolutionarily conserved from humans to zebra fish but interestingly not in chimpanzee ( figure 4(a ) ) .
the rpe65 mutant had a nonfunctional rod erg response due to the lack of 11-cis - retinal production in the rpe and showed disorganized rod outer segments .
another targeted allele mimicking a human r91w mutation was found in leber congenital amaurosis ( lca2 ) patients ( rpe65 ) , and a spontaneous model rpe65 showed a similar disease progression to that observed in rpe65 mutants .
photoreceptors degenerated progressively in homozygous rpe65 mouse from 1 month to 1 year of age , the latest time point examined ( figure 4(b ) ) . at 1 month of age , os and is lengths were approximately 50% shorter than controls with no obvious thinning of the onl .
the photoreceptor nuclei were reduced in thickness by ~20% at 4 months and ~60% by 1 year of age . like the three previously reported mouse models , rpe65 exhibited severely impaired rod ergs and relatively spared cone ergs .
cone b - wave ergs were comparable to controls at 4 weeks of age but by 17 weeks , the amplitudes were reduced compared to controls ( figures 4(c)4(d ) ) .
these mutations offer some advantages for the study of human genetic diseases and basic gene function over mutations obtained by homologous recombination .
first , these mutations are generally identified because they cause a clinically relevant phenotype . by starting with a known phenotype , information about the physiological function of the mutant gene and its biomedical relevance
second , the forward genetic approach has the potential for discovery of new genes involved in ocular development and function that were previously unappreciated .
further , spontaneous and chemically induced mutations may better model naturally occurring human genetic conditions .
they produce a full and unbiased array of mutation types single base pair changes or deletions , and in the case of spontaneous mutations , retroviral insertions , repeat sequence expansions , and chromosomal rearrangements .
they can abolish all protein function ( null ) , partially diminish function ( hypomorphic ) , or change function ( dominant negative or gain - of - function ) .
can provide domain specific information about protein function and information on alternatively spliced variants .
biomedically relevant phenotypes associated with some human genetic disorders may be revealed by the different alleles that are not replicated by knockout alleles .
for example , whereas the null alleles of lama1 [ 57 , 58 ] were embryonic lethal , the hypomorphic enu nmf223 allele allowed for the examination of ocular phenotypes in adult mice . in another example , the rd10 allele of pde6b identified by the emr program has a later onset and slower rate of degeneration than the original rd1 allele , thus allowing for the opportunity to test therapeutic strategies .
finally , two phosphodiesterase 6a mutations first described by the tvrm program cause missense mutations that lead to different biochemical outcomes and rates of photoreceptor degeneration , suggesting a difference in the importance of the particular mutant residues to the function of the protein .
it should also be noted that spontaneous mutations occur on a wide variety of strain backgrounds , and chemical mutagenesis can be carried out in different genetic backgrounds .
the observation of altered mutant phenotypes in different genetic backgrounds can provide a means for identifying interacting genes and molecular pathways of pathophysiology .
for example , nr2e3 was observable clinically only in the b6 genetic background , and a number of genetic backgrounds act to ameliorate the disease .
crb1 is observable clinically in the c3h / hej background but not in the b6 background , and the null mutation is phenotypically different on a segregating 129x1/svj and b6 background .
finally , a wide variety of disease phenotypes are observed in rd3 and gnb1 in different strain backgrounds , indicating interactions with genetic background modifiers .
the variation in genetic background enables discovery of modifiers and gene interactions and could be essential to the discovery of important mutant phenotypes and potential targets for therapeutic intervention . in the future , the emr will continue to screen for spontaneous mutations in the large production colonies at the jackson laboratory . the mutants identified in the tvrm program
will be incorporated into the emr distribution colonies as the molecular bases of the mutations are identified .
finally , sensitized chemical mutagenesis screens are planned that will uncover pathways important in retinal development , maintenance , and function . | the need for mouse models , with their well - developed genetics and similarity to human physiology and anatomy , is clear and their central role in furthering our understanding of human disease is readily apparent in the literature .
mice carrying mutations that alter developmental pathways or cellular function provide model systems for analyzing defects in comparable human disorders and for testing therapeutic strategies .
mutant mice also provide reproducible , experimental systems for elucidating pathways of normal development and function .
two programs , the eye mutant resource and the translational vision research models , focused on providing such models to the vision research community are described herein . over 100 mutant lines from the eye mutant resource and 60 mutant lines from the translational vision research models have been developed .
the ocular diseases of the mutant lines include a wide range of phenotypes , including cataracts , retinal dysplasia and degeneration , and abnormal blood vessel formation .
the mutations in disease genes have been mapped and in some cases identified by direct sequencing . here ,
we report 3 novel alleles of crxtvrm65 , rp1tvrm64 , and rpe65tvrm148 as successful examples of the tvrm program , that closely resemble previously reported knockout models . |
each row of the image represents a gene and each column a tissue ( labeled at the bottom ) , with the color indicating the ratio of expression levels .
clusters of genes coordinately regulated in certain tissues correlate with the biological functions indicated on the right . | it is widely supposed that the tissue specificity of gene expression indicates gene function .
now , an extensive analysis of gene expression in the mouse reveals that quantitative measurement of expression levels in different tissues can contribute powerfully to the prediction of gene function . |
after an acute myocardial infarction , women continue to experience higher mortality than men , despite improvements in reperfusion therapy.1 , 2 this worse outcome is attributed , at least in part , to older age at presentation , though a recent metaanalysis of observational studies has reported that women have a higher risk of inhospital mortality even after adjustment for baseline differences.3
over the last 15 years , there has been a progressive improvement in reperfusion therapy for stelevation myocardial infarction ( stemi ) , with a shift from fibrinolytic therapy to primary percutaneous coronary intervention ( ppci ) and an overall increase in reperfusion rates due to the organization of stemi networks .
these improvements in stemi treatment had the potential to reduce the gap in stemi outcomes among women , given that in the lytic era they consistently received less treatment and were more exposed to adverse bleeding events following fibrinolysis . on the other hand , use of ppci virtually eliminates the risk of intracranial bleeding and has been shown to be an independent predictor of survival in women.4
in the present study , we describe the evolution of reperfusion therapy for stemi from 2001 to 2014 in italy , taking into account sex differences in inhospital treatment and outcome across age groups among patients admitted to cardiac care units ( ccus ) with a diagnosis of stemi and enrolled in nationwide registries .
five consecutive prospective registries designed by the italian association of hospital cardiologists ( anmco ) in patients with acute coronary syndromes ( acs ) were conducted in italy between 2001 and 2014 : blitz in 2001,5 inacs outcome ( italian network on acute coronary syndromes outcome ) in 20062007,6 blitz 4 in 2009 and 2010,7 mantra ( management of patients with acs in the real world practice in italy : an outcome research study focused on the use of antithrombotic agents ) in 20092010,8 and eyeshot ( employed antithrombotic therapies in patients with acute coronary syndromes hospitalized in italian cardiac care units ) in 2013 and 2014.9 all surveys were nationwide and included patients with acs consecutively admitted alive to the participating ccus during a prespecified period ( a few weeks for the blitz and eyeshot registries and 1 year for inacs outcome and mantra ) ( table 1 ) .
the methods used for each registry have been described previously.5 , 6 , 7 , 8 , 9 briefly , their primary objectives were to evaluate the clinical characteristics , management , and outcomes of consecutive patients with acs admitted to italian ccus , using a catchment method broad enough to provide data representative of the entire country .
participation in the various registries had been offered to all institutions in blitz and eyeshot , to ccus able to enroll at least 40 acs patients over 10 weeks in blitz 4 , and to a representative sample of ccus ( balanced by geographical region and hospital complexity ) in inacs outcome and mantra .
physicians were instructed that participation in the registries should not affect clinical care or management .
informed consent was obtained from all patients who were aware of the nature and aims of the registries .
local institutional review boards were informed of the study according to the italian rules and approved the protocol .
characteristics of the 5 italian surveys , incidence of stemi , and rate of women among stemi patients excluding 151 patients enrolled in inacs outcome because they have not been admitted in ccus and 264 patients from the blitz4 since they underwent a coronary angiography in another hospital .
acs indicates acute coronary syndrome ; ccus , cardiac care units ; stemi , stelevation myocardial infarction . data on baseline characteristics , including demographics , risk factors , and medical history , were collected as previously described.5 , 6 , 7 , 8 , 9 information on the use of cardiac procedures , including coronary angiography , type of revascularization therapy ( if any ) , use of medications during hospitalization and at hospital discharge , and inhospital major clinical events , were recorded . in the blitz 4 registry ,
door to balloon time was available only for patients presenting to hospitals with onsite ppci facilities .
for the present analysis , only patients with a final diagnosis of stemi on admission were considered .
stemi was defined as stsegment elevation > 1 mm ( 0.1 mv ) in 2 or more contiguous precordial leads or in 2 or more adjacent limb leads , or new or presumed new left bundle branch block associated with symptoms suggestive of acs .
reinfarction during initial hospitalization was diagnosed in the presence of new ischemic symptoms and a reelevation of biochemical myocardial necrosis markers with or without concurrent ecg changes .
major bleeding was classified according to the thrombolysis in myocardial infarction ( timi ) criteria.10 stroke was identified as an acute neurologic deficit lasting > 24 hours and affecting the ability to perform daily activities with or without confirmation by imaging techniques .
cardiogenic shock was defined as systolic blood pressure ( bp ) of 90 mm hg lasting > 60 minutes , nonresponsive to fluid challenge or requiring the administration of inotropic drugs in order to obtain a systolic bp of > 90 mm hg , with clinical signs of peripheral hypoperfusion such as sensorial obnubilation , low urine output ( < 30 ml / h ) , cold sweat , or cyanosis.11
all data were collected at hospital discharge using a case report form at the participating centers and entered in a centralized database located at the anmco research center in florence , italy . by using a validation plan integrated in the data entry software , data were checked with data queries for missing or contradictory entries and values out of the normal range .
if the queries were not answered by the investigators , the data were considered as missing .
categorical variables were reported as numbers and percentages and compared by test , whereas continuous variables were reported as means and sds and compared by t test or anova , if normally distributed , or by mann
all the analyses were performed considering differences in sex and 4 classes of age : < 55 , 55 to 64 , 65 to 74 , and 75 years .
temporal trends were tested using the cochran armitage test for binary variables and the kendall tau rank correlation coefficient with the jonckheere
. a multivariable analysis ( logistic model ) was performed to estimate the risk of inhospital mortality over time adjusted for study cohort , reperfusion therapy , type of hospital , geographic area , and for risk factors and baseline characteristics significantly associated with inhospital mortality at a previous univariate analysis .
these variables were continuous ( age , baseline systolic blood pressure , and heart rate ) , dichotomous ( female sex , smoking , diabetes mellitus , hypertension on treatment , prior angina , prior myocardial infarction , prior heart failure , peripheral artery disease , prior stroke / transient ischemic attack , prior renal dysfunction , killip class iv at entry , atrial fibrillation , anterior myocardial infarction , hospital with cathlab ) and categorical . in the latter case ,
dummy variables were introduced to define a reference group : reperfusion therapy ( no reperfusion [ reference group ] ; ppci ; lysis ) , geographic area ( north [ reference group ] , center , south ) , and the study cohort ( 2001 as reference group ) .
furthermore , a logistic model on inhospital mortality was performed with only age , sex , and their interaction term , considering age in the prespecified classes instead of in continuous .
finally , a logistic model on inhospital mortality was conducted adjusting for sex , study , and their interaction term .
all analyses were conducted with sas system software version 9.2 ( sas institute inc , cary , nc ) .
data on baseline characteristics , including demographics , risk factors , and medical history , were collected as previously described.5 , 6 , 7 , 8 , 9 information on the use of cardiac procedures , including coronary angiography , type of revascularization therapy ( if any ) , use of medications during hospitalization and at hospital discharge , and inhospital major clinical events , were recorded . in the blitz 4 registry ,
door to balloon time was available only for patients presenting to hospitals with onsite ppci facilities .
for the present analysis , only patients with a final diagnosis of stemi on admission were considered .
stemi was defined as stsegment elevation > 1 mm ( 0.1 mv ) in 2 or more contiguous precordial leads or in 2 or more adjacent limb leads , or new or presumed new left bundle branch block associated with symptoms suggestive of acs .
reinfarction during initial hospitalization was diagnosed in the presence of new ischemic symptoms and a reelevation of biochemical myocardial necrosis markers with or without concurrent ecg changes .
major bleeding was classified according to the thrombolysis in myocardial infarction ( timi ) criteria.10 stroke was identified as an acute neurologic deficit lasting > 24 hours and affecting the ability to perform daily activities with or without confirmation by imaging techniques .
cardiogenic shock was defined as systolic blood pressure ( bp ) of 90 mm hg lasting > 60 minutes , nonresponsive to fluid challenge or requiring the administration of inotropic drugs in order to obtain a systolic bp of > 90 mm hg , with clinical signs of peripheral hypoperfusion such as sensorial obnubilation , low urine output ( < 30 ml / h ) , cold sweat , or cyanosis.11
all data were collected at hospital discharge using a case report form at the participating centers and entered in a centralized database located at the anmco research center in florence , italy . by using a validation plan integrated in the data entry software
, data were checked with data queries for missing or contradictory entries and values out of the normal range .
if the queries were not answered by the investigators , the data were considered as missing .
categorical variables were reported as numbers and percentages and compared by test , whereas continuous variables were reported as means and sds and compared by t test or anova , if normally distributed , or by mann
all the analyses were performed considering differences in sex and 4 classes of age : < 55 , 55 to 64 , 65 to 74 , and 75 years .
temporal trends were tested using the cochran armitage test for binary variables and the kendall tau rank correlation coefficient with the jonckheere
. a multivariable analysis ( logistic model ) was performed to estimate the risk of inhospital mortality over time adjusted for study cohort , reperfusion therapy , type of hospital , geographic area , and for risk factors and baseline characteristics significantly associated with inhospital mortality at a previous univariate analysis .
these variables were continuous ( age , baseline systolic blood pressure , and heart rate ) , dichotomous ( female sex , smoking , diabetes mellitus , hypertension on treatment , prior angina , prior myocardial infarction , prior heart failure , peripheral artery disease , prior stroke / transient ischemic attack , prior renal dysfunction , killip class iv at entry , atrial fibrillation , anterior myocardial infarction , hospital with cathlab ) and categorical . in the latter case , dummy variables were introduced to define a reference group : reperfusion therapy ( no reperfusion [ reference group ] ; ppci ; lysis ) , geographic area ( north [ reference group ] , center , south ) , and the study cohort ( 2001 as reference group ) .
furthermore , a logistic model on inhospital mortality was performed with only age , sex , and their interaction term , considering age in the prespecified classes instead of in continuous .
finally , a logistic model on inhospital mortality was conducted adjusting for sex , study , and their interaction term .
all analyses were conducted with sas system software version 9.2 ( sas institute inc , cary , nc ) .
of 28 274 patients with acs enrolled in the 5 registries , 13 235 ( 46.8% ) were classified as having stemi ( 72.1% men and 27.9% women ) .
the overall rates of women with stemi did not change significantly over time ( from 30.1% in 2001 to 28.1% in 2014 ; p for trend=0.06 ) ( table 1 ) .
the prevalence of women increased significantly with age , being 13.0% in the age class < 55 years , 17.5% in the age class 55 to 64 years , 28.0% in the age class 65 to 74 years , and 48.3% in the age class 75 years ( p for trend < 0.0001 ) , resulting in a higher mean age of women compared with men ( 7213 versus 6312 years , p<0.0001 ) .
sex and age differences in clinical characteristics of patients with stemi enrolled during the study period are listed in table 2 . when compared to men in the majority of corresponding age categories , women were less likely to be active smokers , had more hypertension and lower rates of prior myocardial infarction or revascularization procedures .
killip class iv on admission increased significantly with age in both sexes and was slightly more frequent among women in all age classes , even though a significant difference was observed among patients aged < 55 years .
the use of antithrombotic therapies during hospital admission was significantly lower with the increase in age classes for both sexes .
when compared to men , women aged 65 years received significantly less unfractionated heparin , glycoprotein iib / iiia inhibitors , and dual antiplatelet therapy during hospitalization ( table 2 ) .
baseline characteristics of patients with stemi from 2001 to 2014 , according to sex and age classes twosided p values for comparisons across sex for each age category are < 0.05 .
asa indicates acetylsalicylic acid ; bmi , body mass index ; cabg , coronary artery bypass graft ; ccu , cardiac care unit ; dapt , dual antiplatelet therapy ; ecg , electrocardiogram ; gpi , glycoprotein iib / iiia inhibitors ; hr , heart rate ; lmwh , lowmolecularweight heparins ; lvef , left ventricular ejection fraction ; mi , myocardial infarction ; pad , peripheral artery disease ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; stemi , stelevation myocardial infarction ; tia , transient ischemic attack ; ufh , unfractionated heparin .
shown in figure 1 , the use of fibrinolytic therapy decreased from 2001 to 2014 in both sexes and at all ages ( all p for trend < 0.0001 ) , and this trend was mirrored by a dramatic increase in ppci , also affecting both sexes at all ages ( all p<0.0001 ) .
the overall rates of no reperfusion therapy decreased significantly ( p for trend < 0.0001 ) , and the changes were particularly marked among women aged 75 years ( from 64.1% in 2001 to 27.1% in 2014 ) .
the no reperfusion rates remained significantly higher in older women until 2010 , but tended to disappear in 2014 . among patients receiving ppci ,
no significant differences were observed between men and women with regard to door to balloon times ( 2.24.7 versus 1.71.6 hours in 2001 , 2.24.2 versus 2.02.4 hours in 2006 , 0.85.8 versus 0.94.3 hours in 2009 , 2.77.8 versus 2.64.8 hours in 2010 , and 1.14.8 versus 0.95.0 hours in 2014 ) .
data on catheterization access site were available only for the blitz 4 and eyeshot study cohorts , where the radial approach was used more frequently in men than in women ( 31.4% versus 26.4% , p=0.002 , and 72.7% versus 61.7% , p=0.0007 , respectively ) .
inhospital rates of primary pci ( a , b ) , thrombolysis ( c , d ) , and no reperfusion ( e , f ) over time , among men and women , according to age classes .
crude inhospital mortality rates increased significantly with age in both sexes , being significantly higher in women at all ages ( except in patients < 55 years of age , who presented an extremely low inhospital fatality rate ) .
the rates of reinfarction increased significantly with age in men , but not in women , whereas the rates of stroke and cardiogenic shock increased significantly with age in both sexes .
the rates of timi major bleeding were significantly higher in women at all ages compared to men and increased significantly with age in men , but not in women .
inhospital major clinical events of stemi patients according to sex and age classes twosided p values for comparisons across sex are < 0.05 .
number in parentheses are percenteges calculated on males and females within each class of age .
mi indicates myocardial infarction ; stemi , stelevation myocardial infarction . excluding 151 patients enrolled in inacs outcome because they have not been admitted in ccus and 264 patients from the blitz4 since they underwent a coronary angiography in another hospital .
time trends for mortality according to age are shown in figure 2 , with an overall decline in mortality over time affecting both sexes ( p for trend < 0.01 ) and patients aged 75 years ( p for trend < 0.0001 ) .
mortality and reinfarction rates declined significantly over time in both sexes , whereas stroke rates remained fairly stable , being slightly higher among women at all time points ( table 4 ) .
overall cardiogenic shock rates were significantly higher in women ( 8.6% versus 4.8% , p<0.0001 ) , even though statistically significant only in patients aged 65 to 74 years , and declined significantly over time in women and not in men . furthermore , cardiogenic shock fatality rates were higher in women at all time points , except for year 2001 ( figure 3 ) , but were statistically significant only in year 2009 .
major bleeding rates were significantly higher among women , and there was no significant trend over time in both sexes .
inhospital mortality rates over time among men ( a ) and women ( b ) , according to age classes .
inhospital major clinical events in men ( n=9544 ) and women ( n=3691 ) according to year of the survey twosided p values for comparisons across sex are < 0.05 .
mi indicates myocardial infarction . excluding 151 patients enrolled in inacs outcome because they have not been admitted in ccus and 264 patients from the blitz4 since they underwent a coronary angiography in another hospital .
inhospital mortality rates among men ( gray bars ) and women ( red bars ) with cardiogenic shock ( at entry / during hospital stay ) over the observation time .
* twosided p values for comparisons across sex are < 0.05 . on multivariable analysis ,
several variables were found to be significantly related to inhospital mortality ( figure 4 ) , but without a statistically significant interaction between sex and age ( p for interaction=0.61 ) .
many variables remained predictors of mortality even when the model was applied separately to men and women ( cstatistic=0.892 and = 0.837 , respectively ) ( figure 5 ) . in a further model adjusted only for sex and age , considering the 4 prespecified age classes , the interaction between sex and age remained not significant ( p for interaction=0.21 ) . comparing women to men ,
the adjusted odds ratios for inhospital mortality were 0.74 ( 95% ci 0.173.22 ) for age < 55 years , 2.55 ( 95% ci 1.434.55 ) for age 55 to 64 years , 1.80 ( 95% ci 1.222.67 ) for age 65 to 74 years , and 1.48 ( 95% ci 1.211.81 ) for age 75 years .
finally , in the model where only sex and year of the study were considered , no significant interaction between these 2 variables was observed ( p for interaction=0.95 ) , suggesting that the risk of death for women was higher than that for men , and this excess risk did not change over the years .
multivariable logistic regression analysis for inhospital factors related to mortality over time in the overall population .
hr indicates heart rate ; mi , myocardial infarction ; or , odds ratio ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; tia , transient ischemic attack .
multivariable logistic regression analysis for inhospital factors related to mortality over time in men ( a ) and women ( b ) .
hr indicates heart rate ; mi , myocardial infarction ; or , odds ratio ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; tia , transient ischemic attack .
of 28 274 patients with acs enrolled in the 5 registries , 13 235 ( 46.8% ) were classified as having stemi ( 72.1% men and 27.9% women ) .
the overall rates of women with stemi did not change significantly over time ( from 30.1% in 2001 to 28.1% in 2014 ; p for trend=0.06 ) ( table 1 ) .
the prevalence of women increased significantly with age , being 13.0% in the age class < 55 years , 17.5% in the age class 55 to 64 years , 28.0% in the age class 65 to 74 years , and 48.3% in the age class 75 years ( p for trend < 0.0001 ) , resulting in a higher mean age of women compared with men ( 7213 versus 6312 years , p<0.0001 ) .
sex and age differences in clinical characteristics of patients with stemi enrolled during the study period are listed in table 2 . when compared to men in the majority of corresponding age categories , women were less likely to be active smokers , had more hypertension and lower rates of prior myocardial infarction or revascularization procedures .
killip class iv on admission increased significantly with age in both sexes and was slightly more frequent among women in all age classes , even though a significant difference was observed among patients aged < 55 years .
the use of antithrombotic therapies during hospital admission was significantly lower with the increase in age classes for both sexes .
when compared to men , women aged 65 years received significantly less unfractionated heparin , glycoprotein iib / iiia inhibitors , and dual antiplatelet therapy during hospitalization ( table 2 ) .
baseline characteristics of patients with stemi from 2001 to 2014 , according to sex and age classes twosided p values for comparisons across sex for each age category are < 0.05 .
asa indicates acetylsalicylic acid ; bmi , body mass index ; cabg , coronary artery bypass graft ; ccu , cardiac care unit ; dapt , dual antiplatelet therapy ; ecg , electrocardiogram ; gpi , glycoprotein iib / iiia inhibitors ; hr , heart rate ; lmwh , lowmolecularweight heparins ; lvef , left ventricular ejection fraction ; mi , myocardial infarction ; pad , peripheral artery disease ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; stemi , stelevation myocardial infarction ; tia , transient ischemic attack ; ufh , unfractionated heparin .
as shown in figure 1 , the use of fibrinolytic therapy decreased from 2001 to 2014 in both sexes and at all ages ( all p for trend < 0.0001 ) , and this trend was mirrored by a dramatic increase in ppci , also affecting both sexes at all ages ( all p<0.0001 ) .
the overall rates of no reperfusion therapy decreased significantly ( p for trend < 0.0001 ) , and the changes were particularly marked among women aged 75 years ( from 64.1% in 2001 to 27.1% in 2014 ) .
the no reperfusion rates remained significantly higher in older women until 2010 , but tended to disappear in 2014 . among patients receiving ppci ,
no significant differences were observed between men and women with regard to door to balloon times ( 2.24.7 versus 1.71.6 hours in 2001 , 2.24.2 versus 2.02.4 hours in 2006 , 0.85.8 versus 0.94.3 hours in 2009 , 2.77.8 versus 2.64.8 hours in 2010 , and 1.14.8 versus 0.95.0 hours in 2014 ) .
data on catheterization access site were available only for the blitz 4 and eyeshot study cohorts , where the radial approach was used more frequently in men than in women ( 31.4% versus 26.4% , p=0.002 , and 72.7% versus 61.7% , p=0.0007 , respectively ) .
inhospital rates of primary pci ( a , b ) , thrombolysis ( c , d ) , and no reperfusion ( e , f ) over time , among men and women , according to age classes .
as shown in table 3 , crude inhospital mortality rates increased significantly with age in both sexes , being significantly higher in women at all ages ( except in patients < 55 years of age , who presented an extremely low inhospital fatality rate ) .
the rates of reinfarction increased significantly with age in men , but not in women , whereas the rates of stroke and cardiogenic shock increased significantly with age in both sexes .
the rates of timi major bleeding were significantly higher in women at all ages compared to men and increased significantly with age in men , but not in women .
inhospital major clinical events of stemi patients according to sex and age classes twosided p values for comparisons across sex are < 0.05 .
number in parentheses are percenteges calculated on males and females within each class of age .
mi indicates myocardial infarction ; stemi , stelevation myocardial infarction . excluding 151 patients enrolled in inacs outcome because they have not been admitted in ccus and 264 patients from the blitz4 since they underwent a coronary angiography in another hospital .
time trends for mortality according to age are shown in figure 2 , with an overall decline in mortality over time affecting both sexes ( p for trend < 0.01 ) and patients aged 75 years ( p for trend < 0.0001 ) .
mortality and reinfarction rates declined significantly over time in both sexes , whereas stroke rates remained fairly stable , being slightly higher among women at all time points ( table 4 ) .
overall cardiogenic shock rates were significantly higher in women ( 8.6% versus 4.8% , p<0.0001 ) , even though statistically significant only in patients aged 65 to 74 years , and declined significantly over time in women and not in men . furthermore , cardiogenic shock fatality rates were higher in women at all time points , except for year 2001 ( figure 3 ) , but were statistically significant only in year 2009 .
major bleeding rates were significantly higher among women , and there was no significant trend over time in both sexes .
inhospital mortality rates over time among men ( a ) and women ( b ) , according to age classes .
inhospital major clinical events in men ( n=9544 ) and women ( n=3691 ) according to year of the survey twosided p values for comparisons across sex are < 0.05 .
mi indicates myocardial infarction . excluding 151 patients enrolled in inacs outcome because they have not been admitted in ccus and 264 patients from the blitz4 since they underwent a coronary angiography in another hospital .
inhospital mortality rates among men ( gray bars ) and women ( red bars ) with cardiogenic shock ( at entry / during hospital stay ) over the observation time . * twosided p values for comparisons across sex are < 0.05 .
on multivariable analysis , several variables were found to be significantly related to inhospital mortality ( figure 4 ) , but without a statistically significant interaction between sex and age ( p for interaction=0.61 ) .
many variables remained predictors of mortality even when the model was applied separately to men and women ( cstatistic=0.892 and = 0.837 , respectively ) ( figure 5 ) . in a further model adjusted only for sex and age , considering the 4 prespecified age classes , the interaction between sex and age remained not significant ( p for interaction=0.21 ) . comparing women to men ,
the adjusted odds ratios for inhospital mortality were 0.74 ( 95% ci 0.173.22 ) for age < 55 years , 2.55 ( 95% ci 1.434.55 ) for age 55 to 64 years , 1.80 ( 95% ci 1.222.67 ) for age 65 to 74 years , and 1.48 ( 95% ci 1.211.81 ) for age 75 years .
finally , in the model where only sex and year of the study were considered , no significant interaction between these 2 variables was observed ( p for interaction=0.95 ) , suggesting that the risk of death for women was higher than that for men , and this excess risk did not change over the years .
multivariable logistic regression analysis for inhospital factors related to mortality over time in the overall population .
hr indicates heart rate ; mi , myocardial infarction ; or , odds ratio ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; tia , transient ischemic attack .
multivariable logistic regression analysis for inhospital factors related to mortality over time in men ( a ) and women ( b ) .
hr indicates heart rate ; mi , myocardial infarction ; or , odds ratio ; pci , percutaneous coronary intervention ; sbp , systolic blood pressure ; tia , transient ischemic attack .
the present analysis covers a period of time where reperfusion therapy for stemi changed dramatically with the nationwide organization of stemi networks.12 in the present study , we observed a striking increase in ppci , a concomitant reduction in fibrinolysis and , more importantly , a significant reduction in the rates of no reperfusion affecting both women and men and all age classes .
these positive changes in reperfusion therapy resulted in significantly lower mortality rates for both sexes over time , but the worse outcome of women , as compared to men , remained unchanged among all age classes , except for women aged < 55 years .
a second point of interest in the present study was the comparison of men and women across age classes to verify whether the unfavorable treatment and mortality gaps reported for women were , at least in part , dependent on women 's older age .
our data show that , at least until 2010 , the rates of no reperfusion remained significantly higher among women aged 75 years compared to men , and this gap disappeared only in 2014 . on multivariable analysis
, both age and female sex were found to be significantly associated with inhospital mortality after adjustment for other risk factors , but without any mutual interaction . from the present analysis ,
3 causes emerge as possible culprit in persistently higher mortality rates among women with stemi compared to men : ( 1 ) significantly less reperfusion therapy ; ( 2 ) a higher major bleeding rate ; and ( 3 ) more prevalent and more fatal cardiogenic shock .
lower reperfusion rates in women , as compared to men , have been reported since the early thrombolytic era,2 and this gap continues to persist.13 , 14 , 15 in the present analysis , women in any class of age were less likely to receive either ppci or thrombolysis than men , although the overall rate of reperfusion increased over time .
several reasons can be postulated to explain these findings.2 for instance , women with stemi are more likely to have atypical symptoms possibly leading to delayed presentation and underrecognition of stemi at first medical contact , thus precluding early reperfusion strategies.2 , 16 , 17 , 18 despite an overall increase in emergency coronary angiography , lower reperfusion rates in women can also be explained by a higher frequency of alternative etiologies ( eg , spontaneous coronary dissection , coronary vasospasm ) , and different angiographic findings.19 indeed , the octavia ( optical coherence tomography assessment of gender diversity in primary angioplasty ) study , the first prospective controlled study designed to assess in vivo sex differences in the mechanisms of stemi , demonstrated that men and women , when matched for age , shared similar prevalence of ruptured and eroded plaques.20 these findings suggest that disparities in environment , behavior , risk factors , and management , rather than different biological mechanisms and response to treatment , might be more relevant in affecting clinical outcome .
we observed higher rates of major bleeding in women than in men , particularly among those aged > 55 years , even though no significant trend was observed over age
. a higher risk of bleeding in women has been known since the early fibrinolytic era,21 when the isis3 collaboration observed a 70% excess in major bleeding among women , as compared to men , after adjustment for age and other risk factors .
this increased risk persisted after the reperfusion shift to ppci 22 and is independently associated with a 3fold increase in mortality.23 the higher risk of bleeding among women might be partly related to the lack of weight or body mass dose adjustment of most antithrombotic drugs , and also to a lower use of radial access among women who , compared with men , have smaller radial arteries that may be more prone to spasm , which is a major cause of radial procedure failure.24
the rates of cardiogenic shock due to acute coronary syndromes have decreased in our country during the observation period , as well as overall mortality due to cardiogenic shock.11 yet , as shown by the present data , case fatality rates have been declining more significantly among men than among women .
women in our study had higher rates of cardiogenic shock as compared to men at any age , both upon admission and during subsequent hospital stay , even though a significant difference was observed among the 65 to 74 years group , and at least part of the excess mortality among women should probably be attributed to a higher incidence of cardiogenic shock .
both female sex and age have been shown to be independent predictors for the occurrence20 , 25 and mortality26 of cardiogenic shock in stemi , even after the widespread use of acute reperfusion therapy .
longer delay to reperfusion among women may be the major reason for both the higher incidence and case fatality of cardiogenic shock . focusing future research and quality initiatives on these 3 potential causes of worse outcome in women with stemi
system efforts to reduce the time from symptom onset to reperfusion are strongly endorsed by practice guidelines,27 , 28 though the effectiveness from public campaigns has not yet been clearly established . according to our data
, the implementation of stemi networks in italy has improved the rates of reperfusion in both women and men . among the strategies to reduce bleeding ,
probably the most effective was the gradual shift from fibrinolysis to ppci , but also the later shift from the femoral to the radial vascular approach to pci , which has been shown to improve outcomes and reduce bleeding particularly among women.29 finally , according to our data and other allcomers observational studies , mortality in cardiogenic shock complicating acute coronary syndromes remains higher in women than in men .
therefore , further studies are warranted to better define specific treatment strategies in cardiogenic shock.30
despite a dramatic shift from thrombolysis to ppci and an overall reduction in no reperfusion therapy over time , affecting both sexes and all age classes , women continue to show a significantly higher adjusted risk of inhospital mortality , as compared to men .
cardiac societies from both sides of the atlantic have released specific documents on sex differences in presentation , treatment , and outcomes of cardiovascular disease,2 , 31 though prospective studies aimed at investigating the reasons for the persisting worse outcomes of stemi in women are still lacking.32
the studies included in this manuscript were funded by unrestricted grants , as follows : blitz ( boehringer ingelheim , italy ) , inacs outcome ( sanofiaventis and bristolmyers squibb , italy ) , blitz 4 ( merck , sharp&dohme , italy ) , mantra ( glaxosmithkline , italy ) and eyeshot ( astrazeneca , italy ) . the sponsor of the studies was the heart care foundation , a nonprofit independent institution that is also the owner of the databases .
database management and quality control of the data were under the responsibility of the research centre of the italian association of hospital cardiologists ( anmco ) .
the steering committees of the studies had full access to all of the data in the studies and took complete responsibility for the integrity of the data and the accuracy of data analysis .
dr de luca reports personal fees from astra zeneca , bayer , boehringeringelheim , eli lilly , daiichi sankyo , menarini , and the medicines company , outside the submitted work ; dr olivari reports grants and personal fees from sanofi , astra zeneca , eli lilly , daiichi sankyo , boehringeringelheim , merck sharp&dohme , and menarini , outside the submitted work ; dr gonzini , an employee of heart care foundation , which conducted the studies , reports institutional grants from glaxosmithkline , italy , grants from merck , sharp&dohme , italy , outside the submitted work ; dr de servi reports personal fees from astra zeneca , eli lilly , daiichi sankyo , and the medicines company , outside the submitted work ; dr savonitto reports grants and personal fees from eli lilly , novartis , iroko , daiichi sankyo , and pfizer , outside the submitted work . | backgroundage and sexspecific differences exist in the treatment and outcome of stelevation myocardial infarction ( stemi ) .
we sought to describe age and sexmatched contemporary trends of inhospital management and outcome of patients with stemi.methods and resultswe analyzed data from 5 italian nationwide prospective registries , conducted between 2001 and 2014 , including consecutive patients with stemi .
all the analyses were age and sexmatched , considering 4 age classes : < 55 , 55 to 64 , 65 to 74 , and 75 years .
a total of 13 235 patients were classified as having stemi ( 72.1% men and 27.9% women ) . a progressive shift from thrombolysis to primary percutaneous coronary intervention occurred over time , with a concomitant increase in overall reperfusion rates ( p for trend < 0.0001 ) , which was consistent across sex and age classes .
the crude rates of inhospital death were 3.2% in men and 8.4% in women ( p<0.0001 ) , with a significant increase over age classes for both sexes and a significant decrease over time for both sexes ( all p for trend < 0.01 ) .
on multivariable analysis , age ( odds ratio 1.09 , 95% ci 1.071.10 , p<0.0001 ) and female sex ( odds ratio 1.44 , 95% ci 1.071.93 , p=0.009 ) were found to be significantly associated with inhospital mortality after adjustment for other risk factors , but no significant interaction between these 2 variables was observed ( p for interaction=0.61).conclusionsdespite a nationwide shift from thrombolytic therapy to primary percutaneous coronary intervention for stemi affecting both sexes and all ages , women continue to experience higher inhospital mortality than men , irrespective of age . |
depression is one of the most prevalent , disabling , and costly mental disorders worldwide .
according to recent estimates , the 12-month prevalence rate of depression in europe is 6.9%1 and depression accounts for 33% of the total costs of all brain disorders.2 similarly , each year in the uk , an estimated 6% of adults experience an episode of depression , and more than 15% of the population will experience an episode of depression during their lifetime.3 depression is managed predominantly in primary care and accounts for 15% of all general practice consultations in the uk.3,4 in 2007 , the estimated total cost of health care services for depression in england was 1.7 billion , which increased to 7.5 billion when indirect costs associated with lost productivity were included.5 while high quality care for depression in primary care settings is important to minimize its clinical and economic impact on patients and society as a whole , the identification and treatment of depression in primary care is often suboptimal and has been a target for improvement in recent years.3,4,69 a possible reason for poor detection and/or misdiagnosis of depression is that it often occurs together with pain , with reported co - occurrence rates of 30%50%.10,11 many patients with depression initially present with painful physical symptoms ( pps ) ( eg , headache , musculoskeletal pain ) , but as painful symptoms are not a prominent feature of the diagnostic criteria for depression ( dsm - iv or icd-10 ) , this may negatively affect the recognition of depression in primary care.10,12 when the cause of pain is not known ( often termed unexplained pain ) , the prevalence of unrecognized mood disorders has been reported to be as high as 80%.11,13 this may lead to a delay in the time to diagnosis and treatment of depression in primary care , although evidence of this from longitudinal studies is missing.13 more severe pain may cause greater delays in depression diagnosis , which can potentially lead to a more severe or chronic form of depression and poorer outcomes for the patient.10,14,15 this , in turn , is likely to have an impact on health care utilization and related costs .
there is some evidence that concurrent pain and depression are associated with increased use of health care resources and costs compared with either condition alone.13,1619 however , there is no information on the economic impact of a longer time to diagnosis of depression among primary care patients with pre - existing pps .
this study used a retrospective cohort design to assess the impact of pain severity and time to diagnosis of depression on 12-month health care utilization and associated costs before and after diagnosis of depression in uk primary care patients with pre - existing unexplained pain .
we also assessed if costs would increase differentially between the two consecutive 12-month time periods post - depression diagnosis .
data for this study were obtained from the uk general practice research database ( gprd ) , which is the world s largest computerized database of anonymized electronic medical records of patients in primary care.20 the gprd has records for over 13 million patients from 629 primary care practices throughout the uk , and the records for over 5 million currently registered patients meet the gprd standards of acceptable quality for use in research , which is equivalent to approximately 8.5% of the uk population.21 the information collected on the gprd includes demographics , medical diagnoses , clinical events , medication prescriptions , specialist referrals , hospital admission , and treatment outcomes .
recent systematic reviews have confirmed the validity of medical diagnoses and quality of information on the gprd.20,22 patients for this study were identified from the gprd for the 5-year observation period of january 1 , 2005 until december 31 , 2009 .
the study population included all patients aged 18 years who were ever registered at one of the gp practices in the gprd during the 5-year observation period and who had unexplained pain followed by a subsequent diagnosis of depression . only patients with
firstly , we identified patients with pre - existing unexplained pain , which was defined as pps without any probable organic cause , based on the read code classification , which is a system of clinical classification frequently used in the uk national health service ( nhs).23 we adapted the read codes for pps previously defined elsewhere.16 patients with unexplained pain were required to have one or more pps codes during the observation period , but no other diagnostic code ( other than pps or depression ) recorded 90 days on either side of the pps code .
this was to ensure that the pps diagnosis was not likely to be associated with or attributable to any other comorbid condition during this period .
the index date of pps was the first recorded date of pps during the observation period .
secondly , we then identified those patients who had a subsequent diagnosis of depression , which was based on the read codes for this condition as described in a previous study.24 the full list of read codes used to identify patients with pps and depression are available upon request .
for the present analyses , patients were classified into one of four groups based on the severity of their unexplained pain ( milder versus more severe ) and time to diagnosis of depression ( shorter versus longer ) following record of pain symptoms as follows : ( 1 ) milder pain and shorter time to depression diagnosis ; ( 2 ) milder pain and longer time to depression diagnosis ; ( 3 ) more severe pain and shorter time to depression diagnosis ; and ( 4 ) more severe pain and longer time to depression diagnosis . as there is no record of pain severity available in the gprd , we defined milder pain as less than three pain relief prescriptions ( non - opioid analgesics , opioid analgesics , non - steroidal anti - inflammatory drugs [ nsaids ] , and skeletal muscle relaxants ) in the 180 days after the first pain code .
more severe pain was defined as at least three pain relief prescriptions plus use of at least one opioid in the 180 days after the first pain code .
patients with at least three pain relief prescriptions and no opioid use were excluded from the analysis in order to have a clear dichotomy between mild and severe pain .
the index date for depression was the first recorded date of a definite diagnostic code for depression after unexplained pain was recorded in the gprd during the study period .
shorter time to depression diagnosis was defined as less than 1 year from the pps index date , whereas longer time to depression diagnosis was defined as more than 1 year from the pps index date .
the 1-year cut - off was based on the median time to depression diagnosis within our data and on clinical advice , and allowed for groups of approximately equal size .
patients were required to remain in the gprd for at least 2 years after the diagnosis of depression .
the period after diagnosis of depression was examined as two separate but consecutive 12-month periods ( post1 and post2 ) in order to examine potential differences in health care utilization and costs during these two time periods as a result of pain severity and time to depression diagnosis from pain symptoms .
the flow chart of patient selection and classification into the four groups is presented in figure 1 . following selection of the study sample ,
in addition , information on comorbid conditions was collected and the charlson comorbidity index ( cci ) was calculated.25,26 the cci is a summary measure of index , which represents the 1-year mortality for a patient , based on their history of a range of comorbid conditions .
khan et al27 previously defned 17 categories of comorbid conditions using read codes for the calculation of cci , and we adapted their codes and approach .
we first identified any of the records related to the comorbid conditions in the 12-month period before diagnosis of depression ( pre ) , and then weighted them to produce a summary score .
health care utilization ( gp consultations , secondary care referrals , and pharmacotherapy ) and the related costs ( uk unit costs , 2009 values)28 were estimated for the 12-month period preceding depression diagnosis ( pre ) and for the subsequent two 12-month periods after depression diagnosis ( post1 and post2 ) .
the costs associated with gp consultations were estimated from several major types of gp consultation , including clinical consultations , surgery visits , home visits , and telephone consultations .
the list of gp consultations available in the gprd data were classified into these four types of consultation , and the unit costs for each type of consultation were derived from the unit costs of health and social care 2009 ( table 1).28 in the present analyses , secondary care referrals consisted of inpatient referrals , day care referrals , outpatient referrals , as well as accident and emergency referrals that did and did not lead to hospital admission .
the average costs per episode for each of these different types of secondary care referral were derived from the unit costs of health and social care 2009 ( table 1).28 information on prescriptions for medications for pain and depression were collected from the gprd .
this included non - opioid analgesics , opioid analgesics , nsaids , skeletal muscle relaxants , antidepressants , anxiolytics / hypnotics , and anticonvulsants .
the average number of pain relief prescriptions per patient was determined for each 12-month period .
the costs per day of therapy ( dot ) of each medication were derived from the ims padds database.29 descriptive statistics were used to summarize the demographics and pre - index clinical characteristics as well as the health care resource utilization for the 12-month period before depression diagnosis ( pre ) for each of the four groups of patients .
comparisons between the four patient groups were made using chi - square tests or kruskal wallis tests as appropriate .
multivariate analyses were used to estimate the total costs and the costs of each component ( gp consultations , secondary care referrals , and pharmacotherapy ) in the 12 months before depression diagnosis ( pre ) and in the two 12-month periods after depression diagnosis ( post1 and post2 ) .
the models included time period as a main independent variable , adjusting for the following covariates : age , gender , and cci summary score .
all multivariate analyses were carried out using generalized estimating equation models with a loglink function to correct for the skewness of cost data and also take into account correlation within the same subjects across the time periods using ar1 correlation structure .
bootstrap t - tests were used for pairwise cost comparisons ( within - group and between - group comparisons ) .
data extraction and management were carried out using sas 9.2 ( sas institute inc , cary , nc ) , and all costing analyses were conducted using stata se 10 ( statacorp l p , college station , tx ) .
data for this study were obtained from the uk general practice research database ( gprd ) , which is the world s largest computerized database of anonymized electronic medical records of patients in primary care.20 the gprd has records for over 13 million patients from 629 primary care practices throughout the uk , and the records for over 5 million currently registered patients meet the gprd standards of acceptable quality for use in research , which is equivalent to approximately 8.5% of the uk population.21 the information collected on the gprd includes demographics , medical diagnoses , clinical events , medication prescriptions , specialist referrals , hospital admission , and treatment outcomes .
recent systematic reviews have confirmed the validity of medical diagnoses and quality of information on the gprd.20,22 patients for this study were identified from the gprd for the 5-year observation period of january 1 , 2005 until december 31 , 2009 .
the study population included all patients aged 18 years who were ever registered at one of the gp practices in the gprd during the 5-year observation period and who had unexplained pain followed by a subsequent diagnosis of depression . only patients with
firstly , we identified patients with pre - existing unexplained pain , which was defined as pps without any probable organic cause , based on the read code classification , which is a system of clinical classification frequently used in the uk national health service ( nhs).23 we adapted the read codes for pps previously defined elsewhere.16 patients with unexplained pain were required to have one or more pps codes during the observation period , but no other diagnostic code ( other than pps or depression ) recorded 90 days on either side of the pps code .
this was to ensure that the pps diagnosis was not likely to be associated with or attributable to any other comorbid condition during this period .
the index date of pps was the first recorded date of pps during the observation period .
secondly , we then identified those patients who had a subsequent diagnosis of depression , which was based on the read codes for this condition as described in a previous study.24 the full list of read codes used to identify patients with pps and depression are available upon request . for the present analyses
, patients were classified into one of four groups based on the severity of their unexplained pain ( milder versus more severe ) and time to diagnosis of depression ( shorter versus longer ) following record of pain symptoms as follows : ( 1 ) milder pain and shorter time to depression diagnosis ; ( 2 ) milder pain and longer time to depression diagnosis ; ( 3 ) more severe pain and shorter time to depression diagnosis ; and ( 4 ) more severe pain and longer time to depression diagnosis . as there is no record of pain severity available in the gprd , we defined milder pain as less than three pain relief prescriptions ( non - opioid analgesics , opioid analgesics , non - steroidal anti - inflammatory drugs [ nsaids ] , and skeletal muscle relaxants ) in the 180 days after the first pain code .
more severe pain was defined as at least three pain relief prescriptions plus use of at least one opioid in the 180 days after the first pain code .
patients with at least three pain relief prescriptions and no opioid use were excluded from the analysis in order to have a clear dichotomy between mild and severe pain .
the index date for depression was the first recorded date of a definite diagnostic code for depression after unexplained pain was recorded in the gprd during the study period .
shorter time to depression diagnosis was defined as less than 1 year from the pps index date , whereas longer time to depression diagnosis was defined as more than 1 year from the pps index date .
the 1-year cut - off was based on the median time to depression diagnosis within our data and on clinical advice , and allowed for groups of approximately equal size .
patients were required to remain in the gprd for at least 2 years after the diagnosis of depression . the period after diagnosis of depression
was examined as two separate but consecutive 12-month periods ( post1 and post2 ) in order to examine potential differences in health care utilization and costs during these two time periods as a result of pain severity and time to depression diagnosis from pain symptoms .
the flow chart of patient selection and classification into the four groups is presented in figure 1 .
following selection of the study sample , data on patient age and gender were collected from the gprd . in addition , information on comorbid conditions was collected and the charlson comorbidity index ( cci ) was calculated.25,26 the cci is a summary measure of index , which represents the 1-year mortality for a patient , based on their history of a range of comorbid conditions .
khan et al27 previously defned 17 categories of comorbid conditions using read codes for the calculation of cci , and we adapted their codes and approach .
we first identified any of the records related to the comorbid conditions in the 12-month period before diagnosis of depression ( pre ) , and then weighted them to produce a summary score .
health care utilization ( gp consultations , secondary care referrals , and pharmacotherapy ) and the related costs ( uk unit costs , 2009 values)28 were estimated for the 12-month period preceding depression diagnosis ( pre ) and for the subsequent two 12-month periods after depression diagnosis ( post1 and post2 ) .
the costs associated with gp consultations were estimated from several major types of gp consultation , including clinical consultations , surgery visits , home visits , and telephone consultations .
the list of gp consultations available in the gprd data were classified into these four types of consultation , and the unit costs for each type of consultation were derived from the unit costs of health and social care 2009 ( table 1).28 in the present analyses , secondary care referrals consisted of inpatient referrals , day care referrals , outpatient referrals , as well as accident and emergency referrals that did and did not lead to hospital admission .
the average costs per episode for each of these different types of secondary care referral were derived from the unit costs of health and social care 2009 ( table 1).28 information on prescriptions for medications for pain and depression were collected from the gprd .
this included non - opioid analgesics , opioid analgesics , nsaids , skeletal muscle relaxants , antidepressants , anxiolytics / hypnotics , and anticonvulsants .
the average number of pain relief prescriptions per patient was determined for each 12-month period .
the costs per day of therapy ( dot ) of each medication were derived from the ims padds database.29
health care utilization ( gp consultations , secondary care referrals , and pharmacotherapy ) and the related costs ( uk unit costs , 2009 values)28 were estimated for the 12-month period preceding depression diagnosis ( pre ) and for the subsequent two 12-month periods after depression diagnosis ( post1 and post2 ) .
the costs associated with gp consultations were estimated from several major types of gp consultation , including clinical consultations , surgery visits , home visits , and telephone consultations .
the list of gp consultations available in the gprd data were classified into these four types of consultation , and the unit costs for each type of consultation were derived from the unit costs of health and social care 2009 ( table 1).28 in the present analyses , secondary care referrals consisted of inpatient referrals , day care referrals , outpatient referrals , as well as accident and emergency referrals that did and did not lead to hospital admission .
the average costs per episode for each of these different types of secondary care referral were derived from the unit costs of health and social care 2009 ( table 1).28 information on prescriptions for medications for pain and depression were collected from the gprd .
this included non - opioid analgesics , opioid analgesics , nsaids , skeletal muscle relaxants , antidepressants , anxiolytics / hypnotics , and anticonvulsants .
the average number of pain relief prescriptions per patient was determined for each 12-month period .
the costs per day of therapy ( dot ) of each medication were derived from the ims padds database.29
descriptive statistics were used to summarize the demographics and pre - index clinical characteristics as well as the health care resource utilization for the 12-month period before depression diagnosis ( pre ) for each of the four groups of patients .
comparisons between the four patient groups were made using chi - square tests or kruskal wallis tests as appropriate .
multivariate analyses were used to estimate the total costs and the costs of each component ( gp consultations , secondary care referrals , and pharmacotherapy ) in the 12 months before depression diagnosis ( pre ) and in the two 12-month periods after depression diagnosis ( post1 and post2 ) .
the models included time period as a main independent variable , adjusting for the following covariates : age , gender , and cci summary score .
all multivariate analyses were carried out using generalized estimating equation models with a loglink function to correct for the skewness of cost data and also take into account correlation within the same subjects across the time periods using ar1 correlation structure .
bootstrap t - tests were used for pairwise cost comparisons ( within - group and between - group comparisons ) .
data extraction and management were carried out using sas 9.2 ( sas institute inc , cary , nc ) , and all costing analyses were conducted using stata se 10 ( statacorp l p , college station , tx ) .
the mean age of the total study sample of 4000 adults was 48.9 years and 68.2% were female .
table 2 summarizes the characteristics of the total sample and the four patient groups and shows that the demographics ( gender , age ) differed significantly between the four patient groups .
in particular , the two groups of patients with more severe pain were older than the two groups with milder pain and there were fewer females in the more severe group with longer time to depression diagnosis .
the mean cci score for the total sample was 0.12 ( standard deviation 0.45 ) and differed significantly between the four patient groups ( p = 0.015 ) , with the highest score in the group with more severe pain and longer time to depression diagnosis ( mean cci = 0.21 ) .
for the 17 comorbid conditions included in the cci , the percentages of patients with these comorbidities at baseline were very low , ranging from 0% for aids , metastatic tumor , and moderate liver disease to 3.3% for diabetes in the total sample
. there were significant differences between the four patient groups in the frequencies of the following comorbidities : cancer , chronic pulmonary disease , diabetes , renal disease , and rheumatologic disease ( p < 0.05 , chi - square test ) ( data available upon request ) .
table 3 provides a descriptive summary of the 12-month resource use before depression diagnosis ( pre ) in the four groups of patients .
there were significant differences between groups for each of the four types of gp consultation ( p < 0.001 ) , with more gp consultations in the two groups with more severe pain .
the frequencies of secondary care referrals in the 12 months before depression diagnosis was similar across groups , with the exception of a higher number of accident and emergency visits leading to hospital admission in the group with more severe pain and a longer time to depression diagnosis ( p = 0.034 ) .
the average number of pain relief prescriptions in the 12-month period before depression diagnosis differed significantly between the four patient groups for each type of medication , and was higher in the two groups with more severe pain , consistent with the definition used ( table 3 ) .
estimates of the 12-month total health care costs ( adjusted ) for each of the four groups of patients before ( pre ) and after depression diagnosis ( post1 and post2 ) are shown in table 4 .
the total costs were higher for the two groups with more severe pain compared with the two groups with milder pain ( p < 0.001 for all pairwise comparisons ) .
the pattern remained largely similar even when the total costs were estimated without drug costs .
patients with more severe pain and longer time to depression diagnosis had the highest annual total costs both before ( 925 at pre ) and after diagnosis of depression ( 933 at post1 and 988 at post2 ) . when the two groups with more severe pain were compared those with a longer time to depression diagnosis had higher 12-month total costs after depression diagnosis ( post1 andpost2 ) compared with those who had a shorter time to depression diagnosis ( p < 0.05 ) .
likewise , pairwise comparisons between the two groups with milder pain showed that those with a longer time to depression diagnosis had higher total costs at post1 than those with a shorter time to depression diagnosis ( p < 0.05 ) ; cost comparisons at the other time points were not significant .
the mean 12-month cost estimates of the various cost components ( gp consultations , secondary care referrals , and pharmacotherapy ) for the four groups of patients before and after depression diagnosis are presented in figure 2 .
total gp consultation costs ( figure 2a ) were the largest cost component and were highest both before and after depression diagnosis in the group with more severe pain and longer time to depression diagnosis ( p < 0.001 versus the other three groups ) .
secondary care referral costs ( figure 2b ) were highest in the second year after depression diagnosis in the group with more severe pain and longer time to depression diagnosis ( p < 0.05 versus the other three groups at post2 ) .
mean total drug costs per year ( figure 2c ) were higher in the group with more severe pain and longer time to depression diagnosis compared with the two groups with milder pain ( p < 0.001 for all pairwise comparisons ) , but were similar to the group with more severe pain and shorter time to depression diagnosis .
annual total drug costs were similar before and in the two years after depression diagnosis in all four patient groups .
there was no clear pattern of results when assessing 12-month costs over the pre , post1 , and post2 periods within each group .
neither patients with more severe pain nor those with a longer time to depression diagnosis demonstrated consistent increased costs at post1 .
in this study , we identified primary care patients in the uk gprd who had unexplained pain and a subsequent diagnosis of depression and estimated their annual health care utilization costs in the year before depression diagnosis and in the first 2 years after depression diagnosis .
our findings show that unexplained pain and depression impose a significant economic burden on the uk health care system .
patients with more severe pain ( based on prescription of at least three pain relief medications plus opioid use ) and a longer time to depression diagnosis after identification of pain symptoms had higher total costs than those in the other three groups .
more severe pain was the main factor driving costs ; the two groups with more severe pain had higher annual costs in the year before and in each of the 2 years after depression diagnosis than the two groups with milder pain , after adjusting for age , sex , and cci scores .
furthermore , comparisons between the two groups of patients with more severe pain showed that those with a longer time to depression diagnosis had higher annual total costs in the 2 years after depression diagnosis than those with a shorter time to depression diagnosis .
however , a longer time to depression diagnosis was not associated with higher total costs among patients with milder pain except in the first year after depression diagnosis .
several previous studies have shown that comorbid pain and depression is associated with increased use of health care services.13,1619 in a gprd study of adults with depression , 66% of patients also had codes for pps , and patients with depression and concurrent pain had higher health care resource use ( gp consultations , secondary care referrals , and drug use ) than those with no pain.16 no previous studies have examined the impact of pain and depression on health care costs in the same way as our analysis .
all patients in our study had a diagnosis of depression subsequent to a diagnosis of pain and we did not examine the health care costs associated with either condition alone . in a previous population - based study in sweden ,
the average total health care costs ( primary health care costs , hospital costs , and drug costs ) per patient were higher for those with a diagnosis of both depression and back pain ( sek 46,909 ) , compared with patients with a depression diagnosis ( sek 36,904 ) or a diagnosis of back pain ( sek 26,152).30 comorbidity of depression and pain had a negative interaction effect on health care costs , such that the costs were less than additive.30 this negative interaction effect was largely related to lower hospital care costs , while there was actually an increase in the number of general practitioner visits among patients with both conditions.30 primary care consultations were the largest cost component in our study and were highest in the group with more severe pain and a longer time to depression diagnosis .
this group also had higher costs due to pain relief prescriptions and to secondary care referrals in year two after depression diagnosis .
our findings suggest that there may be potential cost savings for the uk nhs with earlier diagnosis of depression in patients presenting with more severe unexplained pain .
pps and depression appear to be closely linked , but the relationship is complex and not yet fully understood : depression may be both a cause and a consequence of pps.10,11,31 moreover , when pain and depression are both present , they impact on each other,11,32 which may refect common underlying neurobiological mechanisms.10,33 however , it has been estimated that about half of all patients with depression may go undiagnosed because they present with somatic or physical symptoms.31 primary care physicians often fail to look for symptoms of depression in patients presenting with pain despite reports that about 50%80% of patients with depression initially have somatic symptoms including pps,34,35 and that there is an increased risk of depression in patients presenting with pain.36 particular attention should be given to unexplained pain as it is associated with increased depression comorbidity and poorer outcomes.33 a recent study in primary care found that 80% of patients presenting with unexplained chronic pain had an undiagnosed depression or anxiety disorder.13 although medication for pain may be initiated , delayed recognition of depression and initiation of antidepressant treatment may negatively affect the course and severity of illness and result in poorer treatment outcomes .
these results highlight the need for education on the role of pain and unexplained painful symptoms in depression for primary care providers .
our study is the first to examine the economic impact of pain severity and time to depression diagnosis in patients presenting initially with pain .
previous studies have found that moderate to severe pain is common in patients with depression.31,37 more severe pain has been associated with more severe depression,38 a worse course of depression,10,39 a lower likelihood of or longer time to response or remission,15,40,41 a poorer health - related quality of life,14 and increased health care utilization.10 recent data extracted from the gprd has shown greater health service use and costs in patients with non - remission of depression compared with those who achieve remission ( defined as successful cessation of antidepressant treatment for at least 6 months).42 we included a large sample of patients from the gprd , which is representative of the uk population , and for which data are recorded during the consultation . also , we included only data from general practices that were considered to be of a suitable standard by the gprd .
this is important because recent data from the gprd had shown that the incidence of depression varies according to age and sex.43 chronic medical illnesses ( eg , osteoarthritis , rheumatoid arthritis , diabetes , coronary artery disease , congestive heart failure , asthma , and copd ) are common comorbidities in patients with depression and pain.44,45 however , the cci score for the primary care patients with pain and depression in the present study was very low , indicating that they have a low risk of mortality as a result of their comorbid diseases.27 the low prevalence of comorbidities at baseline resulted from the exclusion of patients with any diagnoses ( other than depression ) recorded 90 days on either side of the frst pain code . because we selected patients without any comorbid diagnosis to ensure that the unexplained pain was not due to any other medical conditions , the costs in our study may be underestimated .
it is likely that patients with depression and pain with comorbid conditions would have higher medical costs .
first , selection of patients was based on diagnostic read codes and we do not know whether the episode of depression identified by the diagnostic code was related to the pain identified by the pain code .
previous research has indicated that the relationship between pain and depression is bidirectional,11 but our study can provide no information on cause and effect relationships .
second , the dates of diagnosis of pain and depression were based on the first recorded diagnostic codes for pps and depression during the 5-year study period .
although the validity of the medical diagnoses in the gprd has been confirmed,20,22 we did find considerable antidepressant use in the 12 months prior to depression diagnosis which tends to blur the diagnostic picture . this may account for the smaller than expected differences within groups over the time periods ( especially pre vs post1 ) .
however , the statistically significant difference in health care costs between the groups at each time period remains a robust fnding .
moreover , a register - based study in sweden found that almost 30% of patients with a depression diagnosis were treated with antidepressants during the year before diagnosis.46 there may be considerable variation among gps in the specific diagnostic codes used for recording depression and in the date of entry of a diagnostic code .
thus , we used a large number of diagnostic read codes for depression , covering a wide range of depression disorders .
this lack of standardization of diagnostic codes can cause problems of interpretation between comparison groups , but the sample size within each group is sufficiently large to be valid .
we were restricted to capturing the record of depression diagnosis rather than when the depression episode occurred in relation to the pain symptoms .
in addition , severity of depression was not reliably available in the gprd and , therefore , the cut - off for differentiating between a longer and shorter time to depression diagnosis was chosen as 1 year .
another cut - off time period may have produced different results . the 1-year cut - off
was not based on any standardized reference as information on average time from presentation of pps to depression diagnosis is not available .
the cut - off time was based on the median time to depression diagnosis within our data and on clinical opinion that the cut - off should be long enough for pps unrelated to depression to resolve spontaneously , such that we would expect unrelated pps to resolve within less than 1 year , whereas pps lasting for more than 1 year would be a more chronic condition and have implications for nhs health care costs . additionally , using the 1-year cut - off allowed groups of approximately equal sizes .
third , pain severity was classified indirectly , based on the number of pain relief prescriptions , and did not take into account the use of over - the - counter ( otc ) drugs .
therefore , patients who exclusively used otc pain relief were excluded from the study ; such patients are likely to have milder pain .
patients in the milder pain group may have used more than three pain relief medications due to the possible use of otc drugs in addition to prescribed pain medication .
patients with more severe pain are less likely to use otc drugs and we consider that patients with at least three pain relief prescriptions can be regarded as having more severe pain . excluding those with otc drug use / milder pain from the mild pain groups has potentially increased mean resource use , strengthening the between - group differences identified .
moreover , patients in the more severe pain group were required to be prescribed at least one opioid analgesic .
it is important to note that the data refects the prescription of medication and not the dispensing or taking of the medication .
fourth , it is possible that those with unexplained pain may have received a diagnosis outside of the 90 days on either side of the pain symptom record to account for their pain .
this is a more conservative approach than has been used previously ( 30 days on either side of the pain symptom)16 to limit misclassification and results in a relatively small sample size which does not refect the prevalence of pps in patients receiving a depression diagnosis . by eliminating patients with comorbidities in this way in order to have confidence that the recorded pain was not related to an explainable cause
, we may have eliminated patients with more severe or more chronic depression , thus underestimating costs .
more accurate data collection would be achieved if there was a diagnostic code that is uniformly used to record unexplained pain .
finally , we analyzed only direct medical costs for the uk nhs and did not consider other costs ( eg , costs of otc drugs ) , or indirect costs , such as costs due to lost productivity , which are a major component of the total cost of depression.5
our results show that uk primary care patients with more severe pre - existing unexplained pain and a subsequent diagnosis of depression have greater health care service costs , especially drug costs , than patients with milder pain .
pain ( particularly more severe pain ) in combination with longer time from pain symptoms to depression diagnosis contribute to higher overall costs for the uk health care system compared to a shorter time to depression diagnosis .
our findings suggest that earlier diagnosis of depression in primary care patients presenting with unexplained pain may lead to health care cost savings . | purposeto assess the impact of pain severity and time to diagnosis of depression on health care costs for primary care patients with pre - existing unexplained pain symptoms who subsequently received a diagnosis of depression.patients and methodsthis retrospective cohort study analyzed 4000 adults with unexplained pain ( defined as painful physical symptoms [ pps ] without any probable organic cause ) and a subsequent diagnosis of depression , identified from the uk general practice research database using diagnostic codes .
patients were categorized into four groups based on pain severity ( milder or more severe ; based on number of pain - relief medications and use of opioids ) and time to diagnosis of depression ( 1 year or>1 year from pps index date ) .
annual health care costs were calculated ( 2009 values ) and included general practitioner ( gp ) consultations , secondary care referrals , and prescriptions for pain - relief medications for the 12 months before depression diagnosis and in the subsequent 2 years .
multivariate models of cost included time period as a main independent variable , and adjusted for age , gender , and comorbidities.resultstotal annual health care costs before and after depression diagnosis for the four patient groups were higher for the groups with more severe pain ( 819988 versus 565628 ; p < 0.001 for all pairwise comparisons ) and highest for the group with more severe pain and longer time to depression diagnosis in the subsequent 2 years ( p < 0.05 ) .
total gp costs were highest in the group with more severe pain and longer time to depression diagnosis both before and after depression diagnosis ( p < 0.05 ) . in the second year
following depression diagnosis , this group also had the highest secondary care referral costs ( p < 0.01 ) .
the highest drug costs were in the groups with more severe pain ( p < 0.001 ) , although costs within each group were similar before and after depression diagnosis.conclusionamong patients with unexplained pain symptoms , significant pain in combination with longer time from pain symptoms to depression diagnosis contribute to higher costs for the uk health care system . |
on intraoral examination , there was protrusion of both upper and lower anterior teeth with spacing .
the dental history revealed that the spacing is present from the past 3 years [ figure 1 ] .
different treatment options were presented to the patient . because of the large - sized space and completely flared upper and lower anterior teeth , the patient was informed of the guarded prognosis of these teeth [ figure 2a c ] .
he was thus offered the option of extracting the upper incisors and for lower central incisors root canal treatment .
the patient was offered a smile makeover through computer - aided design ( cad ) computer - aided manufacturing ( cam ) ceramic restoration .
a : intraoral frontal b : intraoral right lateral c : intraoral left lateral in order to address the patient 's chief esthetic concerns , the plan included the following elements : development of a diagnostic mock - up on the study casts to evaluate proper tooth morphology and tooth length for better esthetics and proper gingival contours [ figure 3 ] .
this was presented to the patient to assist in determining the course of treatment . as an initial step , tooth no .
teeth preparation was done in relation to 12 , 13 , 22 , 23 , 31 , 32 , 41 and 42 for all ceramic restoration [ figure 4 ] .
after preparation impressions was made using elastomeric impression material with two stage double mix technique for both upper and lower arch [ figure 5 ] .
provisional restorations were fabricated with a tooth colored auto polymerizing acrylic resin and cemented with noneugenol temporary cement [ figure 6 ] .
then dies were made from final impression and dies of prepared crowns were scanned in scanner ceron - eye : dentsply ( australia ) pvt ltd the dimensions and shape of zirconia copings on three dimensional images of maxillary lateral incisors and canines , mandibular central incisors and lateral incisors were modified and finalized with the help of cercon - art .
the saved data was then sent to the milling machine ( cercon - brain ) : aidite high - technical ceramics co. ltd for fabrication of copings for prepared teeth .
final impressions maxillary and mandibular after 2 days , temporary crowns were removed then all - ceramic coping trial was done ; and after 1-week , temporary crowns were removed and internal fit of all - ceramic restorations was evaluated intraorally with a disclosing agent and occlusion was assessed in intercuspal and excursion position .
the crowns were cemented with self - etch unicem luting cement [ figure 7 ] .
patient was satisfied with the crown length , width and esthetics which was improved and equal to adjacent natural teeth [ figure 8 ] .
different treatment options were presented to the patient . because of the large - sized space and completely flared upper and lower anterior teeth , the patient was informed of the guarded prognosis of these teeth [ figure 2a c ] .
he was thus offered the option of extracting the upper incisors and for lower central incisors root canal treatment .
the patient was offered a smile makeover through computer - aided design ( cad ) computer - aided manufacturing ( cam ) ceramic restoration .
a : intraoral frontal b : intraoral right lateral c : intraoral left lateral in order to address the patient 's chief esthetic concerns , the plan included the following elements : development of a diagnostic mock - up on the study casts to evaluate proper tooth morphology and tooth length for better esthetics and proper gingival contours [ figure 3 ] .
teeth preparation was done in relation to 12 , 13 , 22 , 23 , 31 , 32 , 41 and 42 for all ceramic restoration [ figure 4 ] .
after preparation impressions was made using elastomeric impression material with two stage double mix technique for both upper and lower arch [ figure 5 ] .
provisional restorations were fabricated with a tooth colored auto polymerizing acrylic resin and cemented with noneugenol temporary cement [ figure 6 ] .
then dies were made from final impression and dies of prepared crowns were scanned in scanner ceron - eye : dentsply ( australia ) pvt ltd the dimensions and shape of zirconia copings on three dimensional images of maxillary lateral incisors and canines , mandibular central incisors and lateral incisors were modified and finalized with the help of cercon - art .
the saved data was then sent to the milling machine ( cercon - brain ) : aidite high - technical ceramics co. ltd for fabrication of copings for prepared teeth .
final impressions maxillary and mandibular after 2 days , temporary crowns were removed then all - ceramic coping trial was done ; and after 1-week , temporary crowns were removed and internal fit of all - ceramic restorations was evaluated intraorally with a disclosing agent and occlusion was assessed in intercuspal and excursion position . the crowns were cemented with self - etch unicem luting cement [ figure 7 ] .
patient was satisfied with the crown length , width and esthetics which was improved and equal to adjacent natural teeth [ figure 8 ] .
heffernan et al . suggested that zirconia restoration would be better suited to match opaque , high - value teeth . in this study ,
the translucency of several all - ceramic systems was compared under transmitted light , and the zirconia system was found to be as opaque as the metal ceramic .
one of them is dcs smart - fit is a cad / cam system that utilizes fully sintered yttrium - tetragonal zirconia polycrystals ( y - tzp ) under the hot isotonic pressure .
this result in an extremely hard and dense ceramic that require mill time of around 24 h for coping .
in contrast dens system crown employ partially sintered y - tzp milling blocks that are weak but easy to mill .
usa ) employs a glass - infiltrated 35% zirconia 65% alumina material for fabrication of posterior crown and fixed dental prostheses that can be slip cast or milled from presintered blocks .
its flexural strength is about 620 mpa , which is lower than that reported for densely sintered zirconia .
it is brought about by a feeling of well - being , security and confidence in one 's self .
the dentist should render the patient , the confidence that none of these fine senses will be in jeopardy .
the development of a pleasing oral and facial expression for the patient depends upon the dentist ability to replace the missing teeth , both in contour and color especially for the anterior teeth . | the demand for the dentist to achieve excellence in esthetics and function has driven modern advances in materials and restoration fabrication .
the development of various casting alloys and precise casting systems has contributed to the successful use of metal - based restorations . however , patient requests for more aesthetic and biologically safe materials that have led to an increased demand for metal - free restorations .
the following case presentation illustrates a successful aesthetic and functional application of this exciting computer - aided design / computer - aided manufacturing - digital zirconia - based system for a natural smile . |
we analyzed the dynamics of cumulative severe acute respiratory syndrome ( sars ) cases in singapore , hong kong , and beijing using the richards model .
the predicted total sars incidence was close to the actual number of cases ; the predicted cessation date was close to the lower limit of the 95% confidence interval . | |
a dietary supplement is a product , which contains a dietary ingredient intended to supplement the diet . in general , the natural dietary supplements for male sexual potency consist of different herbal extracts such as ginseng root , lychee seed , barbary wolfberry fruit , longan aril , and aweto .
these dietary supplements could improve male sexual potency without causing danger , even when overdose occurred .
recently , chemically synthetic phosphodiesterase-5 ( pde-5 ) inhibitors have been used to improve erectile dysfunction ( ed ) , one of the most popularly used is vardenafil hydrochloride ( levitra ) , 2-[2-ethoxy-5-(4-ethyl - piperazine-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3h - imidazo[5,1-f]-triazin-4-one monohydrochloride , trihydrate ( figure 1 ) .
vardenafil , approved by the united states food and drug administration ( fda ) in 2003 , is a new oral , potent , highly selective phosphodiesterase-5 ( pde5 ) inhibitor marketed for the improvement of ed in man .
studies have shown that doses of vardenafil ( 1040 mg ) are rapidly absorbed following oral administration , reaching maximum plasma concentration in some men within 15 minutes .
nevertheless , it has been proved that vardenafil poses a serious health risk , such as headache , low blood pressure , flushing , dyspepsia , and nasal congestion or rhinitis .
thus , vardenafil could be legally obtained only with a doctor 's prescription . however , some illegal dealers add vardenafil into dietary supplement products for the sake of profit . for purposes of quality control and health safety , establishing sensitive and selective methods to detect illegal vardenafil addition in dietary supplements are necessary . as shown in the literatures ,
several methods have been reported for vardenafil determination in dietary supplements such as lc - ms and gc - ms method , lc - esi - ms method [ 1 , 4 ] , lc - esi - ms / ms method , mrm - esi - ms - ms method , and fticrms method .
ms provides sensitive and reliable results , but it also implies highly sophisticated apparatus , careful control of conditions and relatively high operation cost .
thus , simpler , automated and less expensive methods are still necessary . taking the advantages of perfect versatility and easy fluidic manipulation , flow injection ( fi )
the integration of fi and high - performance liquid chromatography ( hplc ) has also showed the power for the complex samples analysis .
cl - based technique as a means of detection for hplc aroused the analysts interest in recent years [ 12 , 13 ] , although cl detection is not as universal as fluorescence detection or ultraviolet visible detection , it is more selective for fewer interfering species observed in the chromatogram and sensitive for a large cl signal - to - noise ratio ( s / n ) and wide linear working range .
the combination of hplc high resolution , cl sensitive detection and fi manipulation would be one of the plausible techniques for a sensitive , precise , and facile determination of trace levels of compounds in the complex mixture of substances .
recent studies witness rapid progress in hplc - cl method , which is widely used to perform diverse qualitative and quantitative analytical problems [ 1518 ] .
our experiments indicated that in alkaline medium the cl of luminal - k3fe(cn)6 could be greatly enhanced by the presence of trace vardenafil .
based on above phenomenon , an automated hplc - cl method for vardenafil determination in dietary supplements was developed .
the proposed method exhibited the advantages of automated operation , high analytical throughput , simple instrumentation , wide linear range , and reagent - saving and was applied to oral liquid , wine , and capsule samples analysis .
all the other reagents were of analytical reagent grade unless specified otherwise , and deionized and doubly distilled water was used throughout .
vardenafil hydrochloride trihydrate ( purity , 99.6% ) was purchased from qiyu biology co. , ltd .
other reagents including k3fe(cn)6 , h3po4 , ethylenediaminetetraacetic acid disodium salt ( na2edta ) , and naoh were supplied by xi'an chemical reagent factory ( xi'an , china ) .
mol / l ) was prepared by ethanol and stored at 20c in a refrigerator .
standard series from 8.0 10 ~ 1.0 10 mol / l were prepared daily by sequentially diluting stock solution with mobile phase solution .
a 1.0 10 mol / l k3fe(cn)6 stock solution was prepared by dissolving 164.6 mg of k3fe(cn)6 in 50 ml water , and it was protected against light and stored at 4c in a refrigerator . a 1.0 10 mol / l stock solution of luminol was prepared in 0.1 mol / l naoh solution .
the mobile phase consisting of ethanol and an aqueous solution containing 1.0 10 mol / l h3po4 and 2.0 10 mol / l na2edta ( 25 : 75 , v / v% ) was prepared .
mobile phase was filtered through 0.45 m millipore membrane of polytetrafluoroethylene ( ptfe ) and further degassed in an ultrasonic bath before use .
health food samples including one herbal oral liquid , one herbal medicated wine , and one herbal capsule claimed being capable of enhancing sexual performance for men that were under suspicion of adulteration , were purchased from local drug store and market .
a portion of oral liquid and wine sample were , respectively , 10-fold diluted with mobile phase solution , filtered through a 0.45 m ptfe membrane filter ( shanghai xinya purifier devices factory , shanghai , china ) and finally degassed prior to use . for capsule sample preparation ,
one capsule weight powder was transferred to a 50 ml beaker , and vortically extracted with 10 ml mobile phase solution for 1 h. a portion of the extraction solution was filtered through a 0.45 m ptfe membrane filter and diluted with mobile phase solution for further analysis . as illustrated in figure 2 , the hplc - cl experimental setup consisted of a cl analyzer ( xi'an remex analyse instrument co. , ltd , xi'an , china ) equipped with a hamamatsu r456 pmt ( tokyo , japan ) and a negative high voltage ( nhv ) generator , a planar coiled transparent ptfe flow cell , a 1.8 ml / min constant flow rate cl pump ( shanghai instrument electric motor factory , shanghai , china ) , a lc-10at liquid chromatography ( shimadzu , tokyo , japan ) equipped with a rheodyne 7725i syringe - loading sample valve ( 20 l - loop , cotati , ca , usa ) , a waters c18 column ( i.d . : 4.6 mm 150 mm , particle size : 5 m , usa ) and a dt-230a column oven ( tianjin do - chrom technology co. , ltd , tianjin , china ) .
stream a containing 1.0 10 mol / l luminol , 2.0 10 mol / l na2edta , and 1.0 mol / l naoh and stream b containing 1.0 10 mol / l k3fe(cn)6 delivered by cl pump merged in the t1 .
then , the reagent mixture merged with mobile phase in the t2 and generated a stable cl baseline . subsequently , when the sample valve was switched to the injection position , 20 l standard or sample was injected into the mobile phase .
outflow vardenafil reacted with the reagent mixture and generated the enhanced cl signal in the flow cell .
the cl signal was recorded as a function of time and cl intensity by the remex analyzer with the pmt operated at 400 v. a microcomputer equipped with remex software running under windows xp was employed for data treatment .
vardenafil peak identification was carried out by the standard addition method and the retention time ( tr ) of vardenafil .
the peak height , i ( i = is i0 , where i0 was the cl of baseline , is was the peak cl of vardenafil ) was used to quantify vardenafil content by means of the related calibration equation .
for vardenafil cl determination , we considered several known cl reactions including alkaline luminol - h2o2 , alkaline luminol - k3fe(cn)6 , alkaline luminol - kio4 , acidic kmno4 , acidic kmno4-hcho , acidic kmno4-na2so4 , and naclo - naoh . to prevent the possible interference from metal ions , 2.0 10 mol / l na2edta was used in all luminol concerned reactions .
experimental results indicated that alkaline luminol - k3fe(cn)6 reaction contributed the strongest cl response to 1.0 10 mol / l vardenafil presence .
the kinetic curve of the alkaline luminol - k3fe(cn)6-vardenafil cl reaction was studied with a static injection method .
alkaline luminal and k3fe(cn)6 solution were mixed in the beaker placed in front of the pmt window . after a stable baseline
was obtained , 1.0 ml 1.0 10 mol / l vardenafil standard was injected into the beaker by a syringe . the kinetic curve recorded as a function of time and cl intensity , and it could be concluded that vardenafil greatly enhanced luminol - k3fe(cn)6 cl , which reached a maximum within 1.5 s. thus , the studied cl reaction was a fast reaction . as a result , the t2 was placed as near as possible to the flow cell inside the dark box as shown in figure 2 . to obtain the sensitive and fast vardenafil detection with the described hplc - cl system , a series of experiments were conducted to establish optimum analytical performance .
1.0 10 mol / l vardenafil standard was used in all optimization experiments . both mobile phase and
luminol and k3fe(cn)6 flow rates were controlled at 1.8 ml / min , respectively .
experimental optimum analytical performances were selected based on the biggest cl s / n values and on the average of three injections for each test when the relative standard deviation ( rsd ) of each test point was less than 5.0% , unless specified otherwise .
for hplc - cl analysis , the mobile phase should be , not only suitable for vardenafil separation in complex matrix but also compatible with cl detection . acetonitrile - acidic aqueous buffer [ 1 , 4 , 11 , 19 ] and methanol - acidic aqueous buffer [ 8 , 20 ] were the most commonly used mobile phases for vardenafil separations on c18 column .
when investigating the compatibility of acetonitrile with cl detection , it was found that acetonitrile brought about very high cl background .
similar experiments showed that when methanol and ethanol were used , relatively low cl backgrounds and smooth baselines could be obtained .
thus , methanol and ethanol were selected to constitute the mobile phases for the posterior study .
the effect of organic phase volume percentage ( v / v% , organic phase volume / organic + aqueous phase volume ) on tr , s / n value and column pressure ( p ) , were investigated in the range of 40%60% methanol and 20%30% ethanol , respectively . observing the experimental results listed in table 1 , it could be concluded that the higher the v / v% , the shorter the tr , and 30% ethanol contributed the biggest s / n value .
however , 30% ethanol also caused relatively high p. thereupon , as a compromise among the bigger s / n value , shorter tr and suitable p , 25% ethanol was chosen in subsequent experiments .
a number of organic or inorganic acids were employed to constitute mobile phase to improve vardenafil separation [ 1 , 4 , 8 , 11 , 19 , 20 ] .
considering the possible reaction between organic acid and cl reagents , inorganic acid was considered to be more suitable for cl detection .
experiment indicated that h3po4 could effectively suppress the peak - tailing , and a relatively narrow and symmetric chromatographic peak could be achieved ( figure 3 ) .
what is more , as expected , the tr was shortened . with respect to obtaining the biggest cl s / n value ,
the optimum h3po4 concentration was investigated by varying its concentration in the range of 1.0 10 ~ 1.0 10 mol / l .
experiments showed that s / n value increased with h3po4 concentration increasing . when h3po4 concentration was 1.0 10
mol / l , the ph of mobile phase was about 2.0 , the biggest s / n value was obtained . according to the column supplier 's suggestion , h3po4 concentration higher than 1.0 10 mol /
1.0 10 mol / l h3po4 concentration was employed as the optimum . as could be seen the experimental results summarized in table 2 , by varying the flow rate from 0.3 to 0.9 ml / min , the effect of mobile phase flow rate upon cl detection was investigated .
it was found that the higher the mobile phase flow rate , the shorter the tr and the higher the p.
lastly , a flow rate of 0.8 ml / min was chosen as a compromise between the biggest s / n value and relatively shorter tr .
as was well known , the alkaline medium was necessary for luminal - k3fe(cn)6 reaction . in our experiments ,
the effect of naoh concentration on the cl detection was examined in the range of 0.2~1.3 mol / l .
experiments showed that the s / n value increased with the naoh concentration increasing , and a leveling off could be found when the naoh concentration was higher than 1.0
the effect of luminol concentration on cl detection was evaluated in the range of 1.0 10 ~ 1.3 10 mol / l .
it was found that the s / n value increased with luminol concentration increasing and reached maximum at 1.0 10
mol / l , whereas the s / n values decreased with further luminol concentration increasing .
consequently , 1.0 10 mol / l luminol concentration was selected . as an oxidant of cl reaction , k3fe(cn)6 affected the cl signal directly .
the effect of k3fe(cn)6 concentration on cl detection was assessed in the range of 1.0 10 ~ 3.0 10 mol / l .
it was found that the s / n value increased with k3fe(cn)6 concentration increasing in the range of 1.0 10 ~ 1.0 10 mol / l .
based on above results , 1.0 10 mol / l k3fe(cn)6 was selected .
a series of standard solutions ( 1.0 10 ~ 3.0 10 mol / l ) was used to determine the analytical figures of merit under the optimum conditions .
experiments showed that i to vardenafil concentration ( c ) was linear in the range of 8.0 10 ~ 1.0 10 mol / l with the regression equation of i = 3.17c + 9.91 ( c : 10 mol / l , n = 5 , r = 0.9980 ) and the detection limit was 5 10 mol / l ( 3 ) .
the reproducibility was investigated by injecting 2.0 10 mol / l vardenafil standard for 11 times , which was demonstrated by the rsd .
intraday and inter - day precisions of the described method were examined using the oral liquid sample spiked with 2.0 10 mol / l standard .
the intraday precision , defined as the rsd of 5 times determinations to the same sample within one day , five injections one time , was less than 3.5% .
the inter - day precision , defined as the rsd of five days determinations to the same sample , 5 injections one day , was less than 2.3% .
it was concluded that the described hplc - cl method presented relatively good precision . following the procedures detailed in the experimental section ,
the proposed hplc - cl method was applied to determine vardenafil in oral liquid , wine , and capsule samples . the original and vardenafil standard spiked chromatograms of oral liquid ,
it could be seen from figure 4 that vardenafil was not detected in all three samples and there was no interfering from the endogenous substances at the tr of vardenafil .
it was noticed that the tr of vardenafil was 6.4 min , which was much shorter than the tr reported in relative literatures [ 1 , 4 , 8 , 9 , 11 , 19 ] .
this shorter tr also implied the time and mobile phase saving . to evaluate the applicability of the proposed hplc - cl method for the determination of vardenafil in oral liquid , wine , and capsule , recovery experiments were performed .
these experiments were conducted by spiking 3 levels of vardenafil content into oral liquid and wine and adding identical levels of vardenafil content into capsule as shown in table 3 .
the recovery percentages were estimated by comparing the concentrations detected from spiked samples with the nominal concentrations added . from experimental results summarized in table 3 , it was concluded that the proposed method afforded good precision and accuracy when applied to oral liquid , wine , and capsule samples .
we had carried out the automated hplc - cl method on vardenafil determination in oral liquid , wine , and capsule samples .
based on the obtained experimental results , the following conclusions could be drawn . a new mobile phase that is composed of 25% ethanol and 75% aqueous
was found to be nontoxic , environment friendly , and compatible with the alkaline luminol - k3fe(cn)6 cl detection .
furthermore , the cl detector was cheap , simple and no sophisticated technique required . in a word ,
our proposed method might be adopted as an alternative method for vardenafil determination of in dietary supplement . | a flow method of high - performance liquid chromatography ( hplc ) seperation and chemiluminescence ( cl ) detection for sensitive vardenafil analysis in dietary supplements was developed .
the vardenafil separation was achieved on a c18 column at 30c using ethanol - h3po4 and ethylenediaminetetraacetic acid disodium salt ( na2edta ) aqueous solution ( 25 : 75 , v / v% ) as mobile phase . the followed continuous cl detection was conducted based on the strong cl enhancement by the presence of vardenafil to luminol - k3fe(cn)6 reaction in alkaline medium . at the flow rate of 0.8 ml / min , the vardenafil retention time ( tr ) was 6.4 min .
factors that affected the hplc resolution and cl detection were studied and optimized .
the calibration curve obtained for vardenafil standard was linear in concentration range of 8.0 107 ~
1.0 104 mol / l .
the relative standard deviations ( rsd ) of intraday and interday precision were less than 3.5% .
the proposed method was applied to the vardenafil determination in oral liquid , wine , and capsule samples . |
measurement and quantification
of protein protein interactions
at low and high protein concentrations as a function of solution conditions
is important for understanding biological processes , as well as the development and optimization of biotechnology products . in concentrated protein solutions
, intermolecular interactions may
lead to concerns regarding opalescence , solubility , aggregation , viscosity ,
and phase separation , which pose a challenge for product formulation .
similarly , in physiological
conditions where protein concentrations can reach as high as 300500
protein
interactions and nonidealities influence biochemical reactions and transport properties and are related to
a number of diseases and disorders involving phase separation and
aggregation .
experimental techniques such as surface plasmon
resonance , fluorescence resonance energy
transfer , and affinity purification mass
spectrometry have traditionally been
used to study strong ,
specific lock - and - key protein
however , these techniques are limited to relatively
low protein concentrations and do not reflect the full range of interprotein
interactions that can occur both in vivo and in vitro . in that regard ,
high - concentration protein interactions include not only specific
interactions ( e.g. , protein protein binding ) but also weak
long- and short - range nonspecific interactions resulting from changes
in solution conditions ( e.g. , ph , ionic strength , or protein concentration ) ,
as well as amino acid composition and localized surface features .
such weak interactions at high protein concentrations have been measured
for a number of different proteins via techniques such as osmometry , sedimentation equilibrium , viscometry , nmr methods , and small - angle scattering . among these
experimental methods , scattering techniques including static light
scattering ( sls ) , dynamic light scattering ( dls ) , and small - angle
x
- ray and neutron scattering ( saxs and sans , respectively ) are potentially
powerful and versatile tools to study protein protein interactions
and their influence on the thermodynamics , kinetics , and the structure
of proteins in solution over a wide range of protein concentrations . two main approaches
protein
interactions from scattering methods , and other experimental techniques ,
to the behavior of proteins in solution : ( i ) inferring high - concentration
behavior from protein protein interactions measured at low
concentrations and ( ii ) the use of simplified models for protein
interactions , so as to regress model parameters from scattering data
at high concentrations .
protein interactions at dilute conditions are frequently characterized
via the osmotic second virial coefficient b22 ( measured from sls , osmometry , chromatography , and sedimentation equilibrium ) or the interaction
parameter kd ( obtained from dls ) .
b22 and other virial coefficients
play central roles in both qualitative and quantitative models and
theories relating colloidal protein
protein interactions to
protein crystallization and fluid fluid phase behavior , as well as protein aggregation .
similarly , kd has been used as a phenomenological predictor
of viscosity and protein stability for high - concentration protein
solutions . however , from a biophysical standpoint , the use of b22 or kd is anticipated to
be limited from the perspective of being generally or globally predictive
of high - concentration behavior .
this follows because the qualitative
and quantitative behavior of high - concentration solutions or suspensions
can be dramatically different from that observed at dilute conditions ,
and crowding effects and other thermodynamic nonidealities that are
prominent in concentrated systems can significatively alter the net
interactions when averaged over many neighboring particles . when considering data obtained
directly at high concentrations ,
several approximations have been used to interpret experimental colloidal
interactions and thermodynamic behavior , ranging from considering
higher - order virial expansions to using simplified
but analytical models for the functional form of the potential of
mean force ( pmf ) .
the use of such approaches is limited by model approximations and/or
questions of statistical validity of expansions or models that involve
a large number of parameters . quantifying interactions via small - angle
scattering often relies on regression of models for protein
protein
interactions and is limited to the availability of suitable models
and simple protein geometries to capture the entire set of experimental
conditions ; in addition ,
it can be difficult to directly reach the low - q scattering
limit .
this can result in fitted pmfs that correlate
poorly with changes in protein / cosolute concentration or are restricted
to a limited subset of experimental conditions .
for instance , niebuhr and kotch used
a two - yukawa potential model with attractions and repulsions to successfully
describe low- and high - concentration saxs by lysozyme solutions at
moderately repulsive interacting conditions , but it was found to not
translate well to conditions where attractive interactions were important .
recently , a statistical mechanical description of sls , specifically
rayleigh scattering , was presented that rigorously treats protein
protein
and protein solvent / cosolute interactions in multicomponent
systems based on kirkwood buff ( kb ) solution theory and eliminates
the need to assume an implicit solvent or an underlying model for
these interactions .
although that result
was only applied for low - concentration protein solutions in previous
work , it has the potential to quantify protein interactions for high - concentration
systems where nonidealities are more prominent .
the present work includes
an extension of the earlier work to experimental sls data and analysis
to quantify intermolecular interactions spanning from low to high
protein concentrations , as well as comparing quantitative and qualitative
differences between what one obtains from different scattering methods
and approaches used to characterize these interactions .
specifically ,
sls , dls , and sans were used to determine changes in protein
protein
interactions for bovine -chymotrypsinogen a ( acgn ) at acidic
conditions over a range of protein concentrations and solution ionic
strength .
acgn is a natively monomeric , globular protein of
25.7 kda molecular
weight and approximately 4 nm in diameter .
the conformational stability and aggregation
behavior of this protein have been extensively characterized at acidic
ph , where it forms amyloid polymers upon unfolding at elevated temperatures
but remains as a monomer at 25 c .
previous studies have shown that acgn forms soluble aggregates at
acidic conditions ( ph < 4 ) and at low to moderate ionic strengths
( 10100 mm ) , but the aggregation pathway shifts with increased
ionic strength or ph .
empirically correlated the
behavior of these aggregates with b22 ,
suggesting that the dominant aggregation pathway depended on the magnitude
of repulsive and attractive colloidal interactions .
furthermore , it
was shown that at acidic ph and low to moderate salt concentrations , b22 ranges from strongly repulsive to mildly
attractive behavior .
for parity with prior work characterization of concentration - dependent protein interactions
provided in this report focused on acgn solutions at ph = 3.5 and
a temperature of 25 c , as well as salt concentrations below
100 mm , in order to ensure that the protein remains stable as a monomer
while allowing a reasonably wide range of ionic strength conditions
and protein concentrations to be tested .
the remainder of the
article is organized as follows . in the materials
and methods section , the procedures and
protocols used for preparing the different acgn solutions for light
scattering and sans
protein
interactions as a function of protein concentration from these experimental
techniques , including a brief description of a new analysis based
on a local taylor series approach that quantifies concentration - dependent
protein protein interactions from sls data without the need
to assume a pmf model .
the interactions are quantified in terms of
the kb integral ( g22 ) that is the analogue
of b22 , except that g22 accounts for interactions between multiple proteins
simultaneously .
g22 is concentration - dependent ,
while b22 holds for only dilute protein
concentrations .
the supporting information provides additional details regarding the implementations of these
approaches , as well as a statistical analysis of the intrinsic error
from applying the local taylor series .
the results section first presents the sls behavior of acgn as a function of
nacl concentration at low protein concentration , where b22 is expected to be a reasonable descriptor of protein
the sls behavior is then considered as the protein concentration
( c2 ) is increased beyond the dilute regime ,
where different analysis techniques are illustrated for the sls data ,
and the results are compared with those from sans experiments at selected
nacl concentrations and c2 values .
it
is shown that g22 , rather than b22 , is the more appropriate descriptor of protein
the results section finishes with a comparison
of acgn interactions probed by dls and sls , from low to high / intermediate c2 for the same series of nacl concentrations .
the results from all of these techniques are then considered together
in the discussion section , which highlights
strengths and weaknesses of the different experimental techniques
and analysis methods for quantifying protein protein interactions .
in all cases , the results indicate that the interactions between acgn
molecules are dominated by electrostatic repulsions that may be short-
or long - ranged , and the nonidealities that result from such strong
interactions are not well captured by available , simple pmf models
if one considers more than small ranges of protein and nacl concentrations .
the connections between g22 , concentration - dependent
protein interactions , local molecular fluctuations , and thermodynamics
of protein solutions are then briefly reviewed in the context of the
results for acgn .
finally , the discussion section
also highlights the difficulties in quantitative interpretation of
protein protein interactions from dls measurements when one
considers higher c2 values than the dilute
regime .
the 10 mm sodium
citrate buffer
stock solutions for ls measurements were prepared by dissolving anhydrous
citric acid ( merck kgaa , darmstadt , germany ; acs grade ) in millipore
superq water and titrating to ph 3.5 with 1 m sodium hydroxide solution
( merck kgaa ) . in the case of buffer stock solutions for sans , citric
acid was dissolved in d2o ( sigma - aldrich ) and titrated
with a 1 m sodium hydroxide - d2o solution to ph 3.1 in order
to account for the 0.4 units of difference between ph and pd .
stock salt solutions at 0.01 , 0.05 , and 0.1
m of nacl ( acs grade ; merck kgaa ) were prepared by the same procedure
as the citrate buffers except with the gravimetric addition of the
respective salt prior to dissolution and ph adjustment .
all buffer
solutions were stored at 28 c and used within 1 week
of preparation .
solutions of acgn were prepared gravimetrically from
5 crystallized lyophilized acgn ( worthington biochemical corp .
lakewood , nj ) dissolved in aliquots of buffer stock solutions to yield
a protein concentration of 20.0
samples for light scattering measurements were
4 dialyzed using 10 kda molecular mass cutoff spectra / por 7
dialysis tubing ( spectrum , the netherlands ) against the same citrate
buffer stock to eliminate residual salt impurities in the protein
powder .
buffer exchange and the concentration
of samples ( as needed ) for sans samples was done via membrane centrifugation .
each centrifugation step was carried out for 10 min at 14000 g and 25 c with a 10 kda cutoff filter unit ( amicon
ultra-10 , millipore ) .
after dialysis / buffer exchange , all of the acgn
solutions were concentrated by centrifugation at 12000 g and 25 c with a molecular weight cutoff of 10 kda ( amicon
ultra-10 ) to yield a final protein stock solution at a concentration
of at least 40.0
protein samples were prepared by diluting
stock protein solutions in the remaining buffer after the last dialysis
step to obtain protein concentrations ranging from 1.0 to 40.0
solutions for light scattering were further filtered directly into
a given cuvette through 0.2 m millex - lg syringe filters . in
all ls experiments ,
the lack of both dust and residual aggregates
was checked by preliminary dls measurements .
protein concentrations
were determined by absorbance at 280 nm using an extinction coefficient
of 2.0 l/(g cm ) .
sls and dls measurements were
performed by using a brookhaven bi200-sm goniometer equipped with
with either a he ne laser ( = 632.8 nm ) or a solid - state
laser ( = 532 nm ) .
the temperature of the cell compartment
was controlled within 0.05 c using a thermostated recirculating
bath . the scattered light intensity and its time autocorrelation function
absolute values of scattered intensity ( rayleigh ratio r90 ) were obtained by normalization with respect
to toluene via1where k is an optical constant
and is given by k = 4nna1. na is avogadro s number , and ntol ( = 1.4910 and 1.4996 at = 632.8 or 532 nm ,
respectively ) and n (= 1.333 ) denote the refractive
index of toluene and the solution .
dn / dc ( = 0.192 l / g ) is the differential index of refraction for the sample
and effectively independent of salt concentration.r90tol is the rayleigh ratio of toluene and at 632.8 and 532 nm
was taken as 14.0 10 and 28.0 10 cm , respectively.i , i0 , and itol denote the intensity of the sample , buffer ,
and toluene , respectively .
intensities in sls were obtained by time - averaging
the collected intensities over a time window of 3 min for a given
sample .
at least three replicates of each salt and protein concentration
were measured to reduce statistical uncertainties in the resulting r90 values .
additional sls measurements at angles
different than 90 were performed over selected samples , with
no angular dependence observed in the resulting rayleigh ratios .
sls data were fit against the classical expression for ls analysis to obtain information about the osmotic second virial coefficient b22 via2where mw is the
apparent molecular weight of the protein and c2 is the protein concentration . fitting was performed only
at the low - concentration regime to ensure the accuracy of the fitted
parameters .
b22 is formally related to
protein protein interactions in the limit of low protein concentration ,
averaged over the spatial degrees of freedom of the solvent and any
cosolute or cosolvent species , that is , the pmf w22 in a grand - canonical ensemble via3where kb is the
boltzmann constant , t is the absolute temperature ,
and r denotes the distance between centers - of - mass
of two proteins .
additionally , the kb model for ls was
also used to regress r90/k versus c2 data .
protein kb integral ( g22 ) as a function of protein concentration by applying
a local taylor series approach over small concentration windows , whereby4 formally , g22 is related to the orientation - averaged
protein
notably , g22 depends on the number distribution of proteins and
fluctuations in the scattering volume and is mediated by factors such
as protein and cosolute concentrations as well as protein
thus , the value of g22 is
sensitive to the same factors as b22 ,
but it can change with c2 and can provide
valuable information about thermodynamic nonidealities beyond dilute
colloidal protein protein interactions , such as molecular crowding
and other net attractive or repulsive interactions involving multiple
proteins simultaneously . in the limit of c2
0 , g22(r ) in eq 5 can be
replaced with the boltzmann factor of w22 in the dilute limit , and thus , g22 =
2b22 in the limit of dilute protein
concentrations . because of the sign difference between how b22 and g22 are defined
mathematically ( cf .
eqs 3 and 5 ) , negative g22 values ( positive b22 values ) indicate repulsive conditions and
vice versa . equation 5 also provides a familiar
form for the zero - q limit for a pseudo - one - component
system in sas as 1 + c2g22 is equivalent to the
zero - q static structure factor ( s(q 0 ) ) in a grand - canonical ensemble . in the local taylor series approach ,
one considers a series of
small windows of c2 and
only locally fits g22 for a given c2 window , such that g22 is effectively constant in that c2 range .
thus , one recovers g22 as a function of
protein concentration without a need to assume the functional form
for g22 or intermolecular interactions .
details about the implementation of eq 4 via
the local taylor series approach , as well as a statistical analysis
of the intrinsic error from applying this method are provided in the supporting information . for statistical
reasons and given that r90/k data were collected in two - fold increments of
protein concentration , the regression was performed in a base-2 logarithmic
scale for the independent variable ( i.e. , protein concentration ) in
order to have evenly spaced concentration points during fitting .
furthermore ,
given that a weak concentration dependence of mw is expected and there was no
evidence of aggregate formation ( see also below ) , its value was held
fixed and assumed to be equal to the value obtained from fitting to
eq 2 for each series of concentration windows .
thus , fits to sls data were performed over small concentration windows ,
where the size of the concentration window was selected based on the
local number of data points , provided that the range of concentrations
used for fitting did not exceed 10
although different
window sizes were tested ranging from 3 to 7 data points , no differences
were observed between the resulting fitted g22 values within the statistical uncertainty ( data not shown ) .
therefore , the g22 values shown in the
figure correspond to those that provide the smallest confidence intervals .
the supporting information provides a more
detailed description regarding the selection of the size for the concentration
window , as well as the uncertainties associated with the use of the
local taylor series approach to obtain g22 . for dls measurements , the
measured intensity autocorrelation function g(t )
was analyzed via the method of cumulants.g(t ) was nonlinearly regressed against6where is
an average baseline intensity ,
is the amplitude of g(t ) at t 0 and is an instrument
constant , and q is the magnitude of the scattering
vector , with q = 4n sin(/2)/
and = 90. dc is the average
collective diffusion coefficient and in sufficiently dilute conditions
is related to the average hydrodynamic radius rh of the protein via the stokes
einstein equation ( i.e. , dc = kbt/(6rh ) , where kb is the boltzmann constant , t is the
temperature , and is the viscosity of the solvent ) .
as expressed
in eq 6 , dc represents
the first cumulant of the underlying distribution of diffusive decay
times , and corresponds to the second cumulant ( i.e. , the second
moment around the average ) for the same underlying distribution .
these
two quantities can be related to the reduced second moment or polydispersity
index ( p2 ) , defined as7where p2 is a
dimensionless parameter that gives an experimental measure of the
width of the underlying distribution of decay times . in the limit of negligible interactions between proteins ,
einstein equation to
the distribution of hydrodynamic radii if the system is not greatly
polydisperse .
dls measurement of intermolecular interactions
often relies on a series expansion in terms of protein concentration
of the collective ( or mutual ) diffusion coefficient ( dc ) , in which the first - order term of this expansion is
related to protein protein interactions . that is8where d0 = kbt/(3 )
is the diffusion coefficient at infinite dilution , is the
protein diameter , and kb and are
defined above .
kd is the slope on a dc versus c2 curve
as c2 approaches zero and corresponds
to the so - called interaction parameter .
qualitatively , positive ( negative )
values of kd are taken to indicate net
repulsive ( attractive ) protein interactions .
however , given the nature of eq 8 , the use
of kd is limited to dilute protein conditions
and , in that regard , is analogous to b22 . in order to characterize protein interaction away from the
dilute
regime
, one needs to realize that dc is
the result of two different effects , thermodynamic or so - called direct
protein protein interactions and indirect hydrodynamic
interactions .
the latter refers to the forces that a protein feels
via the time - dependent response of the fluid between proteins and
due to the motion of the other proteins and solvent / cosolute molecules
in solution ( i.e. , the flow field ) .
this leads to expressing dc in the low - q limit as9where h(q 0 ) is the hydrodynamic factor
and ds ( = d0h(q 0 ) ) is the self - diffusion
coefficient .
the rightmost
expression in eq 9 comes from eq 4 and recalling s(q
0 ) = 1 + c2g22 . while the structure factor provides information about direct protein
protein
interactions , the hydrodynamic factor captures the nonequilibrium
or transport effects ( e.g. , fluid dynamics for a primarily incompressible
solvent ) . thus , by measuring s(q ) or r90 and combining it with dc , one can quantify the effects of both thermodynamic
nonidealities ( s(q 0 )
1 ) and hydrodynamic forces ( h(q
0 ) 1 ) on the dynamic and thermodynamic behavior of proteins
in solution as one increases protein concentration . physically ,
h(q 0 ) 1 ( or ds d0 ) ,
where larger deviations from 1 ( or d0 )
indicates stronger hydrodynamic interactions .
notably , if one expands h(q
0 ) and 1/s(q 0 ) in their
corresponding taylor series with respect to protein concentration
and takes the limit as c2 0 ,
eq 9 yields10where h1 is the
derivative of the hydrodynamic factor with respect to protein concentration
in the limit c2 0 ( = dh(q 0)/dc2 ) .
equation 10 follows because s(q 0 ) = 1 + g22c2 , which in the limit of
dilute conditions equals 12b22c2 .
equation 10 is equivalent to eq 8 and shows the relation
between the interaction parameter kd and b22 .
sans
measurements were
performed with the 30-m ng7 and 10-m ngbi instruments at the national institute of standards and technology at
gaithersburg , md . neutrons with a wavelength of 6 were used ,
and a range of scattering angles was achieved by using three different
sample - to - detector distances ( 1 , 4.5 , and 13 m ) .
titanium cells with
quartz windows and a 5 mm path length were filled following a similar
procedure as that for ls samples .
the resulting protein scattering
profile was normalized by incident beam flux , and the raw intensities
were placed on an absolute scale using direct beam measurements .
the
igor software , specifically the nist module , was also used for data
reduction .
the scattering intensity
from protein solutions , i(q ) , is
proportional to the product of the structure factor s(q ) and the form factor p(q)11where m is the protein molecular
weight ( = 25.7 kda for acgn ) , v is the molecular volume
of the protein , and is the difference between the scattering
length density of the protein solution and that of the d2o buffer ( i.e. , = pro buf ) .
buf was taken here as the scattering
length density of d2o alone ( = 6.35 10 ) , whereas pro was assumed as 3.0 10 , which corresponds to a typical scattering
length density value for proteins.q is also the magnitude of the scattering vector and is
related to q by q = q / n , with n being the refractive
index of the solution .
that is , q = 4 sin(/2)/ ,
with denoting the scattering angle and the wavelength .
note that eq 11 inherently assumes that the
sample is monodisperse and composed of identical , homogeneous particles
or proteins .
the form factor is a q - dependent orientation - averaged
function that provides information about the size and shape of the
proteins .
following previous sans analysis of acgn , it was taken here as that of a spherical particle .
the
structure factor gives information about the orientation - averaged
protein protein interactions and the distribution of the proteins
in solution as it is the fourier transform of the radial distribution
function .
protein interactions with respect to the protein / cosolutes
concentration and media conditions is typically not known a priori ,
and therefore , several analytical models for s(q ) were tested here .
protein
interactions as hard - sphere repulsions , screened columbic repulsions , or a
combination of two - yukawa functions ( one attractive and one repulsive ) .
nonlinear regression of sans data to these analytical
models was performed using the igor analysis software , specifically
the nist module for data analysis .
specific
details about the fitting of i(q ) to each of these models are provided in the supporting information .
in order to characterize concentration - dependent
interprotein interactions , rayleigh scattering data for acgn at 0 ,
10 , 50 , and 100 mm added nacl were obtained from sls experiments as
described in the materials and methods section .
these salt concentrations were selected to evaluate the behavior of
intermolecular interactions as a function of protein concentration
( c2 ) at conditions where electrostatic
interactions range from almost unscreened ( 0 added nacl ; 10 mm sodium
citrate buffer ) to moderately screened ( 100 mm added nacl ) .
dls measurements collected for all of the samples confirmed the presence
of only monomeric protein ( see the discussion section ) .
note that in the case
of no added salt , r90/k presents a pronounced downward curvature , reflecting strongly repulsive
conditions . on the other hand , at the highest salt concentration , r90/k versus c2 is nearly linear over the range of protein concentration
evaluated here , suggesting more nearly ideal conditions in terms of
protein protein interactions .
rayleigh scattering ( r90/k ) as a function of protein concentration
for -chymotrypsinogen
at ph = 3.5 and different salt concentrations .
symbols represent the
different nacl concentrations evaluated here : ( blue circles ) 0 ; ( red
squares ) 10 ; ( green diamonds ) 50 ; and ( gray triangles ) 100 mm .
error
bars ( 95% confidence intervals ) are smaller than the size of the symbols . the data in figure 1 were first fitted against
the classical model for analyzing ls data ( eq 2 ) to capture the strength of protein
fitting was performed over those data points with
protein concentrations between 1 and 7 g / l as signal - to - noise ratios
were too small for r90 below 1
g / l for
this system . on the basis of work elsewhere and the analysis of eq 2 provided in the supporting information
, the product of c2 and b22 must be
small compared to 1 ( e.g. , |c2b22| 0.05 ) in order for eq 2 to be reasonably valid ; otherwise , one can significatively
overestimate the magnitude of repulsive protein protein interactions .
the range of c2 used here to measure b22 was chosen to satisfy this criterion for
most of the salt conditions . for comparison , the same data were also
regressed against eq 4 to obtain g22 in the low - concentration regime .
quantitative differences
between g22 and 2b22 should be within the statistical uncertainty of the
regressed values in order to consider b22 to be accurate ( recall , g22 = 2b22 as c2
0 ) .
figure 2a shows the resulting values of b22 , as well as g22 for this low - concentration
regime , as a function of salt concentration .
these values are reported
relative to the hard - sphere second virial coefficient ( i.e. , b22 * = b22/b2hs and g22 * = g22/2b2hs ) , in keeping with increasingly common
practice .
defining b22 * and g22 * in this way gives them the same sign and assures that they are numerically
equal to one another in the limit of c2 0 .
the hard - sphere second virial coefficient was calculated
as b2hs = 2/3 , with being the
hard - sphere protein diameter and assumed equal to 4 nm for acgn . on this scale , values of b22 * larger than 1 indicate repulsive interactions beyond just steric
repulsions , while values smaller than 1 represent net attractive conditions
relative to purely steric repulsions .
osmotic second virial coefficient , b22 , or protein protein kb integral , g22 ( panel a ) , and apparent molecular weight
( mw ) ( panel b ) for -chymotrypsinogen
at ph = 3.5 as a
function of nacl concentration . b22
and mw are obtained by fitting r90/k versus c2 to eq 2 for c2 7
g / l , while g22 is obtained
by fitting the same data to eq 4 .
b22 and g22 are reported relative
to the hard - sphere second virial coefficient ( i.e. , b22 * = b22/b2hs and g22 * = g22/2b2hs ) .
the dashed line in panel b indicates
the true value for the protein molecular weight .
the results in figure 2a correlate
well
with previously published b22 data for
the same protein . when no salt is present , the
second virial coefficient is large and positive ( 15 times b2hs ) , indicating strongly repulsive electrostatic interactions , as was
anticipated from the theoretical net charge of acgn at this ph ( + 20
net charge or valence , based on literature pka values for free amino acids and the amino acid composition
of acgn ) . as the nacl concentration increases ,
charge
charge interactions become screened , yielding a large
decrease for the fitted b22 values .
this
behavior indicates that repulsive electrostatic interactions are the
dominant force for net protein
a lack of electrostatic attractions
is not surprising as the pi for acgn is 9 , and at ph = 3.5 ,
only a few of the acidic side chains are expected to be deprotonated .
furthermore , comparison between b22 * and g22 * suggests that
the dilute concentration regime has been reached at all the salt conditions
as these values are not statistically different within 95% confidence
intervals .
additionally , the analysis of ls data in the dilute
regime allows
one to measure the apparent molecular weight mw in solution ( figure 2b ) . theoretically ,
deviations of mw from the true molecular
weight are due to protein
however , for systems where no protein oligomerization
occurs and solute solvent nonidealities are not large , it is
expected that these deviations may be negligible within experimental
or statistical uncertainty , and mw is
effectively the molecular weight of the monomeric protein .
the values of mw obtained here agree , within 95% confidence intervals of the fits ,
with the molecular weight based on the amino acid composition of acgn ( dashed line in figure 2b ) , consistent with a lack of aggregation under these solution conditions
and temperature . in order to characterize protein
protein
interactions at
concentrated conditions , r90/k versus c2 data were also locally fitted
against eq 4 at higher c2 , using the procedure in the materials and
methods section and described in detail in the supporting information .
protein kb integral g22 as a function of c2 . on the basis of
the definition of g22 in eq 5 and g22 * defined above , the larger the positive ( negative )
value of g22 * , the larger the magnitude of repulsive ( attractive )
interactions that a central protein experiences with its neighboring
proteins .
figure 3 shows the fitted g22 values as a function of c2 for acgn at each of the salt conditions considered here .
protein repulsions is a strong function
of c2 over this concentration range if
the salt concentration is not large , while it is a weak function of c2 under electrostatically screened conditions .
furthermore , they show that the strength of these interactions is
reduced as the protein concentration increases .
fitted values of g22 as a function
of protein concentration for -chymotrypsinogen at ph = 3.5
and different salt concentrations .
symbols represent the different
nacl concentrations evaluated here : ( blue circles ) 0 ; ( red squares )
10 ; ( green diamonds ) 50 ; and ( gray triangles ) 100 mm .
g22 is obtained by regressing r90/k versus c2 to eq 4 over concentration windows of 37 data points
using the local taylor series approach .
g22 is reported relative to the hard - sphere second virial coefficient
( i.e. , g22 * = g22/2b2hs ) .
error bars correspond to 95% confidence intervals in the fitted
values . as mentioned above , the local
taylor series approach used here
to capture protein
protein interactions via g22 is potentially advantageous as it does not rely on
an underlying model for the pmf to describe these interactions .
however ,
concentration - dependent protein interactions have traditionally been
characterized from simplified pmf models , which in principle allow
one to relate changes in an experimental observable ( e.g. , g22 , osmotic pressure ) with changes in physical
parameters ( e.g. , effective charge or temperature , ionic strength ) . for comparison here
, different pmf models were tested against the
results shown in figure 3 ( see table s2 in
the supporting information ) .
these models
include a one - yukawa potential ( or equivalently ,
a screened repulsive coulomb interaction ) and a two - yukawa potential .
these pmfs were selected because they can reproduce
net repulsive conditions as well as different effective ranges for
the interactions .
g22 was calculated from these models via the
zero - q limit static structure factor ( s(q 0 ) ) because 1 + c2g22 is equivalent to this quantity .
for a given pmf model , the model was used in an analytical integral
equation solution to calculate s(q 0 ) as a function of c2 for
a given set of model parameters . for a given choice of model pmf
,
the model parameters were regressed against the experimental r90 data for acgn at a given solution condition .
details about these different models and the fitting to g22 ( or s(q
0 ) ) versus c2 are provided in the supporting information .
qualitatively ,
there is a good agreement in the behavior of g22 versus c2 from
these pmf models with that observed for acgn ( cf . figures 3 and s3 , supporting information ) .
however , fits of these models to the experimental data are both
statistically and physically inconsistent .
an example of the former
is the observation that there is a coupling between some of the parameters
in these models ( e.g. , the effective charge and the range of electrostatic
interactions ) , such that there are multiple combinations of the coupled
parameters that provide the same behavior for g22 versus c2 ( see figure s3 in
the supporting information ) . in many cases ,
the statistical confidence intervals for the fitted parameters
are
larger than the fitted values themselves , reflecting the difficulty
in finding a single set of parameters that was effective in describing
the full range of c2 for the sls data .
an example of the latter issue is that some of the fitted parameters
exhibit a nonphysical behavior with c2 or with changes in salt , such as an increase in the effective charge
or screening length with increasing nacl concentration ( see table
s2 in the supporting information ) .
intensity profiles ( i(q ) ) were obtained from sans experiments
for -chymotrypsinogen solutions at 10 mm citrate buffer and
ph 3.5 and three protein concentrations ( 2 , 10 , and 40
g / l ) and four
nacl concentrations ( 0 , 10 , 50 , and 100 mm ) .
the scattering data from
these samples are shown in figure 4 . for monomeric
protein solutions , qualitative features of protein
protein
interactions in the i(q ) curves
can be identified for low and intermediate q regions
( i.e. , q 0.1 and
lower ) as long as both the strength of the interactions and protein
concentration are large enough for s(q ) 1 . in the case of net attractive conditions ,
high scattering intensities
should be observed at low q as a consequence of s(q
0 ) >
for net repulsive interactions , these qualitative
features include a low value of i(q ) in the low - q limit due to s(q 0 ) <
1 , as well as the presence of a so - called
interaction peak at intermediate q ( e.g. , in the present case for q 0.06
) .
the height of this maximum or peak , relative
to i(q 0 ) , can be related
to the strength of the interaction between neighboring proteins due
to both pairwise protein protein interactions and solution
nonidealities from multibody effects . for both high q and low c2 , the behavior of i(q )
depends only on the molecular dimensions
and geometric structure of the protein ( i.e. , the form factor p(q ) ) , which in the case of acgn is sufficiently
close to that of a spherical particle . sans scattering intensities
as a function of the wave vector q from -chymotrypsinogen
solutions at ph 3.5 and
different salt concentrations : ( a ) 0 ; ( b ) 10 ; ( c ) 50 ; and ( d ) 100
mm nacl .
symbols correspond to three different protein concentrations :
( circles ) 40 ; ( squares ) 10 ; and ( triangles ) 2
lines represent
the best fitted curves to i(q ) for
the working conditions .
all of the models consider the form factor
as that of a spherical particle and differ by the structure factor s(q ) , with s(q ) given by ( solid line ) a two - yukawa potential ( 2y ) ; ( dashed line )
a screened coulomb repulsion ( sc ) ; and ( dotted - dashed line ) a hard - sphere
potential ( hs ) .
labels in each panel indicate different models . in
cases where models were indistinguishable ( e.g. , panel d ) ,
on the basis of the qualitative description given above ,
figure 4 illustrates similar behavior for protein protein
interactions as a function of protein and salt concentration to that
observed from figure 3 . at low salt concentration
and intermediate to high c2 , the i(q ) curves indicate that there are strong
repulsive protein interactions with a more pronounced effect of solution
nonidealities as the protein concentration increases .
in contrast ,
at high salt concentration and/or low protein concentration , the behavior
of i(q ) versus q is dominated by the form factor because at these conditions , s(q ) is expected to be close to 1 because
the product of c2g22 does not differ greatly from 0 , even though g22 can be greatly nonzero if c2 is sufficiently low .
in addition , at low concentrations , sans provides
effectively only the form factor because proteins are not very strong
scatterers even in d2o solutions , and therefore , obtaining
statistically meaningful s(q
0 ) values that differ from 1 at low c2 is not practical . in order to provide a quantitative
comparison between the results
from sans and those obtained from sls , sans scattering intensities
were fit to different theoretical models to assess s(q ) and
extrapolate it to q
0 , including the models described earlier to analyze g22 versus c2 , while p(q ) was treated as that of spherical particles .
the parameters from
the fits are summarized in table s1 ( supporting
information ) , along with illustrative examples of those from
fitting the same models to sls data as a function of c2 and salt concentration .
interestingly , the net result
in terms of the utility of simplified theoretical models is similar
to that from the analysis of g22 in that
the model parameters depend on c2 and
the trends for the c2 dependence are not
systematic or are nonphysical for some of these parameters .
for instance ,
the screened coulomb model shows that the effective charge and ionic
strength are barely changing with protein and salt concentration and
are not always going in the correct direction ( e.g. , they increase
as c2 or the salt concentration increases ) .
g / l , the two - yukawa model shows that the strength
for the repulsive part of the potential increases between 0 and 10
mm nacl and then decreases between 10 and 50 mm , while the parameters
for the attractive interactions change in an effectively random manner
with respect to nacl concentration .
these trends are physically inconsistent
with a system dominated by electrostatic interactions ( see the discussion ) . obtaining the low - q limit directly in sans
may
not be practical if g22(r ) has a long correlation length , as it does in the present case . in order to determine
the low - q limit of s(q ) for the data in figure 3 ,
the best - fitted
models were used to extrapolate the structure factor to the zero - q limit ( where s(q
0 ) = 1 + c2g22 ) and quantitatively compare the results obtained from both sans
and sls .
figure 5 illustrates this comparison in terms of s(q 0 ) as a function of protein concentration for
the different salt concentrations considered here .
given the statistical
uncertainty in the fitted values of g22 , as well as uncertainties arriving from the use of models to fit s(q ) , figure 5 shows
an excellent agreement between between both techniques and affirms
the use of the local taylor series analysis as a function of c2 to obtain g22 versus c2 .
comparison of of the zero - q limit static structure
factor s(q 0 ) as a function
of protein concentration for -chymotrypsinogen obtained from
sls ( open symbols ) and sans ( close symbols ) .
symbols correspond to
different salt concentrations : ( circles ) 0 ; ( squares ) 10 ; ( diamonds )
50 ; and ( triangles ) 100 mm . in the case of sls ,
the structure factor
was calculated from fitted values of g22 because s(q 0 ) = 1 + c2g22 . the above analysis focused on the use of sls and
sans to quantify
protein protein interactions and the thermodynamics of proteins
for concentrated protein solutions of acgn .
these results also suggested
that in the cases where b22 is large ( i.e. ,
strong protein
protein interactions ) , the effects of solvent
nonidealities on the behavior of proteins are prominent , even at a
moderate protein concentration of 40
the next section focuses
on dls measurements for the same sets of conditions as the sls data
as a function of c2 and added nacl .
the temporal autocorrelation
function ( g(t ) ) was
simultaneously measured with sls experiments for all of the salt and
protein conditions tested here and regressed to eq 6 , as described in the materials and methods section , to obtain the first two cumulants of the distribution of
diffusive decay times ( i.e. , dc and ) .
for all of the conditions assessed here
, the polydispersity index , p2 , remained below 0.3 , and the hydrodynamic
radius , rh , was found to be approximately
2.1 nm at the lowest protein concentration . at high c2 , only those samples at high salt concentration showed
a decrease in dc ( which suggests an increase
in rh ) , but this decrease was not larger
than 20% of the infinite dilution diffusion coefficient ( i.e. , dc(c2 0 )
= d0 ) .
this small decrease , together with
the low value of p2 , suggests that no
protein aggregates were present in the solution , as expected because
these are repulsive electrostatic conditions and the temperature is
far below the unfolding tm for non - native
aggregation to occur on these time scales .
the values of dc , together with the corresponding r90 values ( figure 1 ) , were used
to calculate the self - diffusion coefficient ds ( or equivalently , h(q
0 ) ) via eq 9 .
figure 6 shows the values of dc and ds as a function of protein and salt concentration . for
comparison
, the theoretical self - diffusion coefficient for a system
of suspended hard spheres with the same
diameter of acgn ( = 4 nm ) is also shown in figure 6b as a function of c2 .
in addition ,
the values of dc were used to calculate kd as a function of salt concentration from the
data in figure 6a via eq 8 for protein concentrations below 7
table 1 compares the values of kd and b22 for all the nacl concentrations
considered here .
values of the collective ( or mutual ) diffusion coefficient dc ( panel a ) and the self - diffusion coefficient ds ( panel b ) as a function of protein concentration
for -chymotrypsinogen .
symbols correspond to the different
salt concentrations : ( circles ) 0 ; ( squares ) 10 ; ( diamonds ) 50 ; and
( triangles ) 100 mm .
the dashed line in panel b corresponds
to the theoretical ds for a system of
suspended hard spheres 4 nm in diameter .
qualitatively , the behavior of kd is
equivalent to that displayed by b22 in
that increases of salt concentration give rise to a significant decrease
of the strength of protein repulsions .
however , values of kd at high salt concentration indicate that there
are net attractive protein interactions , which appears to contradict
the results obtained from sls and sans . by plotting b22 versus kd ( see the supporting information ) , one can see that kd scales linearly with b22 , but the intercept is smaller than 0 . on the basis of eq 10
, hydrodynamic interactions may also play a significant
role on the behavior of dc , and thus ,
the discrepancies observed here between kd and b22 are perhaps not surprising ,
but they suggest that the contribution from hydrodynamic interactions
is relevant in the behavior of acgn .
figure 6 illustrates the competition between
protein protein and hydrodynamic interactions , as well as the
effect of the range of protein protein interactions on the
mobility of proteins in solution . at high salt concentration ( short
interaction range ) , the diffusion of proteins is dominated by hydrodynamic
interactions as s(q 0 )
1 ( see figure 5 ) . in contrast , at
low salt concentrations , both s(q 0 ) and h(q 0 )
are large , but the structure factor represents the major contribution
to dc , leading to increase dc as the protein concentration increases .
overall , the
contribution from h(q 0 )
is appreciable for all of the conditions tested here .
three different experimental methods to probe concentration - dependent
protein protein interactions were tested here : sls , sans , and
dls .
direct
or thermodynamic interactions either in the q
0 limit ( via b22 or g22 from sls ) or as a function of the wave vector ( via i(q ) from sans ) .
the results in figures 2a , 3 , and 4 show that the qualitative behavior of protein interactions
for acgn with respect to solution conditions ( i.e. , changes in salt
concentration ) is captured by both sls and sans .
this behavior indicates
that intermolecular interactions are dominated by electrostatic forces
in that strong repulsive forces are exhibited at low nacl concentration ,
and the strength of these interactions decreases as the salt concentration
increases .
similarly , the results also show that the strength of the
repulsions also decreases as a consequence of increasing protein concentration
( e.g. , g22 versus c2 in figure 3 ) .
while the effect of
nacl concentration on protein protein interactions can be intuitively
attributed to screening effects as a consequence of accumulation of
ions on the protein surface , the behavior
of these interactions with respect to protein concentration may be
due to more complex effects .
although molecular crowding , self - buffering ,
and non - negligible concentrations of counterions may affect the strength
of protein protein interactions at concentrated conditions , none of these effects alone can explain the observed qualitative
behavior of protein interactions versus protein concentration at the
moderate range of concentrations tested here ( see the discussion below ) .
on the other hand , dls experiments provide information from both
direct protein interactions and hydrodynamic interactions via dc , ds , or kd .
the results in figure 5 and table 1 show that both types of interactions
play a major role in the dls behavior of acgn at the conditions tested
here . at low protein concentrations , differences in the sign of the
interaction parameter kd and b22 ( cf .
indeed , comparison of eqs 8 and 10 illustrates that for
conditions where hydrodynamic interactions are non - negligible with
respect to the evaluated range of protein concentrations ( i.e. , h1 0 ) , kd may be a poor representation of thermodynamic protein interactions .
the convolution of both types of interactions on the behavior of acgn
in solution is even more evident at high protein concentration .
although
eq 9 suggests that thermodynamic contributions
to the net protein protein interactions primarily affect dc through s(q 0 ) , the results in figure 6 show
that ds is sensitive to salt concentration ,
which is unexpected if all effects of direct protein
however , the hydrodynamic factor ( h(q ) ) also implicitly considers the effect of these
interactions on the mobility of proteins , in that intermolecular interactions
dictate the forces that affect how neighboring proteins move , and
therefore affect the local hydrodynamic field .
that is , stronger repulsive
( attractive ) protein interactions , with respect to a noninteracting
system ( e.g. , a hard - sphere system ) , lead to stronger ( weaker ) hydrodynamic
interactions .
felderhof showed
that by using a
taylor expansion in terms of the inverse of the center - to - center distance ,
one can approximate the hydrodynamic factor as a linear function of c2 , where the slope depends on the effect of
the pmf on the different hydrodynamic forces ( i.e. , hydrodynamic dipole ,
short - range and self - contributions ) .
similarly , more elaborate approximations
have been developed to incorporate the effect of thermodynamic contributions
on dc via pairwise additivity assumptions or decomposition of h(q 0 ) into direct and indirect terms .
although such approximations are shown to be
accurate only at dilute conditions , it illustrates how direct protein interactions can
be expected to play a role in the hydrodynamics of protein solutions .
additionally , the analysis here allows one to test a common practice
in characterizing net protein
although
such models may provide a valuable way to infer some information about
the underlying interactions of the system ( e.g. , consider the sans
data in figure 5 , where reaching q 0 is not practical without extrapolating with the aid of
a pmf model ) , one needs to be careful in interpreting the results
from these models .
as discussed above , the pmf is an ensemble - averaged
quantity that is an implicit function of the solution conditions ( ph , c2 , concentration of cosolutes , temperature ) ,
and therefore , one may anticipate that different pmfs are required
at different conditions ( cf .
protein protein pmfs
in solution are due to at least three main contributions , sterics
or excluded volume ( e.g. , a hard - sphere - like potential ) , short - range
attractions due to dispersion forces and solvophobic interactions ,
and screened electrostatic repulsions / attractions .
short - range attractions
are strong interactions with an effective range of a few angstrms .
the net magnitude of these contributions may be expected to change
with temperature or c2 but shows little
to no change with ionic strength and ph within the range of conditions
here . by contrast ,
electrostatic interactions
are long - ranged in nature ,
where the strength and the range of these interactions is sensitive
to the conditions of the medium ( ph , dielectric constant , ionic strength ) .
at dilute protein conditions , the maximum effective range of the interactions
between charged moieties is expected to range from 5 to 10 nm ( for
ionic strengths of 10 mm ) to a few angstrms ( for ionic
strengths of 500 mm ) . similarly ,
if one
considers that the protein net charge is the result of the sum of
the charges of all titratable side chains that have no greater charge
than 1 , the effective strength ( or effective charge )
of screened electrostatic interactions at low ionic strengths is no
greater than that for short - range attractions at near - contact between
amino acids side chains with center - to - center distances of 5
.
furthermore , one may anticipate that the protein net charge
may diminish at high salt concentrations as a result of ion condensation
effects . at high protein concentration ,
electrostatic interactions
may also be affected by other factors such
as polarizability , self - buffering , and condensation of ions at the
protein surface as a consequence of a non - negligible concentration
of counterions in solution ( e.g. , for acgn at
ph 3.5 , in order to maintain electroneutrality , the number of counterion
molecules in solution is at least an order of magnitude larger than
that of the protein ) .
therefore , for fitted pmfs to be considered
physically meaningful ,
they should at least be able to represent the qualitative behavior
of protein protein interactions with changes in solution conditions .
the above analysis and results show that this qualitative behavior
is not fully captured with the pmf models tested here .
although fits
to these pmfs at a given solution condition do not provide a reason
to be suspect from a statistical standpoint ( e.g. , fits appear reasonable
in figure 4 ) , the results from neither sls
nor sans data were able to provide physically reasonable trends for
the effective strength and range of the different types of forces
( i.e. , short - range attractions , electrostatics interactions ) as a
function of protein or cosolute concentration .
this may be problematic
as the true pmf is never known a priori for any experimental
system and can only be experimentally assessed from fully converged
saxs or sans intensity profiles of proteins with simple geometries
at moderate to high c2 , where s(q ) is the dominant factor if the form
factor does not change with protein concentration .
this poses additional
limitations in terms of the time required to collect data ( unless
one is at rather high c2 ) as well as regular
access to a neutron or x - ray synchrotron facility , which is problematic
for applications such as biotechnology product development .
if one
does not require the spatial information contained within a sans or
saxs profile , then the local taylor series approach to determine g22 is a convenient experimental means to quantify
the thermodynamics and net interactions of protein solutions as a
function of protein concentration and solvent conditions . despite
the issues noted above for determining a robust and quantitative
pmf for protein protein interactions
, one may still quantify
the net interactions and the thermodynamic nonidealities without the
need for a pmf .
g22 is potentially advantageous
in this regard because it provides a rigorous and quantitative relation
between multibody protein interactions and the thermodynamics of proteins
in solution.g22 , as well as other kb integrals , is related to changes in different
thermodynamic variables such as the isothermal compressibility or
partial molar or specific volumes . in the case
of g22 , changes in the protein chemical
potential are given by12where r is the ideal gas
constant , t is the absolute temperature , v is the system volume , and 2 is the protein
chemical potential . note that the subscript in the derivative indicates
that it is taken at fixed t , v ,
and chemical potentials of all of the species in the solution other
than that of the protein ( ) . in this regard ,
this derivative
describes an implicit solvent system such as those observed from osmometry
experiments and described by mcmillan mayer theory .
thus , direct integration of r90 via
eq 12 should be avoided if one is seeking to
integrate along a path of fixed temperature , pressure ( p ) , and cosolute concentration ( c3 ) . while
the set of scattering measurements are commonly performed along a
series of c2 values for a common ( t , p , c3 ) pathway ,
the derivative of 2 obtained from ls is not at fixed
( t , p , c3 ) . as a result ,
it is not straightforward to obtain derivatives of
activity coefficients for proteins or cosolutes from only ls data
alone , and doing so therefore requires additional analysis
if one seeks to relate ls to the location of phase boundaries .
a similar issue arises if one attempts to relate the integrated
2 versus c2 to the pmf as the solvent and solute chemical potentials
change as a function of c2 .
these
issues can potentially be avoided by alternative sample preparation
in order to maintain an equal chemical potential of ( co)solvent and
cosolutes at all protein concentrations .
for instance , by exhaustively
dialyzing each protein sample against the same solvent ( e.g. , buffer
plus cosolutes ) , the chemical potential of all of the other species
will be constant and equal to that of the solvent , and one can correctly
integrate the partial derivative in eq 12 versus c2 to recover 2 as a function
of c2 .
however , this requires that one
perform a separate dialysis preparation for each c2 value , independently , for a given salt concentration .
doing so is not common practice as it is typically not logistically
practical due to the greatly increased requirements of time , protein
material , and/or cost to prepare a large number of samples with different
protein concentrations in this manner .
alternatively , one can
consider the behavior of g22 from a statistical - thermodynamic
standpoint .
as eq 5 shows , g22 is related
to the integral over g22(r ) , and thus , it indirectly allows one to assess local changes
in composition around a given protein molecule ( i.e. , the region where g22(r ) 1 ) . that is , g22 depends
on the local variations of concentration with respect to the bulk
concentration .
likewise , g22 and eq 12 qualitatively and semiquantitatively relate the
local fluctuations in protein concentration to the thermodynamic behavior
of proteins in solution because 1 + c2g22 is proportional to the magnitude
and sign of local fluctuations in protein concentration .
positive ( negative ) deviations from zero for c2g22 indicate large ( small ) variations
in the local density of proteins with respect to an ideal gas mixture .
notably , there is a strict lower bound for the product of c2g22 based on thermodynamic
stability criteria as c2g22 less than or equal to 1 violates stability
criteria .
while there is no rigorous
upper bound for c2g22 ( i.e. , strongly attractive conditions ) , c2g22 1 is indicative
of being in proximity to homogeneous transitions ( e.g. , fluid
such processes yield extremely
large molecular fluctuations as a consequence of long - range density
and composition fluctuations as one approaches a spinodal .
to
a first approximation , when g22 ( in
units of volume per mole of protein ) is negative , then its magnitude
is effectively the excluded volume that the other proteins feel
from a central protein ; similarly , 1 + c2g22 then represents the effective free
volume fraction ( i.e. , the volume that is not excluded by proteins )
of the system under repulsive conditions .
this is also consistent
with it being physically impossible to achieve c2g22 less than 1 for an
equilibrium system .
in contrast , for positive g22 there is no simple excluded volume analogy because excluded
volume is positive by definition . in that case , c2g22 physically represents
the extent of statistical accumulation of protein ( relative to solvent / cosolutes )
in the near - neighbor shells compared to what one would have for ideal
solutions .
if one makes the assumption
that g22(r ) is
only short - ranged when g22 is positive
( attractive conditions ) , then c2g22 roughly represents the average number of
nearest - neighbor proteins in the grand - canonical ensemble . as all
of the conditions investigated here showed negative g22 values , the former physical picture holds . as 1 + c2g22 decreases ,
this indicates a decrease in the effective volume fraction that remains
for other proteins to be added to the system as c2 is increased to larger values ( figure 5 ) . if one were to simply define the effective protein excluded
volume fraction ( eff = c2b2hs ) based on the excluded volume of protein at infinite dilution ,
the largest free volume fraction ( = 1 eff ) that one would reach is 0.89 at 35
comparison with
figure 5 shows that this is a gross underestimation
of eff when long - ranged repulsions are present .
a similar conclusion can be drawn if one considers the effective hard - sphere
diameter ( eff ) that yields the same value of s(q 0 ) or c2g22 at a given protein concentration
via a hard - sphere equation of state such as the carnahan starling
approximation ( data not shown ) . for
instance , at no added nacl , when electrostatic repulsions are the
strongest , eff is 10 nm ( i.e. , 2.5
times larger than the protein diameter ) at low protein concentrations
and decreases to 7 nm at 20
g / l of protein as a consequence
of the decrease on the strength of the repulsions due to thermodynamic
nonidealities .
this change in the effective protein excluded
volume or effective
protein diameter with protein interactions and c2 might suggest that the behavior of acgn solutions , at repulsive
conditions , is effectively equivalent to that of a system experiencing
molecular crowding , even though the volume fraction based on just
the protein molecular volume is far below that where large effects
due to crowding are expected for a hard - sphere system .
this observation
is consistent with the qualitative behavior of the hydrodynamic factor
with respect to protein concentration .
strong hydrodynamic interactions
have typically been associated with molecular crowding , and thus ,
they are only considered important on systems where protein and/or
cosolute concentrations are large .
furthermore , when hydrodynamic interactions are considered , h(q 0 ) versus c2 is typically treated via simple relations , which assume
in most cases that h(q
0 ) is linear with respect to protein concentration . however , as figure 6b shows , the hydrodynamic
contribution as a function of protein concentration is neither simple
nor negligible at even these low concentrations ( i.e. , less than 5
w / v% ) .
comparison of experimental ds values
( figure 6 ) with those of a hard - sphere system
shows that hydrodynamic interactions are more complex than simple
steric effects ( i.e. , based on molecular volume alone ) .
the results
in figure 6b also suggest that the range of
electrostatic interactions plays a key role in the strength of hydrodynamic
interactions , with low ( high ) salt concentration leading to stronger
( weaker ) hydrodynamic interactions .
such effects may be the result
of an increase in the effective excluded protein volume as the range
of charge
quantitatively , the reduction
on ds from d0 observed here lies between 20 ( for 100 mm nacl ) and 50% ( for 0 mm
nacl ) at a protein concentration of 40 g / l .
although similar effects
of the range of electrostatic interaction on the self - diffusion of
proteins have been observed previously , they have been found for protein
volume fractions > 10% , which would correspond to c2 > 120
protein interactions as a function of protein
and salt concentration
were evaluated via scattering methods ( sls , sans , and dls ) for solutions
of monomeric -chymotrypsinogen at acidic ph .
protein
kb integral g22 and the structure factor s(q ) from sls and sans data , respectively .
g22 was obtained by regressing the rayleigh ratio
versus protein concentration to eq 4 using a
local taylor series approach , which allows one to quantify interactions
without biasing the results toward a specific model for intermolecular
interactions ( i.e. , pmf models ) .
g22 versus c2 curves , as well as sans intensity spectra i(q ) , were further analyzed via traditional
methods involving fits to effective intermolecular potentials ( i.e. ,
pmf models ) .
these fits were able to capture the curves of g22 or i(q )
as long as one considered concentration - dependent model parameters
and accepted that the fitted parameters showed unphysical trends in
some cases .
the values of s(q
0 ) from sls and from extrapolating sans data to q 0 were quantitatively consistent . in the dilute regime ,
fitted g22 values
agreed with those obtained via the osmotic second virial coefficient
( b22 ) and showed that electrostatic interactions
dominated the scattering behavior for acgn under these ph and salt
conditions .
protein repulsions decreased , with a more pronounced
effect for those conditions where b22 was
larger .
both sls and sans results indicated that the thermodynamic
behavior of the acgn solution is similar to that observed in systems
under molecular crowding , despite the moderate range of protein concentrations
used here ( c2 < 40
, both the strength
and the range of protein protein interactions modulate this
crowding - like effect , such that strong and long - range protein interactions
led to more noticeable thermodynamic nonidealities .
the zero - q limit hydrodynamic factor h(q 0 ) ( or alternatively the self - diffusion
coefficient ds ) was also assessed to quantify
hydrodynamic nonidealities by combining measurements of the collective
diffusion coefficient ( dc ) from dls data
with measurements of s(q
0 ) via eq 9 .
curves of dc versus c2 illustrated the competition
between equilibrium protein interactions ( probed via g22 ) and hydrodynamic interactions as a consequence of
the range of intermolecular interactions .
while at high salt concentrations h(q 0 ) is the dominant contribution
to dc , at low salt concentration , the
net mobility of proteins is dictated by s(q 0 ) .
nevertheless , the hydrodynamic contribution
was found to be significant for all of the conditions and correlated
with the strength of colloidal interactions such that larger repulsive b22 values correspond to stronger hydrodynamic
interactions .
quantitatively , the reduction of ds due to increased protein concentration was much larger than
what could be expected based on purely steric interactions , highlighting
that the long - range repulsions resulted in a much larger effective
excluded volume contribution to protein protein interactions
probed by s(q 0 ) and by h(q 0 ) . | protein protein interactions
were investigated for -chymotrypsinogen
by static and dynamic light scattering ( sls and dls , respectively ) ,
as well as small - angle neutron scattering ( sans ) , as a function of
protein and salt concentration at acidic conditions . net protein
protein
interactions were probed via the kirkwood
buff integral g22 and the static structure factor s(q ) from sls and sans data .
g22 was obtained by regressing the rayleigh ratio versus protein
concentration with a local taylor series approach , which does not
require one to assume the underlying form or nature of intermolecular
interactions .
in addition , g22 and s(q ) were further analyzed by traditional
methods involving fits to effective interaction potentials .
although
the fitted model parameters were not always physically realistic ,
the numerical values for g22 and s(q 0 ) were in good agreement
from sls and sans as a function of protein concentration . in the dilute
regime ,
fitted g22 values agreed with
those obtained via the osmotic second virial coefficient b22 and showed that electrostatic interactions are the
dominant contribution for colloidal interactions in -chymotrypsinogen
solutions .
however , as protein concentration increases , the strength
of protein protein interactions decreases , with a more pronounced
decrease at low salt concentrations .
the results are consistent with
an effective crowding or excluded volume contribution
to g22 due to the long - ranged electrostatic
repulsions that are prominent even at the moderate range of protein
concentrations used here ( < 40
g / l ) . these apparent crowding effects
were confirmed and quantified by assessing the hydrodynamic factor h(q 0 ) , which is obtained by combining
measurements of the collective diffusion coefficient from dls data
with measurements of s(q
0 ) .
h(q 0 ) was significantly
less than that for a corresponding hard - sphere system and showed that
hydrodynamic nonidealities can lead to qualitatively incorrect conclusions
regarding b22 , g22 , and static protein
protein interactions if one uses
only dls to assess protein interactions . |
a 27-year - old female was referred to gil medical center due to a painful and edematous left lower extremity of 4 days duration after varicose vein surgery at a local clinic .
in addition to the edematous left lower extremity , there were bruises on the left thigh and around the operation wound site .
since deep vein thrombosis was suspected , computed tomography ( ct ) was performed , and the left common femoral vein above the proximal superficial femoral vein was not visualized ( fig .
1a , b ) . iatrogenic femoral vein division , rather than deep vein thrombosis ,
subsequent venography depicted a divided left femoral vein and thrombosis around its stump ( fig .
she was a flight attendant , and the edematous , painful leg caused inconvenience both professionally and cosmetically ; thus , we decided on femoral vein repair .
the patient had undergone saphenous vein ablation in both legs , which meant that no autologous saphenous vein was available for femoral vein repair .
accordingly , left femoral vein repair with a polytetrafluoroethylene ( ptfe ) graft and an arteriovenous ( av ) fistula ( to improve ptfe graft patency ) was chosen . during surgery , the previous surgical wound on the left inguinal area was extended and the hematoma around the left femoral vein was removed ( fig .
2b ) , the left cephalic vein was harvested for the av graft , and the divided left femoral vein was repaired using a 14-mm ringed ptfe graft ( fig . 2c ) .
the av fistula was constructed between the left common femoral artery and the ptfe graft using the harvested cephalic vein .
the patency of the left femoral vein and av fistula was confirmed by ct at 7 days after surgery ( fig .
the patient took warfarin ( initial dosage : 4 mg , target international normalized ratio : 2 ) immediately after surgery in order to keep the graft patent , and the international normalized ratio level was maintained around 2 . at 18 months postoperatively , there was no evidence of edema and doppler ultrasonography confirmed the patiency of the left femoral vein ( fig . 4 b ) .
this study was reviewed by the institutional review board of gil hospital ( irb no .
vein injuries following varicose vein surgery are rare but serious due to risks of morbidity and mortality and medico - legal implications . according to a comprehensive literature review of 87 vascular injuries after varicose vein surgery that included 43 vein injuries , laceration or division of the femoral vein was most common ( 28 cases ) , while partial stripping of the femoral vein accounted for only 4 cases .
the most common symptom of venous injury was heavy bleeding in the groin , and symptoms become obvious after a few hours or days with massive leg swelling in some patients without bleeding .
although primary venous ligation might be a first choice in hemodynamically unstable patients , it often induces severe postoperative edema . on the other hand ,
reconstruction has associated risks of stenosis and occlusion , and when a ptfe graft is interposed the risk of severe infection increases . complex venous reconstruction in patients with traumatic injuries of the lower extremity has been found to have a 30-day patency rate of 50%93% , depending on the type of venous repair , and a patency rate of only 50% when a panel or spiral graft was used . the patency rate of ptfe grafts for surgical reconstruction of iliofemoral veins has been reported to be around 45% . to avoid the postoperative risk of early thrombosis due to the use of a synthetic material and problems associated with size mismatch between the saphenous vein and the common femoral vein , most surgeons recommend vein repair without an interposition graft by end - to - end anastomosis , which is likely to be the best option .
however , it was impossible to interpose the graft by end - to - end anastomosis in our present case , because the segment of femoral vein division resulting from previous surgery was too long to repair with end - to - end anastomosis .
based on considerations of the patient s job , we considered femoral vein repair to be the better route toward relieving edema in the lower extremity , but graft choice was a problem , because the saphenous vein was not available , and the patient did not want an extended surgical incision on the left thigh . to increase long - term ptfe graft patency , we added an av fistula to the ptfe interposition site of the femoral vein .
several techniques can be used to increase prosthetic graft patency , such as vein cuffs , vein patches , and av fistulae .
richardson et al . reported the results of temporary av fistulae for the management of traumatic venous injuries of the lower extremity in 8 patients , and noninvasive doppler studies performed in 4 of the 8 patients at 3 to 8 months post - repair revealed no evidence of deep venous obstruction .
extremity edema was not observed in our patient after femoral vein repair and the graft remained patent .
her vein injury was caused by varicose vein surgery , and she considered the symptom of edema and associated cosmetic problems to be the most important issues . choosing the best graft was found to be challenging .
this case shows that a staged procedure can provide an alternative means of venous reconstruction when autologous vein is unavailable . | a 27-year - old female patient was referred due to an edematous left lower extremity .
both saphenous veins had been ablated with an endovenous laser procedure used to treat varicose veins .
venography revealed that the left common femoral vein had been divided and that thrombosis was present at the site of division .
no veins were available around the thighs .
the patient was treated using a staged procedure . during the first stage ,
a ringed polytetrafluoroethylene graft was used to repair the common femoral vein , and an arteriovenous fistula was constructed from the femoral artery to the graft using a short segment of cephalic vein to increase graft patency .
the edema was relieved postoperatively and the graft was patent . during the second stage , which was performed 6 months later , the fistula was occluded by coil embolization .
the staged procedure described herein provides an alternative for venous reconstruction when autologous vein is unavailable . |
the prevalence of crohn 's disease ( cd ) in north america ranges from 26.0 to 198.5 cases per 100,000 persons .
clinical symptoms as well as endoscopic aspects of cd are nonspecific , even if some lesions can be highly suggestive .
diffuse gastric adenocarcinoma with peritoneal carcinomatosis ( pc ) is much less frequent but can mimic certain aspects of cd [ 2 , 3 ] .
gastric adenocarcinomas , which represent about 85% of gastric malignant tumors , may be subdivided into two categories : diffuse type , in which cell cohesion is absent so that individual cells infiltrate and thicken the stomach wall without forming a discrete mass , and intestinal type , characterized by cohesive neoplastic cells that form glandular - like tubular structures .
diffuse carcinomas occur more often in younger patients , develop throughout the stomach , result in a loss of elasticity of the gastric wall ( so - called linitis plastica or leather bottle appearance ) and carry a poorer prognosis . studies also show that pc has a stronger association with this type of cancer ( frequency of pc in patients with poor histology against other subtypes : 40 vs. 13% ; p = 0.0001 , relative risk 3.07 ) , whereas visceral metastases are more frequent with the intestinal type .
in contrast to intestinal type carcinomas , which tend to predominate in high - risk geographic regions , the incidence of diffuse carcinomas is similar in most populations .
we present two clinical cases initially diagnosed as cd , but were later on revealed to be suffering from diffuse gastric cancer with pc .
we stress out similarities which can exist between the presentation of a patient with cd and a patient with malignant disease .
these cases show that repeated diagnostic evaluation with multilevel biopsies is essential in case of unconfirmed cd in order to confirm or dismiss this hypothesis .
a 44-year - old caucasian male was hospitalized in our institution for further diagnostics and evaluation of suspected cd .
the patient had presented to a different institution 1 month earlier with a history of recurrent upper abdominal cramps , accompanied by a 10 kg weight loss over a period of 6 months .
there was no change in stool frequency or consistency , no fever or night sweats .
family history revealed that his father had died of an undefined cancer at the age of 56 years .
laboratory testing in the external hospital revealed minor normochromic , normocytic anemia ( hb : 13.5 g / dl , normal : 1417.5 ) .
upper endoscopy showed mucosal edema with luminal narrowing in the duodenal bulb and gastric antrum .
biopsies showed low - grade chronic inflammation , no signs of malignancy and were positive for helicobacter pylori .
colonoscopy revealed segmental , superficial , partially ulcerated , about 3 cm long polypoid lesions in the ascending colon and the transversum .
multilevel biopsies showed signs of chronic inactive enteritis , segmental mucosal edema and crypt hyperplasia , but no granulomatous lesions .
multislice ct depicted a small diffuse quantity of ascites as well as segmental concentric wall thickening of the colon .
ascites puncture fluid contained elevated cellularity ( 798/l , normal : 0100 ) , but no signs of acute inflammation or neoplastic cells . after negative mendel - mantoux testing for tuberculosis ,
the patient was released on a 1 mg / kg body weight prednisolone therapy , associated with vitamin d and calcium supplements , for suspicion of inflammatory bowel disease .
one month later , the patient presented to our institution with right flank pain and a swollen scrotum . on physical examination
he had an altered general appearance and was in a cachectic nutritional status ( 51 kg/176 cm , bmi 16.4 kg / m ) . during abdominal examination he indicated diffuse abdominal tenderness ; bowel sounds were normal .
laboratory testing showed elevated ca19 - 9 ( 56 u / ml , normal : < 37 ) ( cea , -fp and psa were normal ) , mild hypocalcemia ( 2.12 mmol / l , normal : 2.152.65 ) and minor normochromic , normocytic anemia ( hb : 13 g / dl , normal : 1417.5 ) .
abdominal ultrasound revealed right hydronephrosis , perihepatic , perisplenic and interenteric ascites with a 10 mm wall thickening of the gastric antrum .
the antrum , pylorus and duodenal bulb were swollen and deformed with superficial erosions , and colonoscopy showed circular edema ( fig .
endoscopic ultrasound ( eus ) showed thickening of the anterior wall and the lesser curvature up to 10 mm and extension of the process into the serosa with disruption of individual wall layers ( fig .
biopsies taken in the antrum , corpus and fundus showed signs of invasive , poorly ( g3 ) differentiated adenocarcinoma with signet cell infiltration and linitis plastica appearance .
ct imaging of the abdomen revealed bilateral hydroceles ( probably due to venous obstruction by ascitic fluid ) , right hydronephrosis , wall thickening of the stomach and colon ( fig .
3 ) and a high suspicion of pc . a diagnostic ascites puncture confirmed this hypothesis .
the diagnosis of a primary diffuse ( lauren classification ) gastric adenocarcinoma ct4n m1g3 ( stage iiib ) , her-2/neu - negative , with pc was made and flot ( fluorouracil , leucovorin , oxaliplatin and docetaxel ) palliative chemotherapy was started .
after 6 cycles of chemotherapy the patient showed a marked clinical response ( reduction of initial symptoms ) with tumor markers decreasing and undetectable tumor in re - staging thoracoabdominal ct .
due to recurrent ascites , intraperitoneal treatment with the trifunctional anti - epcam antibody catumaxomab was started . for the time being , the patient is without any further chemotherapy .
a 58-year - old caucasian male presented to our institution for further diagnostics and evaluation of suspected cd due to ongoing emesis after meals .
the patient had been hospitalized in a different institution 6 weeks earlier with a history of nausea and mild abdominal pain accompanied by a 16 kg weight loss over the last 5 months .
colonoscopy revealed segmental stenosis in the ascending colon and several aphthous lesions in the rectum .
the patient was discharged with local - acting budesonide under suspicion of cd . due to clinical deterioration
he had a decreased nutritional status ( 59 kg/170 cm , bmi 20.4 kg / m ) and showed icteric sclerae .
laboratory testing showed elevated ca72 - 4 ( 150.3 u / ml , normal : < 4 ) and minor normochromic , normocytic anemia ( hb : 13.9 g / dl , normal : 1417.5 ) as well as increased liver enzymes ( alt 277 u / l , normal : < 45 ; ast 154 u / l , normal : < 50 ; direct bilirubin 8.2 mg / dl , normal : < 0.3 ) with normal liver synthesis parameters .
abdominal ultrasound revealed ascites , signs of mechanical cholestasis with slightly dilated peripheral bile ducts but a normal width of the central bile duct .
upper endoscopy revealed a swollen distal esophagus without obvious tumor and fluid in the dilated esophagus .
after passage into the stomach , the gastric folds were swollen without ulcerations or protruding tumor .
passage into the duodenum was not possible due to a narrowed pylorus . due to the stenotic distal gastroesophageal region
biopsies taken all over the stomach depicted only chronic inflammation , but the biopsies taken from the gastroesophageal region revealed undifferentiated pas - positive , her-2/neu - negative adenocarcinoma with a high proliferation rate of 90% ( ki67 ) .
colonoscopy confirmed the external finding and biopsies revealed no malignancy as ascites puncture was without detection of malignant cells . due to the increased liver enzymes including mechanical cholestasis ,
systemic chemotherapy was introduced using docetaxel and cisplatin without 5-fu in a 50% reduced dose on a two - weekly rhythm . following chemotherapy , liver enzymes including bilirubin decreased to normal levels and weight increased to 68 kg without parenteral nutrition .
re - staging with ct after 3 months showed no more ascites and a marked decrease in the thickening of the gastric as well as the colonic wall without further ascites .
after 1 year of systemic therapy , the patient is in good clinical condition and complete remission based on ct findings .
although the classical presentation of cd mostly does not cause diagnostic problems , in some cases cd presents with ascites accompanied by only slightly increased stool frequency .
colonoscopy showed segmental , polypoid , superficial erosions and inflammation of the ileocecal valve with circumferential narrowing .
one month later we first followed a protocol to confirm a diagnosis of cd and characterize the activity .
however , endoscopic aspects of gastric and colonic lesions had evolved and became clearly indicative of a neoplastic process .
as biopsies had been negative for malignancy before , eus presented as a useful diagnostic tool to further evaluate the risk of gastric carcinoma .
a study by gins et al . demonstrated that the enlargement of deep layers is an independent predictor of malignancy in patients with large gastric folds found in endoscopy without signs of malignancy in gastric biopsies . in this study ,
the probability of having a malignant process increased from 34 to 94% in case of thickened deep layers , whereas in the absence of this finding the likelihood decreased from 34 to 4.7% . in both described cases ,
distal esophageal stenosis prevented eus in the second case . the primary tumor breaching the serosal layer , proof of carcinomatous invasion of the peritoneal cavity by positive cytology and histological findings in gastric biopsies led to the diagnosis of a diffuse gastric adenocarcinoma ct4n m1g3 .
as curative resection ( r0 ) was impossible in this case of pc , the primary standard of care was chemotherapy .
gastric linitis plastica often presents with weight loss , vomiting and/or early satiety and abdominal pain .
lower intestinal symptoms such as diarrhea , constipation or rectal bleeding are less common . in a case of suspected active cd ,
absent history of diarrhea a major symptom of cd should lead to further investigations to rule out or confirm this hypothesis . as predominant upper gastrointestinal tract manifestations are less common , these should initiate additional investigations in unconfirmed cases .
presented the case of a 41-year - old male suffering from abdominal fullness , anorexia and weight loss diagnosed with diffuse type gastric carcinoma in which colonoscopy revealed flat elevated lesions with superficial erosions . in this case
other reports on patients with linitis plastica show colonoscopic findings including granular and friable mucosa , occasional submucosal nodularity ( resembling the typical cobblestone appearance of cd ) , superficial erosions , pseudopolyps and luminal narrowing mimicking cd .
for all strictures in a patient with suspected or confirmed inflammatory bowel disease it is essential to assess evidence of malignancy .
the most specific microscopic lesions in cd are noncaseating granulomas , but these are seen in less than 25% of mucosal biopsies .
furthermore , the typical transmural inflammation with monocellular cell infiltrate , fibrosis , ulcers and fissures can be absent in case of inactive cd . at the time of presentation at our institution , rigidity of the gastric wall ,
a wall thickness exceeding 6 mm , and a poor demarcation between layers seen in eus , combined with histological findings , proved diffuse gastric cancer described as linitis plastica by endoscopy in our first patient .
linitis plastica with intestinal metastases , pc and cd can present as diffuse concentric wall thickening with segmental stricture pattern on ct .
metastatic polypoid lesions in the intestine usually originate from melanoma or spindle cell renal carcinoma , but can also result from poorly differentiated gastric carcinoma [ 11 , 12 , 13 ] . in most of these cases repeated deep biopsies are necessary to eventually detect neoplastic cells and firmly diagnose malignancy . whereas different other cases of patients with gastric linitis report a certain macroscopic resemblance to cd , the first patient was one of the rarely described cases where
colonoscopic appearance in a patient with gastric linitis and pc combined multiple characteristics of cd : skip lesions , strictures and ulcers .
the clinical presentation of the two patients was atypical and supported repeated diagnostic evaluation in order to rule out malignancy ( fig .
4 ) . initially this led to a false suspicion which delayed the actual diagnosis and therapy of an aggressive neoplasm .
the central aim of this report was to illustrate how metastatic diffuse gastric cancer can imitate endoscopic aspects of cd .
the clinician should be alarmed by atypical clinical presentations such as a missing history of diarrhea or predominant upper gastrointestinal tract manifestations and constantly be aware that malignant diseases can present in insidious ways . considering the possible major therapeutic implications , suspicion of cd
should always be confirmed by further diagnostic testing , especially since pc or intestinal metastases of gastric cancer , inter alia , can mimic cd .
repeated biopsies are often necessary to diagnose linitis plastica because of the diffuse character of neoplastic cell infiltration . in case of macroscopic suspicion of gastric malignancy in endoscopy , eus may be a useful minimally invasive diagnostic tool to further evaluate the patient 's risk of gastric cancer .
exact staging of gastric cancer is very important for the selection of patients for whom r0 surgery might be indicated . at this point , palliative chemotherapy is the standard treatment in cases of pc .
intraperitoneal application of catumaxomab appears to be a valuable tool to help patients with recurrent ascites and may be a further step in improving outcome of patients with pc due to gastric cancer .
s.d . designed the case report , participated in the literature review and drafting of the manuscript as well as oncologic follow - up and treatment .
| in some cases the diagnosis of gastric cancer is difficult and the endoscopic presentation may be misleading .
diffuse type gastric carcinoma with peritoneal metastasis may present primarily with abdominal pain , colonic infiltration and/or diarrhea , thus other differential diagnoses like crohn 's disease ( cd ) may be considered at first .
therefore intensive diagnostic work - up is important .
we report two cases of gastric cancer with ascites due to peritoneal carcinomatosis who were first diagnosed as cd .
the patients were hospitalized in different institutions for weight loss , abdominal pain and nausea .
the first colonoscopy , upper endoscopy with multiple biopsies and ascites puncture were negative for malignant disease , but macroscopic lesions resembling cd were described .
both patients were released on a prednisolone - based treatment for suspected cd .
they presented to our hospital for further evaluation due to persistent symptoms .
neither lower nor upper endoscopy were suggestive of cd and endoscopic ultrasound was suspicious of malignancy in one case .
histology was diagnostic and showed gastric infiltration by a poorly differentiated adenocarcinoma .
diffuse type gastric cancer ( gastric linitis plastica ) with peritoneal metastasis may mimic certain clinical , endoscopic and ct imaging features of cd . repeated biopsies and endoscopic investigations are often necessary to confirm a malignant process , especially in case of an inconclusive clinical and endoscopic picture .
endoscopic ultrasound may be useful to evaluate the risk of malignancy in patients with macroscopic suspicion of malignancy and negative biopsies . |
mass spectrometry ( ms ) based glycomics techniques are broadly used to analyze free oliogsaccharides , glycosaminoglycans as well as the glycan portions of glycoproteins , proteoglycans and glycolipids .
both matrix - assisted laser desorption - ionization ( maldi ) and electrospray ionization ( esi ) are commonly applied .
ms may be used as a stand - alone technique , or coupled online to separation methods such as hplc [ 14 ] and capillary electrophoresis ( ce ) [ 57 ] .
carbohydrate and glycoconjugate analysis by maldi - ms has been comprehensively reviewed by harvey [ 8 , 9 ] .
other useful review articles , which cover a range of analytical techniques including tandem ms ( ms / ms ) of glycoconjugates have appeared in recent years [ 815 ] .
this review aims at giving a concise overview of ms based glycomics technology , together with selected applications in clinical research .
protein - linked n - glycans and o - glycans are typically released by enzymatic and chemical methods , respectively .
also glycosaminoglycans are generally degraded by chemical or enzymatic means for subsequent analysis [ 5 , 17 ] .
analysis of released ( or free ) glycans may be achieved by a variety of techniques such as mass spectrometry , hplc of reductively aminated glycans employing fluorescence or uv detection and capillary gel electrophoresis with laser - induced fluorescence detection ( cge - lif ) of labeled glycans [ 16 , 1821 ] .
ms is particularly advantageous for analyzing very complex glycan mixtures containing unusual oligosaccharide structures for which the standardized migration positions in hplc or cge - lif have not yet been determined .
importantly , the mass of the analyzed glycan when determined with sufficient accuracy or accompanied by a tandem ms experiment will directly provide information on the glycan composition in terms of hexoses , n - acetylhexosamines , deoxyhexoses , etc .
by contrast , this direct link between the observed glycan species and its molecular composition is not inherently present for hplc and cge - lif experiments and additional efforts are required such as the use of glycan standards or exoglycosidase treatments for the determination of terminal monosaccharides [ 22 , 23 ] . on the other hand ,
separation - based methods for glycan analysis will often resolve structural isomers such as the 6-arm and 3-arm isomers of monogalactosylated biantennary glycans , while their distinction is not easily achieved by ms and requires additional efforts such as tandem ms analysis .
therefore , while very complex pools of oligosaccharides can be analyzed by ms(/ms ) without separation , many researchers choose to perform glycan analysis by lc - ms [ 13 ] or - less frequently - ce - ms coupling [ 57 ] .
( normalized ) retention and migration times , precursor masses and fragmentation spectra may then be used for structural elucidation as in the case of o - glycan alditol analysis by porous graphitized carbon ( pgc ) hplc coupled online to ms [ 2729 ] .
pgc - hplc appears to have a particularly high power in separating oligosaccharide structural isomers , which makes this method very useful for in - depth structural analysis of complex oligosaccharide mixtures .
another popular separation technique hyphenated with ms for oligosaccharide analysis is hilic , which likewise features isomer separation [ 24 , 30 , 31 ] .
high - performance anion - exchange chromatography ( hpaec ) coupled with online - desalting and online - esi - ms is another approach which is particularly useful for the analysis of underivatized oligosaccharides .
for example , oligosaccharides may be reduced to alditols resulting in a 2 da mass tag on the innermost monosaccharide which facilitates fragment assignment in tandem ms .
analysis of o - glycan alditols obtained by reductive beta - elimination may be achieved by porous graphitized carbon ( pgc ) hplc coupled via online , negative - mode electrospray ionization to ion trap - tandem mass spectrometry ( ms / ms ) [ 27 , 28 ] . an online database has been made available by the unicarb - db partners allowing structural assignment of o - glycan alditols on the basis of ms and ms / ms spectra in addition to retention times ( http://www.unicarb-db.com/ ) .
similarly , n - glycans may be structurally assigned on the basis of mass and retention time in pgc - esi - ms
. this approach has been introduced by altmann and coworkers . within the range of mass spectrometric techniques , negative - mode ms of glycans
has recently obtained increased attention , both for maldi and esi ionization [ 33 , 34 ] .
negative - mode ionization is particularly effective for acidic glycan structures . in this respect ,
labeling of glycans at the reducing end with an acidic tag such as 2-aminobenzoic acid ( anthranilic acid ; aa ) is advantageous , as it confers acidic properties to all glycans including neutral species , thereby allowing the efficient detection of both sialylated and non - sialylated aa - labeled oligosaccharides in negative - mode maldi - time of flight ( tof)-ms .
in addition , negative - mode ms / ms of oligosaccharides has attractive features , for example that the glycosidic linkages of fucose are rather stable , in contrast to their labile behavior in positive - ion mode .
harvey has described several diagnostic ions , which are observed in negative - ion mode ms / ms of n - glycans and allow the elucidation of antenna compositions as well as the differentiation between the 6-branch and 3-branch of the glycan [ 36 , 37 ] .
moreover , the carboxylic acid groups of sialylated glycans are protected by methyl esterification , which stabilizes the sialic acids and enables maldi - tof - ms profiling of permethylated neutral and acidic glycans simultaneously . by contrast ,
sialic acids are labile when analyzing native glycans , leading to the observation of degradation products in maldi - tof - ms spectra [ 16 , 33 ] .
analysis of the sodium adducts of permethylated glycans by tandem ms is a very useful approach for detailed structural characterization as - next to cleavages of glycosidic bonds - diagnostic cross - ring cleavages are observed , which reveal linkage positions .
these analyses may be performed by high - energy collision - induced dissociation ( cid ) maldi - tof / tof - ms resulting in very complex yet informative fragmentation spectra .
maldi - ion trap - ms of permethylated n - glycans released from total plasma glycoproteins has recently been established by guillard et al . .
this approach allows in - depth analysis of glycans by multistage - tandem mass spectrometry as exemplified in fig . 1 : ms2 ( fig .
1b ) provided evidence for the occurrence of a sialyl - lewis x structure on a plasma n-glycan.fig .
1permethylated serum n - glycans measured by maldi - linear ion trap - ms ( a ) .
fragments at m / z 1022 , 1143 , and 2690 are indicative of antenna fucosylation , whereas fragments at m / z 1113 , 1317 , and 2516 mark core fucosylation ( b ) .
the inset in ( b ) shows an ms3 experiment of m / z 1022 confirming the proposed structurewith terminal sialic acid and antenna fucosylation .
filled square , glcnac , empty circle , galactose ; filled circle , mannose ; filled triangle , fucose ; filled diamond , n - acetylneuraminic acid . taken from with permission permethylated serum n - glycans measured by maldi - linear ion trap - ms ( a ) .
fragments at m / z 1022 , 1143 , and 2690 are indicative of antenna fucosylation , whereas fragments at m / z 1113 , 1317 , and 2516 mark core fucosylation ( b ) .
the inset in ( b ) shows an ms3 experiment of m / z 1022 confirming the proposed structurewith terminal sialic acid and antenna fucosylation .
filled square , glcnac , empty circle , galactose ; filled circle , mannose ; filled triangle , fucose ; filled diamond , n - acetylneuraminic acid . taken from with permission analysis of permethylated glycans in combination with esi - ion trap - ms
when this approach is combined with multistage fragmentation of permethylated glycans , the combination of various characteristic fragmentation spectra of sub - structures of the precursor oligosaccharides allows the unambiguous structural assignment of large oligosaccharide structures as impressively demonstrated by reinhold and coworkers [ 25 , 26 ] .
for example reductive amination or permethylation using deuterated or c13-labeled versions of the tag / chemicals have been shown to be advantageous for oligosaccharide quantification and the detailed comparison of glycan profiles .
it has to be noted , however , that most of these isotope labeling strategies have not yet been applied to clinical glycomics research questions .
in addition to the analysis of released glycans studying protein glycosylation at the level of glycopeptides is rapidly gaining importance [ 4044 ] . the peptide portion may be seen as a tag , which potentially allows the assignment of the glycan to a specific n- or o - glycosylation site on a specific protein . however , this approach is complicated by several obstacles .
first , proteolytic cleavage is often hindered in highly glycosylated proteins , resulting in very large , highly and heterogeneously glycosylated peptide moieties , which are hardly accessible for ms analysis .
second , a variety of glycans are generally found attached to one specific glycosylation site ( microheterogeneity of glycosylation ) , and different n - glycosylation sites on one protein often have different glycan patterns .
therefore , glycopeptides generally occur substoichiometrically , making them difficult to analyze by ms in the presence of a majority of non - glycosylated peptides . various enrichment techniques including lectin affinity chromatography are available to purify glycopeptides for ms analysis [ 3 , 46 ] .
a very promising technique for enriching n - glycopeptides is hydrophilic interaction liquid chromatography - solid phase extraction ( hilic - spe ) , which may be performed using silica - based or carbohydrate - based stationary phases [ 30 , 31 , 47 , 48 ] .
third , depending on the size of the glycan moiety and the chosen ms / ms approach , it is often hard to obtain peptide sequence information , which is in most cases needed for unambiguous assignment of the glycan to a specific protein .
popular approaches are electron capture dissociation ( ecd ) and electron transfer dissociation ( etd ) of glycopeptides as well as various types of ( multistage ) cid [ 4 , 43 , 44 ] . in ecd and etd the glycan portion is generally stable , and peptide backbone cleavages tend to provide ( some ) peptide sequence information .
single stage low - energy cid ( as occurring on an ion trap ) is generally characterized by fragmentation of glycosidic bonds , and peptide backbone cleavages are usually minor , if detectable at all .
fragmentation of the peptide portion may be achieved by performing ion trap - multistage ms / ms , and has been successfully applied in various cases for the identification of glycosylated proteins and glycosylation sites [ 41 , 43 ] . alternatively , fragmentation of glycopeptides at elevated energies in maldi - tof / tof - ms and maldi- or esi - quadrupole - tof - ms has been reported to provide peptide sequence information next to information on glycan composition and structure .
it has been observed that under these conditions glycan moieties may undergo rearrangements in ms / ms , of which prominent examples are the migration of fucoses between n - glycan antennae , or from the core to outer portions of the n - glycan structure [ 49 , 50 ] .
these rearrangements may not only be observed for n - glycopeptides , but also for o - glycopeptides . obviously , awareness of these processes is required for avoiding misinterpretation of glycopeptide fragmentation spectra .
the major bottle - neck in glycopeptidomics - based proteomics of complex samples is data analysis .
software supporting data analysis is desperately needed , and several promising approaches have recently been reported ( and references cited therein ) . yet additional , concerted efforts in developing data analysis tools are needed to boost the impact of this analytical approach . analyzing protein glycosylation at the glycopeptide level
the analysis of the intact mass of glycoproteins , together with a bottom - up analysis , often allows the detailed structural assignment of protein species such as monoclonal antibodies [ 51 , 52 ] .
in addition , top - down glycoproteomics , i.e. , the ms analysis of intact glycoproteins followed by their tandem ms analysis for the characterization of posttranslational modifications including glycosylation , has high potential but needs to be further developed [ 53 , 54 ] .
igg glycopeptide profiling by maldi - tof - ms has been performed to determine the changes in igg1 and igg2 fc glycosylation features with pregnancy and rheumatoid arthritis as well as with longevity and healthy aging .
maldi - fticr - ms was likewise evaluated for igg fc glycopeptides profiling and was found to be particularly useful for analyzing changes in sialylation .
maldi - fticr - ms analysis of igg fc glycopeptides is characterized by reduced losses of sialic acid , which is most probably due to the higher pressure in the source and the resulting collisional cooling , in combination with the lower extraction voltages as compared to maldi - tof - ms . recently , using a sheath - flow esi sprayer , a robust nanolc - ms method for igg fc glycosylation profiling was established ( fig . 2 ) .
notably , the sheath - flow esi sprayer setup was found to significantly increase the long - term stability of the system while keeping the sensitivity of the system in the same range as with conventional nano - esi - ms .
high - sensitivity igg fc glycosylation analysis is particularly valuable when analyzing affinity - purified , antigen - specific iggs , which may be present at low concentrations . for the most common applications , however , such as glycosylation analysis of total plasma igg and biotechnologically produced
igg the available sample amounts are generally plenty and sensitivity is not an issue . the sheath flow setup was used in combination with trifluoracetic acid containing running solvents resulting in the coelution of sialylated and non - sialylated igg fc glycopeptides . in contrast , conventional nano - lc - ms with formic acid - containing running solvents features early - eluting glycopeptides with neutral glycans and late - eluting ones with sialylated glycans .
it was found that galactosylation , sialylation were increased whilst fucosylation and the incidence of bisecting glcnac were decreased during pregnancy .
the observed glycosylation changes may contribute to the immune suppression occurring during pregnancy in order to protect the fetus from alloimmune reactions of the mother .fig .
long - term stability of ms detection in nanolc - ms is achieved using a sheath - flow esi spray with a sheath flow of 2 l / min 50 % isopropanol , 20 % propionic acid ( a ) .
the extracted ion chromatograms of igg1 , igg4 and igg2/3 tryptic fc glycopeptide species ( b ) , and the mass spectrum of the igg1 fc glycopeptides elution range is shown in ( c ) .
glycopeptide signals observed below m / z 1200 are triple protonated , and signals above m / z 1200 are double protonated .
the excellent repeatability of the overall sample preparation and analysis method is demonstrated in ( d ) for igg1 .
blue square , n - acetylglucosamine ; red triangle , fucose ; green circle , mannose ; yellow circle , galactose ; purple diamond , n - acetylneuraminic acid igg fc - glycosylation profiling by lc - quadrupole - tof - ms .
long - term stability of ms detection in nanolc - ms is achieved using a sheath - flow esi spray with a sheath flow of 2 l / min 50 % isopropanol , 20 % propionic acid ( a ) .
the extracted ion chromatograms of igg1 , igg4 and igg2/3 tryptic fc glycopeptide species ( b ) , and the mass spectrum of the igg1 fc glycopeptides elution range is shown in ( c ) .
glycopeptide signals observed below m / z 1200 are triple protonated , and signals above m / z 1200 are double protonated .
the excellent repeatability of the overall sample preparation and analysis method is demonstrated in ( d ) for igg1 .
blue square , n - acetylglucosamine ; red triangle , fucose ; green circle , mannose ; yellow circle , galactose ; purple diamond , n - acetylneuraminic acid
next to glycoproteins , glycolipids play an important role in cellular interaction and cellular differentiation . the majority of glycolipids observed in humans have a ceramide portion ( sphingoid base carrying an amide - linked fatty acid ) and are , therefore , categorized as glycosphingolipids .
they occur mainly in the outer leaflet of the plasma membrane and also in the inner membranes .
glycosphingolipids show a marked tissue- and cell type - specific expression pattern [ 59 , 60 ] .
this is for example reflected by the fact that various human cluster of differentiation ( cd ) markers , which are differentially expressed between leukocytes , are glycolipids such as cd60a ( gd3 ; neu5ac(2 - 8)neu5ac(2 - 3)gal(1 - 4)glc(1 - 1)ceramide ) , cd60b ( 9-o - acetyl gd3 ) , cd60c ( 7-o - acetyl gd3 ) , cd77 ( gb3 ; globotriaosylceramide ; gal(1 - 4)gal(1 - 4)glc(1 - 1)ceramide ) ) ( http://www.hcdm.org/ ) .
recently , chip - based approaches for glycosphingolipid analysis have been reviewed [ 59 , 62 ] .
importantly , there is also technology available to combine the most commonly applied separation technique in lipid , as well as glycolipid analysis , high - performance thin layer chromatography ( hptlc ) , with ms .
for example , glycosphingolipids separated by hptlc can be probed by overlay detection using carbohydrate - binding proteins such as lectins , bacterial toxins , and antibodies , followed by the ms analysis of positive bands , either directly from the hptlc plate or after lipid extraction , as reviewed recently by meisen et al . .
alternatively , glycolipids may be analyzed by hilic - nano - lc - ms using slightly adjusted solvent conditions when compared to methods used for glycan and glycopeptide separation . using this approach ,
it has been demonstrated that 2 - 3-sialylated and 2 - 6-sialylated isomers of lactoneotetraosylceramides can be baseline separated from complex mixtures and characterized individually by tandem ms .
the importance of the above described techniques is illustrated by their application in clinical studies .
glycosylation changes play important roles in the cellular mechanisms of health and disease , and glycans have a great potential as biomarkers for different types of cancer [ 66 , 67 ] .
there is a vast range of studies of human glycobiology in healthy and diseased people employing ms , and some selected examples will be presented demonstrating the potential of mass spectrometric approaches for clinical glycomics .
established an approach that relies on n - glycan release from total plasma , permethylation , and maldi - ion trap - ms measurement , allowing in - depth analysis of glycans by tandem ms ( fig . 1 ) .
this approach was applied to determine plasma n - glycan profiles of congenital disorder of glycosylation ( cdg ) type ii patients , as well as controls .
a total of 38 peaks were assigned in terms of molecular composition , and changes in the n - glycan profiles were found to be useful to distinguish between the patient groups .
the authors also successfully addressed the challenge of differentiating cdg type ii diseases from other diseases with secondary causes of underglycosylation .
this method is now being successfully applied in clinical research , including research on patients with defects in 1 - 4-galactosyltransferase i ( b4gat1 ) , which leads to the expression of largely truncated glycans on plasma proteins .
lysosomal defects of glycoconjugate degradation may lead to the secretion of glycopeptides , glycolipids or oligosaccharides in patient urine .
molecular analysis of these degradation products by ms often directly pinpoints to the genetic defect . in schindler
s disease , which is a hereditary n - acetylhexosaminidase deficiency , characteristic o - glycosylated amino acids and o - linked glycopeptides were detected from patients urines . in fabry s disease ,
the causative enzymatic defect leads to elevated levels of globotriaosylceramide and lyso - globotriaosylceramide species in urine and plasma , which can be detected by lc - ms with good diagnostic sensitivity and specificity [ 71 , 72 ] . a very powerful approach for the analysis of urinary oligosaccharides is hpaec , which was applied in capillary - scale with online - desalting and esi - ion trap - ms / ms analysis to study urinary oligosaccharides of patients with gm1-gangliosidosis and galactosialidosis [ 32 , 73 ] . on the basis of literature knowledge of n - glycan biosynthesis
, this approach allowed the structural assignment of chromatographically separated isomeric n - glycan degradation products in gm1-gangliosidosis ( fig .
the observation of n - glycans with terminal galactose residues points to a deficiency of -galactosidase activity .
when the same analytical setup was applied to study urinary glycans in galactosialidosis , novel degradation products were observed such as glycolipid - derived oligosaccharides , both in reducing form and with c1-oxidation of the innermost glucose .
these results indicate the presence of an alternative glycolipid degradation pathway in galactosialidosis patients involving a hitherto not described endoglycoceramidase activity.fig .
3high performance - anion exchange chromatography with online ms detection of urinary oligosaccharides of a gm1-gangliosidosis patient ( a ) .
next to the total ion chromatogram ( tic ) specific extracted ion chromatograms are given labeled with the composition of the oligosaccharide in terms of hexoses ( h ) and n - acetylhexosamines ( n ) .
the ion trap tandem mass spectra obtained for the two detected h3n2 isomers are shown in ( b ) and ( c ) .
green circle , mannose ; yellow circle , galactose ; blue square , n - acetylglucosamine .
taken from with permission high performance - anion exchange chromatography with online ms detection of urinary oligosaccharides of a gm1-gangliosidosis patient ( a ) .
next to the total ion chromatogram ( tic ) specific extracted ion chromatograms are given labeled with the composition of the oligosaccharide in terms of hexoses ( h ) and n - acetylhexosamines ( n ) .
the ion trap tandem mass spectra obtained for the two detected h3n2 isomers are shown in ( b ) and ( c ) .
green circle , mannose ; yellow circle , galactose ; blue square , n - acetylglucosamine .
taken from with permission the analysis of protein degradation products from bio fluids has repeatedly led to the identification of glycopeptides , thereby shedding new light on protein glycosylation .
apolipoprotein ciii - derived o - glycopeptides were found in the urine of schistosoma mansoni infected individuals .
remarkably , these glycopeptides did not exhibit the sialylated t - antigen glycan structures found on apolipoprotein ciii from human serum , but instead carried larger o - glycan structures with a high degree of sialylation . in another study ,
an o - glycosylated peptide stemming from the c - terminus of the fibrinogen -chain was found to be increased in the urine during urinary tract infection with escherichia coli .
recently , o - glycosylated amyloid -peptides representing a potential disease biomarker were characterized from cerebrospinal fluid of alzheimer patients using both cid and ecd fragmentation .
cancer glycomics biomarker discovery has recently been reviewed [ 66 , 67 ] , and ms is becoming an important research tool in this field .
novotny and mechref with coworkers chose to analyze serum n - glycan profiles after permethylation using maldi - tof - ms . using this approach , they demonstrated vastly different n - glycan profiles in metastatic prostate cancer as compared to healthy tissue .
a variety of mainly fucosylated , complex - type n - glycans were found to be increased in cancer vs. control . in another
study the relative abundances of a set of 8 complex - type serum n - glycans were found to be indicative of the progression of breast cancer .
for example , maldi - ms of 2-aminobenzoic acid - labeled n - glycans showed that the n - glycan fucosylation of -1-acid glycoprotein is significantly increased in ovarian cancer .
notably , most of the reported cancer glycomics studies focus on the analysis of the total plasma or serum n - glycome or certain acute - phase proteins [ 66 , 67 ] .
while these approaches are promising , an increase in sensitivity and specificity may be expected when tumor - derived antigens isolated from body fluids are characterized together with their specific glycosylation profiles .
still another glycomics application area for ms is the study of the genetic and environmental regulation and dysregulation of protein glycosylation in health and diseases .
for example , various novel aspects of the regulation of immunoglobulin g fc glycosylation have only recently been revealed by high - sensitivity glycosylation profiling at the glycopeptide level . employing this analysis technique , in vitro studies
have shown that soluble factors such as cytokines and toll - like receptor ligands modulate the degree of igg fc galactosylation , sialylation and the incidence of bisecting glcnac .
likewise , fucosylation of igg fc glycans appears to be regulated in humans : igg fc glycan fucosylation in humans is known to be generally above 90 % , yet recently pathogenic alloantibodies with a low degree of fucosylation ( 50 % and below ) have been described for patients with fetal and neonatal alloimmune thrombocytopenia ( fnait ) .
figure 4 shows the total serum igg1 fc glycosylation profile of a patient and the corresponding profile of the pathogenic anti - human plate antigen ( hpa ) 3a alloantibodies .
while the total serum igg1 shows 9 % afucosylated structures ( a ) , the afucosylation is 38 % for the alloantibodies of this patient ( b ) .
low fucosylation has been associated with enhanced cellular cytocoxicity , whilst high degrees of sialylation confer anti - inflammatory properties to iggs .fig .
fc glycosylation of total serum igg1 ( a ) and anti - hpa3a alloantibodies from a patient with pregnancy complications ( fetal and neonatal alloimmune thrombocytopenia ; fnait ) ( b ) .
glycopeptides were detected in triple protonated form by nanolc - esi - ion trap - ms carrying neutral n - glycan chains ( left panels ) and acidic n - glycan chains ( right panels ) . in
blue square , n - acetylglucosamine ; yellow circle , galactose , green circle , mannose ; red triangle , fucose ; purple diamond , n - acetylneuraminic acid ; pep , tryptic peptide moiety ; asterisk , non - glycopeptide signal . taken from with permission low core - fucosylation of anti - hpa-3a alloantibodies .
fc glycosylation of total serum igg1 ( a ) and anti - hpa3a alloantibodies from a patient with pregnancy complications ( fetal and neonatal alloimmune thrombocytopenia ; fnait ) ( b ) .
glycopeptides were detected in triple protonated form by nanolc - esi - ion trap - ms carrying neutral n - glycan chains ( left panels ) and acidic n - glycan chains ( right panels ) . in
blue square , n - acetylglucosamine ; yellow circle , galactose , green circle , mannose ; red triangle , fucose ; purple diamond , n - acetylneuraminic acid ; pep , tryptic peptide moiety ; asterisk , non - glycopeptide signal . taken from with permission
in the coming years the field of mass spectrometric analysis of protein glycosylation is expected to show an increase in measurement sensitivity and precision as well as sample throughput , allowing the in - depth analysis of biological systems , with data analysis being a major challenge . despite the limitations mentioned in this review , glycoproteomics approaches focusing on the glycopeptide level will gain in popularity .
mass spectrometric analyses of ( tryptic ) glycopeptides are rewarding as they have intrinsically the potential of assigning specific glycan structures to a specific site on a specific protein .
this information is often of utmost importance , as the primary role of glycans is modulating the properties ( such as function , activity , stability , targeting ) of their carrier proteins .
notably , approaches based on the analysis of released n - glycans often fail to provide this information on protein- and site - specificity and are , therefore , of limited value .
another important aspect is the analysis of intact glycoproteins by ms , which is expected to gain importance in the next years . on the one hand ,
ms analysis of intact glycoproteins allows the integration of the information obtained at the glycopeptide and released glycan level to obtain an overall view of protein glycosylation . on the other hand intact protein analysis may be accompanied by top - down tandem mass spectrometric analysis for characterization of posttranslational modifications , including glycosylation .
it is anticipated that the concept of a specific protein having specific functions will undergo refinement , and specific proteins will be perceived as an assembly of isoforms ( including glycoforms ) that are caused by a variety of posttranslational modifications including glycosylation . defining such
protein species is of utmost importance for functional proteomics supporting systems biology and will require bioinformatics tools and databases to facilitate posttranslational modification analysis at the glycopeptide level . | mass spectrometry plays an increasingly important role in structural glycomics .
this review provides an overview on currently used mass spectrometric approaches such as the characterization of glycans , the analysis of glycopeptides obtained by proteolytic cleavage of proteins and the analysis of glycosphingolipids .
the given examples are demonstrating the application of mass spectrometry to study glycosylation changes associated with congenital disorders of glycosylation , lysosomal storage diseases , autoimmune diseases and cancer . |
optical
sensing of solid - state nanopores is a relatively new approach
that can enable high - throughput , multicolor readout from a collection
of nanopores .
it is therefore highly attractive for applications such
as nanopore - based dna sequencing and genotyping using dna barcodes .
however , to date optical readout has been plagued by the need to achieve
sufficiently high signal - to - noise ratio ( snr ) for single fluorophore
sensing , while still maintaining millisecond resolution .
one of the
main factors degrading the optical snr in solid - state nanopores is
the high photoluminescence ( pl ) background emanating from the silicon
nitride ( sinx ) membrane in which pores
are commonly fabricated .
focusing on the optical properties of sinx nanopores we show that the local membrane
pl intensity is substantially reduced , and its spectrum is shifted
toward shorter wavelengths with increasing e - beam dose .
this phenomenon ,
which is correlated with a marked photocurrent enhancement in these
nanopores , is utilized to perform for the first time single molecule
fluorescence detection using both green and red laser excitations .
specifically , the reduction in pl and the concurrent measurement of
the nanopore photocurrent enhancement allow us to maximize the background
suppression and to detect a dual color , five - unit dna barcode with high snr levels . | |
use 0.5 m circular , closed mesocosms to prevent emigration or immigration of animal species ( figure 1 ) . construct mesocosms using 2.4 m lengths of 1.5 m high " mesh aluminum fence as a scaffolding . cover the fencing with 2.5 m lengths 1.75 m high aluminum window screening folded over the top and bottom of the fencing and stapled together along fold . join the fencing ends to form a closed circle and then staple the overlapping window screening together to create a seal . set the mesocosm into the soil in the field by digging a 10 cm deep by 4 cm wide trench around the base of the mesocosom , sink the mesocosm into the trench and then tamp the sod of the trench around the sunken part of the mesocosm .
plot locations should be selected to match the plant species identity and plant relative cover .
sink cages 10 cm into the ground at the plot site . using a sweep net ,
collect early ( 2 ) instar grasshopper nymphs and stock them into the mesocosms at natural field densities . using a sweep net ,
capture individuals of a dominant sit - and - wait hunting ( not web weaving ) spider predator species .
glue shut the spider chelicerae ( mouthparts used to subdue prey ) with fast - drying cement to decoupled risk effects from actual survival selection favoring individual grasshoppers with better abilities to evade spider predation .
collect all individuals from the cages and randomly assign individuals from each cage to one of three subsequent assay groups : ( 1 ) validation of physiological stress state ; ( 2 ) validation of shift in body elemental stoichiometry ; ( 3 ) microbial decomposition .
measure grasshopper standard metabolic rate ( smr ) , as the rate of carbon dioxide emission ( ) in an incurrent flow - through respirometry system with an air flow rate of 200 ml / min .
remove water vapor by passing flowing air through a drying agent . following food deprivation of 16 hr
( water should be available ) , weigh individual grasshoppers ( 0.1 mg ) , and place them in transparent 50 ml ( 9.2 cm l x 2.0 cm d ) respirometer chambers and allow them to recover from handling for at least 10 min before measurements commence . under constant average ambient temperature ( temperature 1% standard error variation ) within the respirometer chamber ,
analyze respired air using an infra - red co2 analyzer ( e.g. qubit s151- 1 ppm resolution ) .
the analyzer provides fractional co2 concentration ( parts - per - million ) , yet smr should be reported as a rate , so one must transformed the recordings as where
assumed equal to 0.85 in herbivorous animals . evaluate carbon : nitrogen ( c : n ) content of a sample of grasshoppers collected from the field mesocosms .
reduce variation in c : n due to recent food consumption by removing grasshopper gut contents under a dissecting microscope .
freeze - dry the empty gut and body for 48 hr and then grind the individual carcass and gut to a homogeneous powder .
, inserted ~4 cm into the soil ) in the field site ( figure 3c ) .
these collars are used for decomposition measures . additionally , establish a set of pvc collars across the field site to act as c natural - abundance controls ( see below ) , to which neither grasshoppers nor grass - litter are added .
to one collar in each pair add 2 intact , freeze - dried carcasses of grasshoppers ( record the biomass added ) reared with predator risk as described above using field - cages . to the other collar in each pair add 2 intact freeze - dried carcasses reared without predator risk .
cover the pvc collars with a screen to prevent grasshopper removal by scavengers from the plots and let the grasshopper carcasses decompose for 40 days .
while carcasses are decomposing , label grass - litter with c. construct a clear plexiglass chamber ( 60 cm x 60 cm x 1.5 m ) with an inlet and outlet valve ( figure 3b).sink a square 60 cm x 60 cm wooden frame with a rubber seal coated with silicon grease 5 cm into the ground ( figure 3b).slide the chamber on top of the wooden frame so that the chamber becomes sealed by the rubber ( figure 3b).connect the chamber inlets to compressed gas cylinders containing 99 atom% co2 .
plants inside the chamber will be labelled with c , where co2 concentrations are maintained at ambient levels ( because elevating concentrations alters plant carbon partitioning ) .
also , chamber temperatures are monitored and chambers are removed if temperatures reach 5 c above ambient.one week after labeling , compare c of the grass - litter with natural abundance values collected from a random sample of identical grass species using a thermo deltaplus isotope ratio mass spectrometer ( thermo , san jose , ca , usa ) .
construct a clear plexiglass chamber ( 60 cm x 60 cm x 1.5 m ) with an inlet and outlet valve ( figure 3b ) . sink a square 60 cm
x 60 cm wooden frame with a rubber seal coated with silicon grease 5 cm into the ground ( figure 3b ) .
slide the chamber on top of the wooden frame so that the chamber becomes sealed by the rubber ( figure 3b ) .
plants inside the chamber will be labelled with c , where co2 concentrations are maintained at ambient levels ( because elevating concentrations alters plant carbon partitioning ) .
also , chamber temperatures are monitored and chambers are removed if temperatures reach 5 c above ambient .
one week after labeling , compare c of the grass - litter with natural abundance values collected from a random sample of identical grass species using a thermo deltaplus isotope ratio mass spectrometer ( thermo , san jose , ca , usa ) .
after 40 days , add 10 g of air - dried c - labeled grass - litter to each collar that had previously been amended with grasshopper carcasses .
monitor the mineralization rate of the grass - litter in situ across 75 days by capping each collar and tracking both total soil respiration and the respiration of co2 .
this is accomplished using a flow - through chamber technique where gas samples from each collar are monitored , in real time , for 8 min each using cavity ring - down spectroscopy ( crds ; picarro inc . , santa clara , ca , usa ; model : g1101-i ) .
estimate the contribution of c - labeled grass - litter to total soil respiration using isotope mixing equations .
the amount of grass - litter derived co2 is calculated as follows : cgrass - litter derived = ctotal ( crespiration- cnat.abn.)/ ( cgrass - litter- cnat.abn ) , where ctotal is the total amount of c respired during a given measurement , crespiration is the c of respired - c for collars amended with labeled grass litter , cnat.abn . is the mean c of respired c in the three natural abundance collars ( i.e. those that were not amended with litter ) , and cgrass - litter is the c of the grass litter added to the collars .
monitor both soil temperature and moisture across the experiment using hand - held probes to correct for differences in soil respiration due to differences in temperature and moisture .
although intended for field use , the cavity ring - down spectroscopy instrument ( picarro inc . ,
santa clara , ca , usa ; model : g1101-i ) readings are sensitive to movement . therefore , one should erect a base measurement station central to all of the plots containing pvc collars , and connect the instrument to the collars with lengths of pvc tubing .
use 0.5 m circular , closed mesocosms to prevent emigration or immigration of animal species ( figure 1 ) . construct mesocosms using 2.4 m lengths of 1.5 m high " mesh aluminum fence as a scaffolding . cover the fencing with 2.5 m lengths 1.75 m high aluminum window screening folded over the top and bottom of the fencing and stapled together along fold . join the fencing ends to form a closed circle and then staple the overlapping window screening together to create a seal . set the mesocosm into the soil in the field by digging a 10 cm deep by 4 cm wide trench around the base of the mesocosom , sink the mesocosm into the trench and then tamp the sod of the trench around the sunken part of the mesocosm .
plot locations should be selected to match the plant species identity and plant relative cover .
sink cages 10 cm into the ground at the plot site . using a sweep net ,
collect early ( 2 ) instar grasshopper nymphs and stock them into the mesocosms at natural field densities . using a sweep net ,
capture individuals of a dominant sit - and - wait hunting ( not web weaving ) spider predator species .
glue shut the spider chelicerae ( mouthparts used to subdue prey ) with fast - drying cement to decoupled risk effects from actual survival selection favoring individual grasshoppers with better abilities to evade spider predation .
collect all individuals from the cages and randomly assign individuals from each cage to one of three subsequent assay groups : ( 1 ) validation of physiological stress state ; ( 2 ) validation of shift in body elemental stoichiometry ; ( 3 ) microbial decomposition .
measure grasshopper standard metabolic rate ( smr ) , as the rate of carbon dioxide emission ( ) in an incurrent flow - through respirometry system with an air flow rate of 200 ml / min .
remove water vapor by passing flowing air through a drying agent . following food deprivation of 16 hr
( water should be available ) , weigh individual grasshoppers ( 0.1 mg ) , and place them in transparent 50 ml ( 9.2 cm l x 2.0 cm d ) respirometer chambers and allow them to recover from handling for at least 10 min before measurements commence . under constant average ambient temperature ( temperature 1% standard error variation ) within the respirometer chamber ,
analyze respired air using an infra - red co2 analyzer ( e.g. qubit s151- 1 ppm resolution ) .
the analyzer provides fractional co2 concentration ( parts - per - million ) , yet smr should be reported as a rate , so one must transformed the recordings as where
assumed equal to 0.85 in herbivorous animals .
evaluate carbon : nitrogen ( c : n ) content of a sample of grasshoppers collected from the field mesocosms .
reduce variation in c : n due to recent food consumption by removing grasshopper gut contents under a dissecting microscope .
freeze - dry the empty gut and body for 48 hr and then grind the individual carcass and gut to a homogeneous powder .
place replicated pairs of pvc collars ( 15.4-cm dia . , inserted ~4 cm into the soil ) in the field site ( figure 3c ) . remove all vegetation within them via clipping at the soil surface .
these collars are used for decomposition measures . additionally , establish a set of pvc collars across the field site to act as c natural - abundance controls ( see below ) , to which neither grasshoppers nor grass - litter are added .
to one collar in each pair add 2 intact , freeze - dried carcasses of grasshoppers ( record the biomass added ) reared with predator risk as described above using field - cages . to the other collar in each pair add 2 intact freeze - dried carcasses reared without predator risk .
cover the pvc collars with a screen to prevent grasshopper removal by scavengers from the plots and let the grasshopper carcasses decompose for 40 days .
while carcasses are decomposing , label grass - litter with c. construct a clear plexiglass chamber ( 60 cm x 60 cm x 1.5 m ) with an inlet and outlet valve ( figure 3b).sink a square 60 cm x 60 cm wooden frame with a rubber seal coated with silicon grease 5 cm into the ground ( figure 3b).slide the chamber on top of the wooden frame so that the chamber becomes sealed by the rubber ( figure 3b).connect the chamber inlets to compressed gas cylinders containing 99 atom% co2 .
plants inside the chamber will be labelled with c , where co2 concentrations are maintained at ambient levels ( because elevating concentrations alters plant carbon partitioning ) .
also , chamber temperatures are monitored and chambers are removed if temperatures reach 5 c above ambient.one week after labeling , compare c of the grass - litter with natural abundance values collected from a random sample of identical grass species using a thermo deltaplus isotope ratio mass spectrometer ( thermo , san jose , ca , usa ) .
construct a clear plexiglass chamber ( 60 cm x 60 cm x 1.5 m ) with an inlet and outlet valve ( figure 3b ) . sink a square 60 cm x 60 cm wooden frame with a rubber seal coated with silicon grease 5 cm into the ground ( figure 3b ) .
slide the chamber on top of the wooden frame so that the chamber becomes sealed by the rubber ( figure 3b ) .
plants inside the chamber will be labelled with c , where co2 concentrations are maintained at ambient levels ( because elevating concentrations alters plant carbon partitioning ) .
ambient levels are maintained by only pulsing labeled co2 for short periods of time . also , chamber temperatures are monitored and chambers are removed if temperatures reach 5 c above ambient .
one week after labeling , compare c of the grass - litter with natural abundance values collected from a random sample of identical grass species using a thermo deltaplus isotope ratio mass spectrometer ( thermo , san jose , ca , usa ) .
after 40 days , add 10 g of air - dried c - labeled grass - litter to each collar that had previously been amended with grasshopper carcasses .
monitor the mineralization rate of the grass - litter in situ across 75 days by capping each collar and tracking both total soil respiration and the respiration of co2 .
this is accomplished using a flow - through chamber technique where gas samples from each collar are monitored , in real time , for 8 min each using cavity ring - down spectroscopy ( crds ; picarro inc . ,
estimate the contribution of c - labeled grass - litter to total soil respiration using isotope mixing equations .
the amount of grass - litter derived co2 is calculated as follows : cgrass - litter derived = ctotal ( crespiration- cnat.abn.)/ ( cgrass - litter- cnat.abn ) , where ctotal is the total amount of c respired during a given measurement , crespiration is the c of respired - c for collars amended with labeled grass litter , cnat.abn .
is the mean c of respired c in the three natural abundance collars ( i.e. those that were not amended with litter ) , and cgrass - litter is the c of the grass litter added to the collars .
monitor both soil temperature and moisture across the experiment using hand - held probes to correct for differences in soil respiration due to differences in temperature and moisture .
although intended for field use , the cavity ring - down spectroscopy instrument ( picarro inc . , santa clara , ca , usa ; model : g1101-i ) readings are sensitive to movement .
therefore , one should erect a base measurement station central to all of the plots containing pvc collars , and connect the instrument to the collars with lengths of pvc tubing .
an example plot of grasshopper standard metabolic rates in stress and stress free conditions are presented in figure 2 . due to body mass differences among individual grasshoppers , and
the fact that metabolic rate varies with body mass , plots should present metabolic rates in relation to grasshopper body mass .
parallel trends for the different treatments indicate that metabolic rate rises as a constant multiple of standard metabolic rate ( i.e. there is no body mass x metabolic rate interaction ) for all stressed individuals .
grasshopper body c and n elemental contents in risk and risk free conditions are presented in table 1 .
it is noteworthy that there is a very small ( 4% ) difference in body c : n ratios between treatments .
nevertheless , these small differences can translate into large differences in grass litter decomposition by the soil microbial pool ( figure 3 ) . adding grass litter to pvc collars previously amended with stressed or stress - free grasshoppers leads to different degrees of litter decomposition , as reflected in the curves describing cumulate co2 release from the soil due to microbial respiration ( figure 3 ) .
comparison of the chemical content of grasshopper herbivore carcasses from conditions in which they faced predation risk ( stress ) and in which predation risk was absent ( stress free ) .
illustration of the design of the field mesocosms used in the experiment and overall scheme of the experimental evaluation of risk effects on litter decomposition .
figure 2 . a plot of herbivore standard metabolic rate in relation to herbivore body mass .
the data are divided into two classes according to experimental treatment : grasshoppers from mesocosms containing predators ( predation ) to induce stress , and mesocosms without predators ( control ) and hence no induced stress .
curves describing cumulative co2 release by the microbial pool while decomposing experimental grass litter inputs in pvc collars . plotted values are mean 1 standard error .
the graph demonstrates that soils primed with stressed grasshopper carcasses ( predator ) result in 19% lower ( anova f1,6 = 9.06 , p < 0.05 ) plant litter decomposition rates than soils primed with stress free grasshopper carcasses ( control ) .
the sequence of methods presented here should allow systematic measurement of the way stress in species comprising above - ground food webs can prime soil microbial communities in ways that lead to alteration of subsequent decomposition of plant litter .
the methods are ideal for studying ecosystems comprised of arthropod consumers and herbaceous plants because intact food webs can be spatially circumscribed and contained within mesocosms .
spatial variability may exist due to gradients in background soil moisture , soil temperature , plant nutrient content , etc .
the study design allows one to array mescosms and pvc collars to block along spatial environmental gradients and thereby account for such environmental variation when analyzing for effects . although intended for field use , the cavity ring - down spectroscopy instrument ( picarro inc . ,
santa clara , ca , usa ; model : g1101-i ) readings are sensitive to movement .
therefore , one should erect a base measurement station central to all of the plots containing pvc collars , and connect the instrument to the collars with lengths of pvc tubing .
soil litter decomposition has traditionally been measured by enclosing known quantities of litter into fiberglass mesh bags , depositing the bags onto the soil surface in the field and periodically re - measuring the bags to quantify litter disappearance rate ( decomposition ) .
the limitation of this method is that one is unable to trace the fate of the decomposed matter or determine the contribution to co2 mineralization of the soil amendment ( added litter ) from background soil co2 mineralization .
ecosystem ecology and biogeochemistry have operated under the working paradigm that because uneaten plant - litter comprises the majority of detritus , belowground ecosystem processes are only marginally influenced by biomass inputs from higher trophic levels in aboveground food webs , such as herbivores themselves . however , there is growing evidence that species in higher trophic levels of ecosystems can have a profound influence on belowground processes .
the method presented here stands to enhance quantification of the contribution of higher trophic levels , either directly through biomass from carcass deposition ( e.g.
) or excretion and egestion ( e.g.
) or indirectly through alteration of plant community composition ( e.g.
) on ecosystem nutrient cycling .
such quantification can help reveal the mechanisms by which animals control ecosystem dynamics as part of a concerted effort to enhance and revise the current working paradigm of biotic control over ecosystem functioning .
| the quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen ( n ) and carbon ( c ) sequestration1,2 .
plant litter comprises the majority of detritus3 , and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5 .
however , carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6 .
a physiological stress response by herbivores to the risk of predation can change the c : n elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient - limited systems forces herbivores to shift their consumption from n - rich resources to support growth and reproduction to c - rich carbohydrate resources to support heightened metabolism6 .
herbivores have limited ability to store excess nutrients , so stressed herbivores excrete n as they increase carbohydrate - c consumption7 .
ultimately , prey stressed by predation risk increase their body c : n ratio7,10 , making them poorer quality resources for the soil microbial pool likely due to lower availability of labile n for microbial enzyme production6 .
thus , decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.we present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes .
we describe how to : induce stress in herbivores from predation risk ; measure those stress responses , and measure the consequences on microbial decomposition .
we use insights from a model grassland ecosystem comprising the hunting spider predator ( pisuarina mira ) , a dominant grasshopper herbivore ( melanoplus femurrubrum),and a variety of grass and forb plants9 . |
unlike infections by other human hepatitis viruses , hepatitis c virus ( hcv ) infection of healthy immunocompetent adults is persistent or prolonged in the majority of cases , unlike hepatitis b , where it persist in the minority of cases .
investigators have suggested that viral interactions with tumor necrosis factor / tumor necrosis factor receptor ( tnf / tnfr ) family members may be important in immune evasion .
investigators have reported that hcv core protein binds to the cytoplasmic domain of tnfr1 , specifically the death domain , which may hinder or promote cell death during hcv infection .
in addition , the hcv core protein binds to another tnfr family member , lymphotoxin receptor 's ( ltr ) intracellular domain , which is involved in signal transduction .
investigators have also demonstrated that the hcv core protein potentiates nf6b activation initiated by tnf/tnfr and lt
1
2/ltr interactions , which , in turn may contribute to the chronically activated , persistent state of hcv - infected cells [ 3 , 4 ] .
interference with signaling through members of the tnf family of receptors may affect the chronicity of hcv via affecting either cell death and/or activation of nfb [ 1 , 3 , 4 ] . of interest ,
other investigators have examined the relationship between hcv and the soluble tnf receptors ( stnfr ) in the blood .
these stnfr levels appear to peak 9 hours after the first ifn administration correlating with ifn serum levels .
other investigators have noted higher levels of tnf and stnfrs in hcv+ patients than in hcv ( ) controls prior to treatment and postulated that high levels of stnfr might modify host responses but found no correlation between levels and response to therapy .
tnf receptors are present on the surface of a number of cell subsets and play a role in a variety of functions .
for example , tnf / tnfr interactions are imperative for the optimal proliferation and effector functions of cd8 + t cells [ 7 , 8 ] , whose antiviral effects are essential for the clearance of noncytopathic viruses , such as hcv . in addition ,
ltr - light / lt
1
2 interactions have been implicated in both optimal growth and effector functions as well as costimulation of cd8 + t cells .
similarly , tnfr is expressed on macrophages , peripheral blood monocytes , and antigen presenting cells ( apcs ) , cells known to be important in viral infections .
recently , hcv negative strand rna has been noted in the macrophages of 67% of sustained virological responders to interferon - based therapies .
others have noted the importance of monocytes and dendritic cells in the clearance of hcv [ 1114 ] .
the role of tnfr in hcv viral clearance may involve any of these cell subsets .
the largely chronic nature of hcv infection has been attributed to an attenuated antiviral t - cell response .
it has been postulated that apc 's may become dysfunctional in some way during hcv infection contributing to this attenuation .
specifically , large deficits in ifn production in plasmacytoid dendritic cells ( pdcs ) in hcv - infected patients have been reported in . in the same study
hcv - infected pdc displayed distinct immunophenotypic features including an increased ability to stimulate a mixed lymphocyte response ( mlr ) but lower hla - dr and cd86 expression .
this profile suggested that hcv - infected pdc were at an immature stage of differentiation .
the present studies were designed to quantify the mrna and protein levels of the tnf receptor family members , including tnfr1 and tnfr2 , in pbmcs during ifn-based therapy of hcv+ patients .
the results of these studies reveal increased peak levels of tnfr1 mrna levels in responders to ifn - based therapies .
furthermore , by flow cytometric studies and western blot analysis , upregulation of tnfr1 protein expression was noted in specific pbmc subsets .
the increase in tnfr1 expression was specifically isolated in pdc ( cd11 and cd123 ) of hcv - infected patients who responded to ifn based therapies compared to control patients and nonresponders .
veteran patients > 18 years of age with chronic hcv were recruited to ifn based treatment trials conducted at the dallas veterans affairs medical center from february 2002 through july 2005 .
thirty - seven patients with genotype 1 consented per institutional review board ( irb ) guidelines for blood draws at one or more time points , including 0 , 4 , 8 , 12 , 24 , and 48 weeks of therapy , where the standard therapy ( 48 weeks of weight - based dosing of 1.5 g / kg of pegylated interferon -2b and 13.5 mg / kg of ( ribavirin ) for genotype 1 patients was utilized .
thirteen patients were sustained virological responders ( svr , hcv rna negative 24 weeks after end of therapy ) , 20 patients were nonresponders ( nr , persistently hcv rna positive during therapy ) , and 4 patients were relapsers ( rel , hcv rna negative during therapy but hcv rna positive after end of therapy ) .
similarly , 10 hcv( ) patients and 11 patients with alcoholic liver disease ( ald ) or nonalcoholic fatty liver disease ( nafld ) consented per irb guidelines for a blood draw . in a second set of patients , samples from 13 genotype 1 hcv+ patients ( 5 svr , 6 nr / rel and 2 hcv+ prior to therapy ) and 2 hcv( )
control patients obtained during therapy at < 12 , 1624 , or 2544 weeks were collected for a specialized cell subset analysis .
rna was isolated from pbmc 's at 0 , 4 8 , 12 , 24 , and 48 weeks of interferon / ribavirin ( ifn / r ) treatment trials from 39 hcv+ men , 10 age- and sex- matched hcv( ) controls , and 11 age- and sex- matched controls with either alcoholic liver disease ( ald ) or nonalcoholic fatty liver disease ( nafld ) . initially
pbmcs were then separated by density gradient centrifugation and then stored in trizol reagent to stabilize the rna .
rna was then extracted , precipitated , and subjected to dnase treatment , and then the treated rna underwent reverse transcription . in order to assess whether there were differences in any specific cytokine receptors or tnf receptor family members , 101 cdna samples were utilized in a quantitative real time pcr - based assay assessing the relative quantities of cyclophilin , ngfr , cd30 , tnfr1 , tnfr2 , ltr , il-2r , cd27 , fas , and cd3 .
rna from pdc ( cd11 and cd123 ) and myeloid dendritic cells ( mdc ) ( cd11 + and cd123 ) from a control , svr , and nr were isolated in an identical fashion and utilized in a quantitative pcr assay for tnfr1 .
the ncbi database was first interrogated to find the sequence of the cytokine receptor or other receptors of interest and then entered into the primer express software program in order to generate a primer which was then optimized according to the parameters of the real time pcr ( table 1 ) .
the threshold cycle or c
t value , the lowest cycle in which fluorescence was detected during the exponential phase , was calculated where the exponential phase of the reaction began .
the ct values were standardized using rna isolated from cell lines known to express high levels of the target genes . in order to normalize across experiments , these values
were compared to the same standard for all experiments , that is rna from cell lines expressing high levels of tnfr or ltr ( pha activated lymphocytes and thp-1 , resp . ) .
the resulting cycle threshold values were normalized to endogenous cyclophillin values and a calibrator value for each experiment .
raw cycle threshold values for the no template control within each experiment were significantly different than the experimental values for cyclophillin , cd3 , ngfr , cd30 , tnfr1 , tnfr2 , ltr , fas , il-2r , and cd27 , verifying the presence of cdna for these receptors .
the comparison between control hcv( ) patients and svr , nr , and rel were expressed as fold elevation above control . the control population is defined as both hcv( ) patients with ald or nafld ( n = 9 ) and hcv( ) control subjects ( n = 10 ) .
whole pbmc 's from one svr with a high level of tnfr1 mrna by real time pcr were analyzed by western blot in order to determine protein levels of tnfr1 .
whole pbmc 's were freeze - thawed twice , suspended in pbs , and assayed for protein , and 20 g from one svr and one control as well as 200 ng of recombinant human soluble tnf receptor 1 ( r&d systems , minneapolis / st .
paul , mn ) were prepared for sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) in the presence of 2-mercaptoethanol , resolved on a 10% acrylamide gel , and transferred to a nitrocellulose membrane . after blocking in 5% milk in tbs ( 0.1 mol / l nacl and 0.4 mmol / l tris , ph 7.4 ) , containing 0.05% sodium azide , the blot was exposed to human soluble tnf receptor 1 polyclonal detection antibody ( r&d systems ) .
bound antibody was allowed to react with alkaline phosphatase - conjugated goat antimouse igg ( sigma chemical co. , st .
louis , mo ) . at 44 weeks of therapy , whole pbmcs from a svr and an nr
were isolated from 4060 cc whole blood by density gradient separation . in another experiment ,
pbmcs from an svr at 48 weeks , nr at 24 weeks , and control patient were isolated as previously described for subset analysis .
the pbmc 's from all patients were suspended in staining buffer ( 0.5 l hbss , 0.5 l pbs , 2% bsa , 0.02% sodium azide ) .
cells were labeled with the fitc - labeled anti - tnfr1 ab ( fab225f , r&d systems ) or control fitc igg16 ( 555748l , bd pharmingen ) .
the samples designated for cell subset analysis were then stained with cd33 ( 555748l , bd pharmingen ) , cd14 ( 555748l , bd pharmingen ) , cd11 ( 555748l , bd pharmingen ) , cd123 ( 555748l , bd pharmingen ) , or relative isotype controls .
after mab staining and washing , all samples were fixed in pbs containing 1% paraformaldehyde at room temperature for 10 minutes and stored at 4c until flow cytometric analysis as previously described in [ 16 , 17 ] .
flow cytometric analysis was performed on an facscan ( becton dickinson ) and used to determine the mean fluorescence intensity ( mfi ) of tnfr1 on all labeled cells .
rna from fac sorted plamacytoid dendritic cells ( pdcs ) ( cd11 and cd123 ) and myeloid dendritic cells ( mdcs ) ( cd11 + and cd123 from a control , svrs and nr were utilized in a real time pcr assay for tnfr1 .
descriptive data were reported as a mean for all standard deviation ( sd ) and median and range in figures 1 and 2 .
a p value of less than .05 was considered to be significant .
rna from pbmc of 23 hcv+ patients was isolated prior to the commencement of ifn based therapy . similarly ,
rna from pbmc 's of 10 hcv( ) controls and 11 patients with ald or nafld were also isolated . during the course of 48 weeks of ifn
based therapy , 80 rna samples were isolated from pbmc 's from 13 hcv+ responder [ ( svr and end of treatment responders ( etr ) ] patients and from 24 nr patients .
quantitative real time pcr levels of tnfr1 , tnfr2 , ltr , fasr , cd30 , cd27 , and ngf , as well as cd3 and il-2r mrna were assessed in available samples at 8 , 12 , 24 , and/or 48 weeks of therapy ( table 2 ) . in order to normalize across experiments ,
these values were normalized to the same standard for all experiments , that is , rna from cell lines expressing high levels of tnfr ( pha - activated lymphocytes ) .
as noted , tnfr1 mrna levels of pbmc from hcv+ responder patients were significantly higher than levels from control age- and sex- matched hcv( ) control patients during the course of the ifn based therapy ( n = 26 , p = .033 ) , while tnfr1 mrna levels of pbmc 's from hcv nr patients were not significantly different from controls ( n = 38 , p = .20 ) .
similarly , during the course of ifn based therapy , tnfr2 mrna levels of pbmc from hcv+ responder patients were significantly higher than levels in the control age- and sex- matched hcv( ) patients ( n = 22 , p = .045 ) while no difference was noted relative to levels in hcv nr and controls ( n = 33 , p = .41 ) .
no other significant increases were observed in the other tnfr family members or t - cell - associated mrna ( cd3 or il-2r ) levels of pbmc isolated from responders or nr ( table 2 ) . in summary ,
pbmc 's mrna levels of tnfr1 and tnfr2 from responders to ifn based therapy were statistically different than levels in control hcv( ) age- and sex- matched patients . in light of differences between levels of tnfr1 and tnfr2 mrna in pbmc of responders and nonresponders during the therapy , further analysis of the tnfr1 and tnfr2 levels was performed .
rna was isolated from pbmcs of 23 hcv+ untreated patients , 10 control hcv( ) patients , and 11 patients with ald or nafld .
tnfr1 and tnfr2 mrna levels were similar in both hcv infected and control groups [ 1.02 1.25 ( n = 21 ) versus 2.53 for all 2.71 ( n = 23 ) and 1.95 for all 2.42 ( n = 15 ) versus 2.07 for all 2.45 ( n = 18 ) ] ( figure 1 , panels a and b ) . quantitative real time pcr revealed no difference between initial levels of tnfr1 mrna in the hcv+ genotype 1 patients and control non - hcv+ patients ( p = .08 ) nor between hcv+ genotype 1 patients and patients with non - hcv liver disease ( p = .17 ) . similar results between initial tnfr1 rna levels in control ( hcv negative and non - hcv liver disease ) and initial levels in hcv+ patients were noted [ median 0.59 ( range : 0.025.42 ) ] versus [ median 1.52 ( range : 0.0611.65 ) ] ( figure 1 , panel a ) .
tnfr2 mrna levels were not significantly different between hcv+ patients prior to therapy and either hcv( ) control patients or hcv( ) patients with liver disease ( p = .88 and .95 ) , ( figure 1 , panel b ) . in order to determine whether the elevation of tnfr1 or tnfr2 mrna was above control levels at any point during ifn based therapy ( 4 , 8 , 12 , 24 and 48 weeks ) , 12 genotype 1 hcv+ svr and 25 genotype 1 hcv+ patients nr / rel were evaluated .
tnfr1 and tnfr2 mrna elevations were significantly more frequent in svr versus nr / rel .
the peak tnfr1 mrna levels in pbmc of genotype 1 hcv+ patients at 4 , 8 , 12 , 24 , or 48 weeks were greater than 2 sd ( 2.63 fold elevation ) above mean tnfr1 mrna levels from hcv( ) controls in 7/12 ( 58% ) sustained virological responders ( svr ) , but in only 5/21 ( 28% ) genotype 1 nr and 2/4 ( 50% ) genotype 1 rel .
furthermore , the peak tnfr1 mrna levels from responders were significantly higher than the peak tnfr1 mrna levels in nr and rel ( p = .0023 ) [ svr : median 7.37 ( range : 0.2935.92 ) versus nr / rel : median 1.17 ( range : 0.0815.91 ) ] ( figure 2(a ) ) .
the peak tnfr2 mrna levels expressed in pbmc from genotype 1 hcv+ patients at 4 , 8 , 12 , 24 or 48 weeks were greater than 2 sd ( 8.6 fold elevation ) above mean control tnfr2 mrna levels from hcv( ) patients in 3/12 ( 25% ) svr versus 3/20 ( 15% ) genotype 1 nr and 0/4 ( 0% ) genotype 1 rel .
the peak tnfr2 mrna levels from svr were not significantly higher than the peak tnfr2 mrna levels from nr and rel ( p = .085 ) ( figure 2(b ) ) .
similar results of peak tnfr levels in responders and nonresponders were noted [ median / range 6.1 ( 0.7132.01 ) versus 2.98 ( 0.0118.57 ) in figure 2(b ) ] .
therefore , unlike peak pbmc tnfr1 mrna levels , peak pbmc tnfr2 mrna levels during ifn for all therapy were not significantly different between svr and nonresponders . in order to assess whether tnfr1 mrna levels directly correlated with differential expression of tnfr1 protein in a svr or control pbmcs , whole pbmc from a hcv+ svr and a control hcv( ) patient were lysed , assayed for protein content , and 20 g of protein per sample was loaded on a sds page gel .
detection of the protein of 55 kda molecular weight by anti - tnfr1 was observed in the pbmc of an hcv+ responder patient but was not observed in the hcv( ) control pbmc lysate ( figure 3 ) .
the surface expression of tnfr1 by pbmc in hcv+ patients undergoing ifn / r has not been reported . however
because of the differences noted in the tnfr1 mrna levels between nr and the svr , flow cytometric analysis of tnfr1 expression by pbmc from a svr and a nr was assessed .
whole pbmcs from nr and svr were isolated and stained with an fitc - conjugated anti - tnfr1 ab as described in the methods .
the pbmc of the hcv+ svr patient displayed 52% tnfr1 positive pbmc with mean fluorescence intensity ( mfi ) of 80 .
the non - responding patient displayed 0.0% tnfr1 positive pbmc 's , with mfi of 0 .
as demonstrated by the flow cytometric figure , the increase of tnfr1 levels was noted in cells with light scatter characteristics of peripheral blood monocytes or dendritic cells ( figure 4 ) . in order to further delineate which pbmc subset
had elevated levels of tnfr1 expression , tnfr1 mfi ratios ( tnfr1 mfi / isotype control mfi ) were compared between svr , nr / rel , and controls .
hcv+ patients prior to therapy and uninfected controls were noted to have similar tnfr1 mfi ratios in both the cd33 + ( 1.2 0.01 ( n = 2 ) versus 1.56 0.37 ( n = 2 ) , resp .
p = .19 ) and cd123 + pbmc ( 1.14 0.56 ( n = 2 ) versus 1.40 0.09 , ( n = 2 ) resp .
no difference was observed between the tnfr1 mfi ratios from both cd33 + and cd123 + pbmc of the uninfected controls and the nr ( p = .4 and p = 1.0 , resp . ) . in order to further assess the specific cell subset affected by ifn ,
24 pbmc samples were stained with cd123 , cd33 , and tnfr-1 ( 16 svr and 8 nr / rel ) .
sixteen samples were obtained from 5 svr from weeks 6248 , and 8 samples were taken from nr / rel from weeks 581 . a trend toward a lower tnfr1 expression in cd123 +
dendritic cells subset was noted in the nr / rel group ( 1.25 0.45 versus 1.59 0.42 , p = .052 ) . of interest , a significant difference
was noted between the peak value of tnfr mfi ratio ( highest tnfr1 mfi ratio observed during therapy ) in cd123 + pbmc isolated from svr and nr / rel ( table 3 ) .
further cell subset identification was performed utilizing an analysis by flow sort using three cell markers to identify the two cell subsets of dendritic cells : myeloid ( mdc ) and plasmacytoid dendritic cells ( pdcs ) .
in the first panel , the control patient without chronic liver disease had virtually no tnfr1 expression , in all 3 subsets , cd14+cd123 + , mdc ( cd11 + and cd123 ) , and pdc ( cd11 and cd123 ) ( figure 5(a ) )
. however , in the second panel , after 48 weeks of ifn / r therapy , expression of tnfr1 in mdc and pdc in the svr was higher compared to the control ( figure 5(b ) ) in the non responder group the expression of tnfr1 was similar to controls , low in all three subsets ( figure 5(c ) ) .
hence , upregulation of tnfr1 membrane expression in the dendritic cell subset isolated from the svr was noted . in order to determine when tnfr1 mrna levels increased during ifn for all based therapy , 13 genotype 1 svr and 12 genotype 1 nr or rel were evaluated . in twenty - five genotype 1 hcv+ patients enrolled in ifn / r therapy with pbmc mrna samples collected during the 48 weeks of therapy , patients with svr ( n = 10 ) were more likely to have increases in tnfr1 mrna levels above mean control levels than patients with nr / rel ( n = 3 ) versus 3/12 ( 25% ) nr / rel .
importantly , the peak levels in the nr / rel were lower than in the svr ( tables 4(a ) and 4(b ) ) . of interest ,
4/7 ( 56% ) patients with elevated tnfr1 mrna values during therapy remained above mean control levels weeks after interferon therapy discontinuation .
rna from fac sorted plasmacytoid dendritic cells ( pdcs ) ( cd11 and cd123 ) and myeloid dendritic cells ( mdcs ) ( cd11 + and cd123 ) from a control , svr , and nr at 28 weeks of therapy were utilized in a real time pcr assay for tnfr1 .
tnfr1 mrna levels in pdc isolated from a responder were 6.7 fold higher than the level of the control pdc .
no difference in tnfr1 mrna levels was noted in the control compared to responders mdc , non responders mdc , or non responders pdc .
tnfr 1 rna level is higher in the pdc isolated from patients with svr than patients with nr at 28 weeks ( figure 6 ) .
this study is the first paper detailing the levels of tnfr family member expression in pbmc samples from responders and nonresponders to ifn therapy for chronic hcv over a 12-month period .
both tnfr1 and tnfr2 mrna levels in pbmc isolated during therapy were higher in responders than control hcv( ) patients .
notably , the peak values of pbmc tnfr1 mrna during therapy were higher in sustained virological responders than nonresponders or relapsers .
furthermore , pbmc expression of other members of the tnfr family remained stable during the course of the therapy .
importantly , the tnfr1 mrna upregulation occurred in a subset of pbmc , with cell surface markers consistent with the dendritic cells .
though other investigators have noted an increase in soluble tnfr1 ( stnfr1 ) and soluble tnfr2 ( stnfr2 ) in the serum of chronic hcv patients ( 1821 ) , this study is the first to note that the level of pbmc tnfr mrna rises over the course of ifn based therapy in responders .
furthermore , others have noted that stnfr2 appears to be significantly correlated with the severity of the disease and fibrosis .
as noted in our previous publications , 50%60% of patients treated at the dallas va have stage 3 fibrosis , and in part enhanced tnfr1 expression at baseline may be associated with fibrosis scores . however , there have been no significant differences in fibrosis between responders and nonresponders to therapy in our patient population . though the upregulation of tnfr1 may be associated with prolonged exposure to ifn , patients with multiple sclerosis ( ms ) treated with long term type 1 ifn ( interferon beta ) had stable pbmc tnfr 1 mrna levels ( data not shown ) .
other mechanisms besides prolonged exposure to type 1 ifn , involved in tnfr1 upregulation may include clearance of hcv virus from specific cell types , allowing further signaling from tnfr1 to nf6b , inducing tnfr1 upregulation .
other mechanisms may relate to improved antigen presentation by apc 's in specific patient populations after ifn exposure .
finally , specific patient populations may have different genetic polymorphisms of tnfr1 , which allow for enhanced viral clearance and upregulation of the receptor 's mrna , accounting for differences in response to ifn based therapy .
though there are limitations in the study in that less than half of the patients with svr had both initial and long term followup , there is a suggestion that tnfr1 rna levels may increase in patients that respond and that this increase appears be related to an upregulation of tnfr1 on a dendritic cell subset .
though this represents only a portion of the patients , the authors are suggesting that the findings in this study bear reporting .
the finding that dendritic cell subset appears to be the site of tnfr1 upregulation may provide insight into the cause , these cells have been implicated as the cell among pbmc that contains hcv negative rna strand .
though only a portion of the patients had extensive flow cytometric analysis , the results suggest that the dendritic cell is at least one of the cells , where upregulation of tnfr1 occurs .
while recent reports suggest that pbmc 's from up to 67% of svr patients still express residual hcv rna , these levels are likely greatly reduced from pretherapy levels and relief of hcv - mediated expression of tnfr family signaling may explain rebound enhanced expression of tnfr1 .
furthermore , these apc 's have also been implicated as being critical for viral clearance .
our study supports that within these cells , a cell surface receptor , tnfr1 , is disparately upregulated in patients responding to ifn based therapy .
initially , tnfr1 expression was noted to be upregulated in cd123 + dendritic cells in patients with sustained virological response .
the data supports that at least in some patients the increase in tnfr1 mrna is predominantly found in the plasmacytoid dendritic cells , an avid antigen presenting cell .
importantly , upregulation of tnfr1 surface expression was noted in one svr patient 's myeloid dendritic cell , suggesting that both types of dendritic cells may be important in hcv viral clearance . in summary
while the mechanism of this upregulation has yet to be elucidated , this cell subset may be involved in antigen presentation of viral proteins , and tnfr1 levels may correlate with recovery of effective apc function . | the present studies assessed the level of tumor necrosis factor receptor ( tnfr ) expression in peripheral blood mononuclear cells ( pbmcs ) subsets from patients with chronic hcv undergoing interferon /ribavirin - based therapy ( ifn / r ) . methods .
tnfr family member mrna expression was determined using quantitative real - time pcr assays ( rtpcrs ) in pbmc from 39 hcv+ patients and 21 control hcv patients .
further subset analysis of hcv + patients ( untreated ( u ) , sustained virological responders ( svr ) , and nonresponders ( nr)/relapsers ( rel ) ) pbmc was performed via staining with anti - cd123 , anti - cd33 , anti - tnfr1 or via rtpcr for tnfr1 mrna .
results . a similar level of tnfr1 mrna in pbmc from untreated hcv+ genotype 1 patients and controls was noted .
tnfr1 and tnfr2 mrna levels in pbmc from hcv+ patients with svr were statistically different than levels in hcv( ) patients .
a significant difference was noted between the peak values of tnfr1 of the cd123 + pbmc isolated from svr and the nr / rel .
conclusion .
upregulation of tnfr1 expression , occurring in a specific subset of cd123 + dendritic cells , appeared in hcv+ patients with svr . |
the indian metropolitan city of mumbai was besieged with india 's heaviest downpour of the century in july 2005 , killing nearly 600 people . according to the indian meteorological department , was the heaviest ever rainfall received in a single day , any where in india recording 94.4 cm in the last 100 years .
it broke the record of previous highest rainfall at one place in india at cherrapunjee in meghalaya of 83.82 cm recorded on july 12 , 1910 .
cherrapunjee in the north eastern state of meghalaya is a generally wellknown for being the wettest place in the world . in the same year
, there was another record broken in eastern indian state of orissa , for unusual mercurial rise in summer , june 2005 recorded the highest temperature of 46.3 degree celsius in bhubaneswar of the last 33 years which is 10 degrees above normal , leading to a heatwave .
speaking of heat wave , the 1998 heat wave in orissa was recorded as one of the worst , claiming more than 2000 lives .
extremes of climatic changes surpassing their usual statistical ranges and tumbling records in india should be an early alarm to all of us to sit back and take notice .
we are not insisting that the record - breaking mumbai rain or heat waves in orissa have a direct causal association with global warming /global climate change but at the same time , we should also not ignore them as simple local aberrations .
extreme weather events such as severe storms , floods and drought have claimed thousands of lives during last few years and have adversely affected the lives of millions and cost significantly in terms of economic losses and damage to property . just to take few examples
: floods are an annual feature in bihar , but the 2004 floods were unique for its severity .
andhra pradesh reeled under heat wave in 2003 killing 1,421 people , which is an all - time high in the history of andhra pradesh .
orissa is no stranger to cyclones but the 1999 cyclone was again unprecedented for the sheer severity with wind speed reaching over 300 km per hour leaving nearly 10000 dead and has gone down in history as the super cyclone .
cheerrapunjee , the world 's wettest place is going through a rare rain crisis and is experiencing dry spells .
this year while mumbai was being flooded , cherrapunjee received less than average rain fall in june and july with distressing situation subsequently . according to the meteorological department officials the unusual pattern of rainfall can be attributed to the monsoon trough moving southwards from normal position of the cherrpunjee - assam - bihar belt .
in addition to changing weather patterns , climatic conditions affect diseases transmitted through water and via vectors such as mosquitoes .
diarrhoea , malaria and protein - energy malnutrition alone caused more than 3.3 million deaths globally in 2002 , with 29% of these deaths occurring in the region of africa .
no , we are not even saying that india is the only country taking the entire brunt of global warming .
of course all the countries are facing it , e.g. , the 1995 chicago heat wave killed nearly 600 people , french heat wave of 2004 killed 15 , 000 people in a matter of a weeks .
like - wise in fiji , the 1998 dengue outbreaks are again said to be an evidence of global climate change , as the distribution of the vector ( aedes polyneiensis ) is said to be affected by rise in the sea level , since it breeds in brackish water .
recognition of the existence of the problem is the first step towards solution , rather than dismissing global climate change as conspiracy theory or hype created by environmentalists .
it is important that we have these extreme events on our surveillance radar and verify them for being potential pieces of evidence from india for global climate change .
extreme climate events are expected to become more frequent in the coming years with climate change .
let 's face it ; the major brunt of global climate change in terms of adverse health impact will be mostly borne by poor and developing countries , even though rich and industrialized countries account for maximum green house gas emission .
any region under stress , such as the indian sub - continent , is likely to experience greater effects from these extreme weather events , because of obvious reasons poverty , malnutrition , poor public health infrastructure etc . in its third assessment report ( 2001 ) , the united nation 's intergovernmental panel on climate change ( ipcc ) stated : there is new and stronger evidence that most of the warming observed over the last 50 years is attributable to human activities and concluded that : overall , climate change is projected to increase threats to human health , particularly in lower income populations , predominantly within tropical / subtropical countries .
a brief overview of likely health effects due to climate change ( figure 1 ) is discussed here .
if the overall climate becomes warmer , there will be an increase in health problems .
it is anticipated that there will be an increase in the number of deaths due to greater frequency and severity of heat waves and other extreme weather events .
the elderly , the very young and those suffering from respiratory and cardiovascular disorders will probably be affected by such weather extremes as they have lesser coping capacity .
an extreme rise in the temperature will affect people living in the urban areas more than those in the rural areas .
this is due to the heat islands that develop here owing to the presence of concrete constructions , paved and tarred roads .
higher temperatures in the cities would lead to an increase in the ground - level concentration of ozone thereby increasing air pollution problems .
indirectly , changes in weather pattern , can lead to ecological disturbances , changes in food production levels , increase in the distribution of malaria , dengue and other vector - borne diseases .
fluctuation in the climate especially in the temperature , precipitation and humidity can influence biological organisms and the processes linked to the spread of infectious diseases .
they are focal and their distribution is limited by the ecology of their reservoir , be it arthropod , snail or water .
they usually have a two or three - host life cycle , meaning that in addition to infecting people , they infect a vector and frequently also a wild vertebrate animal host . either the vector or the host or both , are the reservoir .
if the agent and reservoir are successful in the newly warmer climate , the agent can be expected to multiply more rapidly and if the reservoir is an arthropod or snail , it too will develop more rapidly ( it may also have a shorter life ) .
the risk of explosive epidemics due to vector borne diseases is enhanced because of two main properties of the vector - virus relationship .
firstly , within limits viruses multiply more rapidly in mosquitoes at high temperatures than at low ones .
secondly , the mosquito also develops more rapidly at high temperatures than at low ones .
this combination is conducive to a very short incubation period in the mosquito and rapid mosquito population increase .
a short incubation period in the mosquito along with rapid population increase in turn can lead to more rapid and sometimes explosive transmission in the human population .
warmer temperatures also lead to a shorter life span of the mosquito and shorter life means less time to transmit the virus to another person . among vector - borne diseases in india ,
periodic epidemics of malaria occur every five to seven years and the world bank estimates that about 577,000 dalys ( disability - adjusted life years ) were lost due to malaria in india in 1998 . climate change could increase the incidence of malaria in areas that are already malaria - prone and also introduce malaria into new areas .
potential effects on health due to sea level rise include :
death and injury due to flooding;reduced availability of fresh water due to saltwater intrusion;contamination of water supply through pollutants from submerged waste dumps;change in the distribution of disease - spreading insects;health effect on the nutrition due to a loss in agriculture land and changes in fish catch ; and health impacts associated with population displacement
death and injury due to flooding ; reduced availability of fresh water due to saltwater intrusion ; contamination of water supply through pollutants from submerged waste dumps ; change in the distribution of disease - spreading insects ; health effect on the nutrition due to a loss in agriculture land and changes in fish catch ; and health impacts associated with population displacement | extremes weather changes surpassing their usual statistical ranges and tumbling records in india could be an early warning bell of global warming .
extreme weather events like the recent record setting in western indian city of mumbai or all time high fatalities due to the heat wave in southern indian states or increasing vulnerability of easten indian states to flood could all be a manifestation of climate change in the asian subcontinent .
while the skeptics may be inclined to dismiss these events as simple local aberrations , when viewed in an epidemiological paradigm in terms of person , time and space couple with frequency , intensity and fatalities , it could well be an early manifestation of climate change .
global warming poses serious challenge to the health sector and hence warrants emergency health preparedness and response .
climate - sensitive diseases are among the largest global killers , hence major brunt of global climate change in terms of adverse health impact will be mostly borne by poor and developing countries in asia , given the levels of poverty , nutional levels and poor public health infrastructure . |
in a previous issue of critical care , ertmer and colleagues present an experimental study in which they examine the effects of dobutamine when given together with arginine vasopressin ( avp ) in endotoxemic sheep .
avp is an increasingly used supplementary vasopressor in advanced vasodilatory shock states where standard treatment can not stabilize cardiovascular function with an acceptable benefit : risk ratio . due to its strong vasoconstrictive and lack of beta - mimetic effects
preliminary results of a recently accomplished multicenter trial including patients with septic shock suggest a significant survival benefit at 28 and 90 days with a supplementary avp infusion when given to patients with moderate cardiovascular failure ( congress of the european society of intensive care medicine , barcelona , september 2427 , 2006 ) . a potentially deleterious decrease in cardiac index ( ci ) during avp infusion
was reported soon after the first results on the use of avp in septic shock had been published . whereas animal experiments ( mostly applying avp as a single vasopressor ) homogeneously reported a decrease in ci and systemic oxygen transport capacity ( do2i ) , clinical observations ( applying avp as a supplementary vasopressor ) showed heterogeneous responses , with most studies reporting neutral or even beneficial effects of avp on cardiac performance .
a recent analysis demonstrated a decrease in ci during avp infusion in 41% of vasodilatory shock patients .
high ci before avp was the single independent risk factor for a subsequent fall of ci .
while patients with hyperdynamic circulation exhibited a decrease in ci to normodynamic values , patients with hypodynamic circulation experienced a slight improvement of ci .
stroke volume index increased irrespective of the circulatory state , with the most pronounced increase in patients with hypodynamic circulation . in their present study , ertmer and colleagues evaluate whether dobutamine can reverse an avp - induced decrease in ci and do2i .
the highly experienced working group used an ovine endotoxinemicmodel , in which avp has been shown to depress ci and do2i .
the authors ' conclusions are fully supported by the results of their experiment and prove that dobutamine is indeed a useful agent to reverse an avp - associated depression in ci and do2i in this animal model . when taking the results of this experimental study into clinical practice , important limitations need to be considered .
as stated by the authors themselves , their protocol did not analyze end organ perfusion and can , therefore , not prove if the decrease in ci and do2i resulted in organ hypoperfusion .
moreover , avp was applied as a single vasopressor at dosages twice as high as recommended in clinical practice ( 0.04 iu / minute in 35 kg sheep versus 0.04 iu / minute in 70 kg patients ) . a small prospective study infusing high avp dosages
has shown a highly different hemodynamic response to avp than observed with standard recommended dosages .
accordingly , the systemic vascular resistance index ( svri ) during avp infusion in this experimental study reached supranormal values ( approximately 2,000 dynes m cm ) when compared to baseline values .
this unphysiological increase in svri could well explain a baroreceptor - mediated ( although possibly attenuated in endotoxemia ) decrease in ci .
the fact that in clinical practice svri is , at maximum , increased to subnormal values by vasopressors , might be one reason for the avp - induced depression in ci in this experiment .
nonetheless , this study underlines important points of hemodynamic management in vasodilatory shock : systemic blood flow must be ensured to guarantee adequate organ perfusion as reflected by central venous oxygen saturation > 70% or mixed venous oxygen saturation > 65% .
as shown by the authors , inotropes such as dobutamine are a good choice to reverse low systemic blood flow if preload optimization can not adequately increase ci .
what must never be forgotten when infusing inotropic agents and assessing adequate organ perfusion is that , in critical illness , beta - adrenergic stimulation has virtually no positive , but merely adverse effects on the heart itself ( exponential elevation of oxygen demand due to increases in heart rate or triggering of tachyarrhythmias , induction of myocardial stunning at high dosages ) .
recent data revealed that cardiac pathologies made up almost 50% of the causes of death in patients with advanced vasodilatory shock . considering the heart as the obviously most vulnerable link of the cardiovascular chain
, it must be one of our goals to relieve the heart as much as possible , while ' enforcing ' as much systemic blood flow as necessary for peripheral organs .
a demand - based strategy relying on central / mixed venous oxygen saturation is a very useful tool to achieve this critical balance .
a putative mechanism by which supplementary avp infusion may exert beneficial effects is the significant reduction of high , potentially toxic catecholamine dosages .
when reviewing the results of ertmer and colleagues , dobutamine did not only restore hyperdynamic circulation , but also reversed potentially beneficial effects of avp on myocardial oxygen balance .
as shown in figure 1 of the paper , the dobutamine - induced increase in ci was accompanied by an increase in heart rate from approximately 85 to 135 beats / minute . as cited by ertmer and collleagues ,
a recent study has shown an alarmingly high incidence of major cardiac events ( 49% ) in high risk cardiac patients with tachycardia ( > 95 beats / minutes for > 12 hours ) .
finding the delicate balance between pharmacological stress on the heart and adequate organ perfusion is difficult but crucial to guarantee optimal patient outcome in vasodilatory and septic shock .
avp = arginine vasopressin ; ci = cardiac index ; do2i = systemic oxygen transport capacity ; svri = systemic vascular resistance index .
| supplementary arginine vasopressin infusion in advanced vasodilatory shock may be accompanied by a decrease in cardiac index and systemic oxygen transport capacity in approximately 40% of patients . while a reduction of cardiac output most frequently occurs in patients with hyperdynamic circulation ,
it is less often observed in patients with low cardiac index .
infusion of inotropes , such as dobutamine , may be an effective strategy to restore systemic blood flow . however , when administering inotropic drugs , systemic blood flow should be increased to adequately meet systemic demands ( assessed by central or mixed venous oxygen saturation ) without putting an excessive beta - adrenergic stress on the heart .
overcorrection of cardiac index to hyperdynamic values with inotropes places myocardial oxygen supply at significant risk . |
cyclosporin a ( cya ) is a cyclic undecapeptide with strong immunosuppressive activity , since it has a specific inhibitory effect on the t - cell receptor signal transduction pathway [ 1 , 2 ] .
reactive oxygen species ( ros ) have been implicated in the nephrotoxicity and hepatotoxicity caused by cya , either by direct action or by the activation of lipid peroxidation ( lp ) [ 36 ] .
our previous work showed that the effect of cya on the antioxidant defense system ( ads ) is related to lipoperoxidation and liver function .
the results for the chronic and acute treatment , only with 20 mg / kg / day of cya , caused alterations in liver parenchyma histoarchitecture .
in previous works we showed that cya administration to organ transplant patients caused an increase in the levels of serum creatinine and urinary excretion of gamma - glutamyl transpeptidase ( uggt ) .
these functional changes were dose and time dependent , showing structural alterations with doses higher than 15 mg / kg / day cya in chronic treatments .
morphofunctional alterations in the liver parenchyma were observed in rats treated with cya at different doses for 120 days .
doses higher than 15 mg / kg / day cya , equivalent to concentrations in blood of 437 13.40 ng / ml or greater , caused functional alterations in complexes i and ii of the mitochondrial respiratory chain in the liver of treated rats . in an attempt to design and develop therapeutic strategies that could prove effective against the adverse effects induced by cya
vitamin e ( vit e ) is the collective name for a group of liposoluble compounds derived from tocol and tocotrienol , alpha tocopherol being the one with greatest biological activity .
its absorption and distribution are closely related to the digestion , absorption , and distribution of lipids , the liver being its main storage organ , from where it is rapidly mobilized .
the characteristic of biological antioxidant of vit e derives from its molecular structure as its capability of fixing ros types o2 , o2 , and oh [ 11 , 12 ] .
vitamin e prevents oxidation of polyunsaturated fatty acids and lipoproteins and it has been shown that the antioxidant effect of vit e might be beneficial in the prevention of autoimmune , inflammatory and infectious diseases as well as neoplasia [ 1315 ] .
trimetazidine ( tmz ) , 1 - 2,3,4 trimetoxibencil piperazine , is a drug used as a cardioprotector since it prevents the secondary cell death that usually follows transient myocardial ischemia .
tmz is a cytoprotector whose place of action and the mechanism and chronological order of its effects are not yet known in depth .
according to some authors , tmz acts at the mitochondrial level diminishing the beta - oxidation of fatty acids . another beneficial effect described in the existing literature
numerous studies have analyzed the protective effect of tmz against myocardial ischemia - reperfusion injury in different models . among
the mechanisms suggested , the following have been mentioned as the most probable : hemodynamic changes , reduction in ros toxicity , decrease in the inflammatory reaction , optimization of the energetic metabolism , and reduction in the utilization of fatty acids in favor of carbohydrates [ 1720 ] .
our experiments using an in vivo model to study the effect of tmz on the nephrotoxicity induced by gentamicin demonstrated that tmz , when used in a 7-day pretreatment , was able to attenuate the nephrotoxic effect of this aminoglycoside . to find a protective drug against the toxicity induced by cya during chronic treatment ,
we assayed the cardioprotector trimetazidine ( tmz ) and vitamin e , used as nutritional supplements to alleviate oxidative stress .
the experiments were performed with adult male wistar rats of 180 to 200 g body weight housed in standard cages with a 12 h light - dark cycle ( light on at 7:00 a.m. ) at 20c and 60% humidity .
all experimental procedures complied with the regulations of the european union ( 86/60/eec ) and with the recommendations of the federacin de sociedades sudamericanas de la ciencia de animales de laboratorio - fesscal ( federation of south american societies of laboratory animal science).in the treatment of the animals the following drugs were used : cya provided by sandoz laboratories ( germany ) sandimmune .
vitamin e provided by raymos laboratories , tanvimil e 400 mg.trimetazidine provided by servier laboratories , vastarel 20 mg . in the treatment of the animals the following drugs were used : cya provided by sandoz laboratories ( germany ) sandimmune .
group a ( control ) : animals were fed a standard rat - mouse chow for 120 days .
group b ( control vit e ) : animals were fed a standard rat - mouse chow supplemented with 10 mg / kg / day vit e for 120 days .
group c ( tmz control ) : animals were fed a standard rat - mouse chow supplemented with 20 mg / kg / day tmz for 120 days .
group d ( cya control ) : animals were fed a standard rat - mouse chow supplemented with 25 mg / kg / day cya , the drug being orally administered for 120 days .
group e ( cya + vit e ) : animals were fed a standard rat - mouse chow treated with 25 mg / kg / day cya and 10 mg / kg / day vit e , the drugs being orally administered for 120 days .
group f ( cya + tmz ) : animals were fed a standard rat - mouse chow pretreated with 20
mg / kg / day tmz for 20 days and then treated with 20 mg / kg / day tmz and 25 mg / kg / day cya for 120 subsequent days .
group a ( control ) : animals were fed a standard rat - mouse chow for 120 days . group b ( control vit e ) : animals were fed a standard rat - mouse chow supplemented with 10 mg / kg / day vit e for 120 days .
group c ( tmz control ) : animals were fed a standard rat - mouse chow supplemented with 20 mg / kg / day tmz for 120 days .
group d ( cya control ) : animals were fed a standard rat - mouse chow supplemented with 25 mg / kg / day cya , the drug being orally administered for 120 days .
group e ( cya + vit e ) : animals were fed a standard rat - mouse chow treated with 25 mg / kg / day cya and 10 mg / kg / day vit e , the drugs being orally administered for 120 days .
group f ( cya + tmz ) : animals were fed a standard rat - mouse chow pretreated with 20
mg / kg / day tmz for 20 days and then treated with 20 mg / kg / day tmz and 25 mg / kg / day cya for 120 subsequent days .
during the chronic experiment , the animals were studied with daily evaluations of behavior , appetite , and activity .
the body weight of the animals was determined at three points in time : the beginning of the experiment , 60 days after the beginning , and 120 days after the beginning . at the end of the experiment ,
the liver and kidney were excised and weighed . at the beginning and at the end of the experiment ,
blood samples were collected by tail vein punction and by intracardiac punction , respectively , for the biochemical studies .
the nitrogen compounds in blood , urea , and creatinine were determined using the urease method and jaffe 's colorimetric method , respectively .
the reactives were provided by wiener laboratories [ 22 , 23 ] . in blood samples , serum aminotransferases and glutamate dehydrogenase
were determined using optimized uv methods [ 24 , 25 ] . at the end of the experimental design ,
then they were bled and their liver and kidneys were removed and prepared for structural and ultrastructural studies .
small portions of the kidney and liver previously separated were washed with physiological solution , fixed in a 10% formaldehyde solution , and embedded in paraffin .
they were cut into 4 - 5 m thick sections and stained with hematoxylin - eosin .
the histological slices were observed under an axiostar plus zeiss optical microscope . for observation under the electron microscope ,
the liver and kidney portions were processed as follows : they were fixed in a 3.5% glutaraldehyde solution in 0.1 m phosphate buffer at ph 7.40 for 3 h and then placed in 1% osmium tetroxide .
then , the samples were treated with an aqueous solution of 2% uranyl acetate for 40 min .
after fixation , the tissues were gradually dehydrated by a successive passage through a series of increasing alcohol concentrations .
ultrathin sections were treated with uranyl acetate , placed in citrate , and examined at 50 kv with a zeiss em 109 transmission electron microscope belonging to lamenoa - electron microscopy laboratory of northwestern argentina .
analysis of variance and later comparison of means were performed with tukey 's test ( alpha = 5% ) .
during the 120-day chronic assay , the animals in the different groups increased their weight and maintained their healthy status .
hepatosomatic ratio was determined at the end of the treatment , after the animals had been sacrificed .
no significant differences were found in groups b , c , or f with respect to control group a. groups d and e showed a statistically significant decrease in the hepatosomatic ratio with respect to control group a. these results are shown in table 1 .
figure 1 shows the statistically significant increase in the activity of mitochondrial enzymes ast and gldh .
this increase was observed after chronic treatment with 25 mg / kg / day cya .
group f , pretreated for 20 days with tmz and then for 120 days with tmz + cya , showed a protective effect on the liver profile , with enzymatic activity of ast and gldh comparable to that in control group a. however , the therapeutic scheme applied to group e reflected a behavior similar to group d. vitamin e , in these conditions , did no exert a hepatoprotective effect . in order to relate the biochemical changes observed to the histoarchitecture of the liver parenchyma , we studied histological slices with mallory and hematoxylin - eosin staining of the liver parenchyma . in figure 2(a ) , 10x magnification shows hepatocytes in cords disposition around the central vein .
they present a homogeneous cytoplasm with a central nucleus , with no cytological alterations . in figures 2(b ) and 2(c ) , ( 20x ) control groups : b with vitamin e and c with tmz , respectively , shows liver parenchyma histoarchitecture with no alterations . in figure 2(d ) ( 20x ) shows the histological slice of the liver parenchyma of group d , treated with 25 mg / kg / day cya for 120 days .
we can see the central vein and the infiltration of mononuclear elements in the perivascular space . in figure 2(e ) ( 20x ) shows the histological slice of the liver parenchyma of group e , treated with 25 mg cya / kg / day and 10 mg vit
we can see mononuclear infiltrates in the whole liver parenchyma , including the portal spaces . in figure 2(f ) ( 40x ) shows the protective effect on liver parenchyma of pretreatment with tmz for 20 days and then for 120 days with 20 mg tmz / kg / day + 25 mg cya / kg / day .
we can see the conserved liver parenchyma histoarchitecture , similar to the one in animals control group a. portions of the liver were processed for ultrastructural observation , especially of the mitochondria .
figure 3 shows the electron microphotograph of hepatocytes of the different treated groups with 82.640x magnification .
figure 3(a ) corresponds to control group a. we can see a hepatocyte with its nucleus with lax chromatin . at the top a mitochondria with normal size and structure can be observed in a transverse section .
figure 3(b ) shows the electron microphotograph of a hepatocyte from group d. the animals treated with 25 mg cya / kg / day for 120 days show swollen mitochondria with destruction of the inner membrane and of their intramitochondrial crests , where enzyme complexes i and ii are located ( 82.640x ) .
figure 3(c ) shows the electron microphotograph of a hepatocyte from group f. the animals pretreated with 20 mg of tmz and then with 20 mg tmz / kg / day + 25 mg cya / kg / day for 120 days show conserved mitochondrial ultrastructure .
figure 4 shows a statistically significant increase in the levels of serum urea and creatinine in animals from groups d ( avsd ) .
these biochemical findings show the experimental nephrotoxicity induced by the treatment with cya at a dose of 25 mg / kg / day .
the therapeutic scheme used with group f ( pretreatment with 20 mg tmz / kg / day and simultaneous treatment with 20 mg tmz / kg / day + 25 mg cya / kg / day ) did not show significant biochemical changes ( avsf ) , showing the protector effect of tmz on the renal function .
the nitrogen compounds urea and creatinine in group e were within the ranges of group d. vit e in these conditions did not exert a renoprotective effect . in both control and
treated groups diuresis was measured for 24 h , no statistically significant differences being found ( data not shown ) . in order to compare the biochemical changes with the histoarchitecture of the renal parenchyma ,
figure 5(a ) shows the kidney of a rat from control group a ( 10x ) .
the renal cortex with a central glomerulus can be seen as well as a well - defined bowman 's space .
the rest of the parenchyma shows transverse sections of proximal and distal convolute tubules .
part of a conserved glomerulus can be seen ; however , in the tubular epithelium can be seen cell desquamation and hydropic degeneration corresponding to revertible dysplasia .
figure 5(c ) shows the kidney of a rat treated with 20 mg tmz / kg / day ( 20x ) .
figure 5(d ) corresponds to the kidney of a rat treated with 25 mg cya / kg / day ( 20x ) . in the renal medulla
figure 5(f ) shows the kidney of a rat pretreated with 20 mg tmz / kg / day and then with 20 mg tmz / kg / day + 25 mg cya / kg / day belonging to group f ( 10x ) .
the renal cortex with glomeruli and a transverse slice of the conserved tubules can be seen , showing the protector effect of tmz on the renal parenchyma .
however , the therapeutic scheme applied to group e reflected a behavior similar to group d. vit e in these conditions did not exert a renoprotective effect ( figure 5(e ) ) .
sections of the kidney were processed for ultrastructural observation , especially of the mitochondria .
figure 6(a ) shows the normal ultrastructure of the renal tubular epithelium of the male wistar rats ; can be seen cells with apical pole showing brush border conserved . in the apical brush border , can be seen the microvilli typical of the proximal tubular epithelium ( 7.800x ) .
figure 6(b ) shows the tubular epithelium of group d , treated with 25 mg cya / kg / day . in the transverse section of the epithelium
can be seen a tubular cell with intact basement membrane and atrophied apical border , and next cell without nucleus .
both cells show disarranged mitochondria , corresponding to cytoplasmic vacuolization or hydropic degeneration ( 7.800x ) .
figure 6(c ) shows two tubular cells from group e , treated with 10 mg vite / kg / day + 25 mg cya / kg / day .
. however , can be seen mitochondria with altered shapes , sizes , and arrangement in relation to the control group . on the upper right hand side
we can see the apical border totally atrophied and with no microvilli ( 7.800x ) .
figure 6(d ) shows two nucleated tubular cells from group f , pretreated with 20 mg tmz / kg / day and then with 20 mg tmz / kg / day + 25 mg cya / kg / day .
can be seen the intact basement membrane with the perpendicular arrangement of the mitochondria at the basal domain .
cya is the drug most frequently used in transplant surgery because of its potent immunosuppressive action .
however , its clinical use is accompanied by adverse side effects such as hypertension , nephrotoxicity , and hepatotoxicity .
previous studies established that ros production and oxidative stress situation are involved in cya cytotoxicity in cultured rat hepatocytes . in all cases , they attribute to cya the ability to produce oxidative stress . however
, none of them have explained what is the mechanism of formation and accumulation of ros [ 1115 ] .
however , it has been demonstrated in numerous in vivo and in vitro experiments that cya - induced renal failure and increased the synthesis of ros , thromboxane , and lipid peroxidation products in the kidney [ 28 , 29 ] .
different authors published works about the benefits of treatment with vitamin e to attenuate the nephrotoxic and hepatotoxic effects of cya .
most of them have based their conclusions on the antioxidant capacity of alpha tocopherol . in a work published in 2007 [ 28 , 29 ] , oxidative stress and mitochondrial dysfunction were studied in tubular renal cells in vitro .
the authors concluded that the cellular toxicity of cya resulted from important alterations in the mitochondrial physiology and structure , with an increase in ros synthesis and a decrease in antioxidant capacity .
other authors have reported vitamin e as a protector against cya - induced cytotoxicity in a rat hepatocyte culture [ 30 , 31 ] .
one of those reports , with in vitro experiments , concluded that there is an imbalance in antioxidant enzymes due to the direct action of cya on the primary hepatocyte culture and the fact that the simultaneous treatment with cya and vit e reduces oxidative stress , inhibiting lipoperoxidation and restoring antioxidant enzymes . in previous papers
we have experimentally demonstrated that nephrotoxicity and hepatotoxicity are the main secondary effects of cya treatment . these effects are time period and dose dependent [ 79 ] . for the purpose of finding a drug that would exert a protective effect against cya - induced hepato- and nephrotoxicity , we assayed two drugs : vitamin e and tmz .
we conclude that vitamin e does not exert a protector effect against cya - induced nephrotoxicity .
we base these results on the increased levels of serum urea and creatinine in group e , shown in figure 4 .
these results correlate with our structural and ultrastructural findings ( figures 5(d ) and 6(c ) ) .
special attention should be paid to the effect of treatment with 10 mg / kg / day vit e on group e. in figure 5(b ) , we can see part of a conserved glomerulus and in the tubular epithelium cell desquamation and hydropic degeneration can be observed .
these morphological changes correspond to regenerative dysplasia . apparently , vit e by itself is capable of producing morphological changes in the tubular renal cell .
some authors have reported that when the optimal level ( 80 g / dl ) of this antioxidant is surpassed , the risks of toxicity increase . on the other hand , the hepatotoxic effect of cya
is not attenuated by treatment with vitamin e. if we analyze table 1 , we can see that group e shows a statistically significant decrease in hepatosomatic ratio comparable only to group d with treatment with 25 mg cya / kg / day .
this decrease correlates with the increase in the activities of mitochondrial sublocalization enzymes : ast and gldh , in figure 1 .
the increase in ast and gldh is statistically significant with respect to the other control and treated groups .
these results are corroborated by optical microscopy of the liver parenchyma in figure 2(e ) .
we can see infiltrates of mononuclear elements in the whole liver parenchyma , including the portal spaces , a similar histoarchitecture to the one described in group d treated with 25 mg cya / kg / day .
figure 2(d ) . to find a protective drug against those effects we assayed the cardioprotector trimetazidine ( tmz ) .
some authors have reported that tmz exerts a cytoprotective effect on the cardiomyocyte [ 1620 ] .
our previous works have demonstrated the cytoprotection of tmz in an in vivo model of gentamicin - induced nephrotoxicity . in the present work we studied and described the cytoprotective effect of tmz against cya - induced toxicity on the liver and kidney of treated rats .
we demonstrated that pretreatment with 20 mg / kg / day tmz followed by treatment with 20 mg / kg / day tmz + 25 mg
/ kg / day cya for 120 days preserves the morphofunctionality of the tubular renal cell and of the liver cell .
this was clearly manifested by the improvement in all the biochemical variables determining cya - induced hepato- and nephrotoxicity ( figures 1 and 4 ) .
we can see histoarchitecture similar to the one in control group a ( figure 2(f ) ) .
we can see the intramitochondrial cristae , site of the mitochondrial respiratory chain ( 82.640x ) figure 3(c ) . on the other hand
, we demonstrated in an in vivo animal model to the mitochondria as a target for the toxic action of cya .
we reported that chronic treatment with cya at doses above 15 mg / kg / day causes alterations in the mitochondria , by the functional alteration in the mitochondrial respiratory chain in its complexes i and ii , since this dose- and time - dependent effect requires active cell metabolism .
in our study we investigated whether tmz allows inhibition of the mitochondrial alteration induced by cya .
trimetazidine protects the cells against the changes produced by the cya - induced oxygen deficit .
trimetazidine optimizes the energetic metabolism of the ischemic cell through a metabolic exchange ( switch ) between the fatty acid and glucose oxidation . in theory
, tmz would reduce strongly the fatty acid oxidation towards glucose , without affecting the mitochondrial respiratory chain efficiency .
furthermore , tmz increases the production of phospholipids in the mitochondrial membranes , which confers stability to these structures .
we conclude the following.the target of the cytotoxic action of cya is in enzymes of complexes i and ii of the mitochondrial respiratory chain , which results in an alteration in the energy metabolism and leads to cell death by necrosis.tmz exerts a cytoprotective effect both on the liver cells and on the tubular renal cells . among the different mechanisms of action proposed by some authors , pretreatment with tmz
would contribute to optimizing the energy metabolism of the mitochondria treated with tmz + cya at the doses and time periods studied.pretreatment with tmz is essential for the protection of the cell since it confers stability and prevents the incorporation of cya into the inner membrane of the mitochondria.the antioxidant vitamin e does not exert a protective effect since we demonstrated that the cya action in the mitochondrial chain does not involve ros .
the target of the cytotoxic action of cya is in enzymes of complexes i and ii of the mitochondrial respiratory chain , which results in an alteration in the energy metabolism and leads to cell death by necrosis .
tmz exerts a cytoprotective effect both on the liver cells and on the tubular renal cells . among the different mechanisms of action proposed by some authors , pretreatment with tmz
would contribute to optimizing the energy metabolism of the mitochondria treated with tmz + cya at the doses and time periods studied .
pretreatment with tmz is essential for the protection of the cell since it confers stability and prevents the incorporation of cya into the inner membrane of the mitochondria .
the antioxidant vitamin e does not exert a protective effect since we demonstrated that the cya action in the mitochondrial chain does not involve ros . | the immunosuppressant drug cyclosporin a ( cya ) has been used in diseases with immunological basis and in transplant patients .
nephrotoxicity and hepatotoxicity are the main adverse effects of this drug . to find a protective drug against those effects we assayed the cardioprotector trimetazidine ( tmz ) and vitamin e , used as nutritional supplements to alleviate oxidative stress .
six groups of eight male wistar rats each were prepared ( groups a
f ) : a , control ; b , vitamin e ( 10 mg / kg / day ) ; c , tmz ( 20 mg / kg / day ) ; d , 25 mg / kg / day cya ; e , cya and vitamin e ( 25 mg / kg / day cya + 10 mg / kg / day vit e ) ; f , tmz for 20 days ( 20 mg / kg / day ) ; and then cya ( 25 mg / kg / day ) and tmz ( 20 mg / kg / day ) . the experiment lasted 120 days .
the exposure of rats to cya promoted nephrotoxicity and hepatotoxicity with an increase in serum urea , creatinine , and glutamate dehydrogenase ( gldh ) .
structural and ultrastructural studies of liver and kidney were performed .
group d showed adverse effects induced by cya since statistically significant differences were found with respect to the control group ( a ) .
vitamin e ( e ) showed no protective effect .
pretreatment with tmz ( f ) attenuated the adverse effects of cya .
we conclude that cya - induced nephrotoxicity and hepatotoxicity are attenuated by the cytoprotective effect of tmz .
tmz inhibits the reabsorption and , consequently , the accumulation of cya in the cell . the antioxidant capacity of vitamin e did not improve the effect of cya . |
chronic obstructive pulmonary disease ( copd ) is a common , preventable , and treatable disease
characterized by progressive airflow limitation .
associated with airway chronic inflammatory
response and by lung noxiousness to particles or gases , copd is the third leading cause of death in
the us and has an estimated worldwide prevalence of 10% in people over 40 years.1 the annual cost of treatment per copd patient in the us
is $ 4,119 ( usd ) ; additionally , indirect nonmedical care costs of $ 1,527 ( usd ) per patient are
known.2 while other leading causes of death in the
us have declined steadily , copd death rates have doubled since 1970.3 copd disproportionately afflicts those of lower socioeconomic status and older adults , creating
reluctance to diagnose and treat patients.4 the
global initiative for chronic obstructive lung disease report in 2011 , emphasizing the significance
of this disease , confirmed that spirometry is a requirement for copd case definition.5 spirometry has the distinct advantage of being a reproducible measurement of lung function that
is superior to peak expiratory flow because of greater reliability and specificity.6 symptom based and clinical diagnosis contributes to
misdiagnosis and mistreatment.7,8 additionally , spirometry can distinguish between asthma
and copd.9 only one us study has investigated the prevalence of copd misdiagnosis with spirometry.10 however , generalizability of this study was limited :
patients were gathered from primary care practices in colorado and scotland.10 studies investigating copd diagnosis among the uninsured are absent .
the purpose of this study is to evaluate clinically diagnosed copd patients with spirometry to
ascertain copd misdiagnosis in an underserved population .
a descriptive study was conducted at a federally qualified health center following institutional
review board approval .
the retrospective cohort from february 2011 to june 2012 included all
patients who were referred by primary care providers for spirometry testing available at the health
center to confirm a previous copd diagnosis or for better management of uncontrolled copd symptoms .
spirometry was done according to procedures recommended by the american thoracic society using the
burdick spirocard ( cardiac science , hannover , germany).11 spirometry for each patient was performed by personnel who had
undergone training on how to use the spirometry program .
personnel were also trained on device
calibration , as well as recording all procedure relevant data .
a series of questions was asked to
the patients to identify contraindications for spirometry testing .
all referred patients
did not use a short acting beta agonist within 6 hours , a long acting beta agonist within 12 hours ,
ipratropium within 6 hours , tiotropium within 24 hours , or theophylline within 24 hours of the test .
a forced expiratory volume in the first second of expiration to forced vital capacity
( fev1/fvc ) ratio above or equal to 0.7 was designated as no obstruction.5 fev1/fvc ratio below 0.7 required further
evaluation with postbronchodilator testing . for postbronchodilator testing , 400 g of beta2 agonist ( albuterol ) with a spacer was
administered .
following the administration of the beta2 agonist and a 15 minute waiting period ,
postbronchodilator spirometry values were reassessed . increased fev1
> 12% and
> 200 ml when compared with baseline confirmed reversibility.12 patients were categorized as either no obstruction , reversible obstruction , or nonreversible
obstruction .
differences by age and pack year history for each group were assessed using analysis of
variance and tabulation of mean and standard deviation .
the 80 patients referred , 72 had a
previous diagnosis of copd ( 72 out of 444 patients at the clinic ) and eight were on anticholinergic
inhalers but had no previous copd diagnosis ( eight out of 51 patients at the clinic ) .
the majority of patients were self - pay patients ( 71.3% ) ,
caucasians ( 88.8% ) , and females ( 60% ) . across all patients , average pack years was 37.9
26.1 .
five patients in this study never smoked , three had previous copd diagnosis , and two were on
anticholinergic inhalers but had no previous copd diagnosis .
all five patients had spirometry
results consistent with no obstruction . in the group of patients with previous copd diagnosis
, three
patients claimed that copd diagnosis was made prior to the age of 35 years .
spirometry results
showed no obstruction for two of those patients and reversible obstruction for the third patient .
additionally , in the group of patients with previous copd diagnosis
, 18 patients did not remember
when they were first diagnosed with copd , and only 14 patients remembered having spirometry or
pulmonary function testing .
spirometry revealed 34 patients ( 42.5% ) with no obstruction , 18 patients ( 22.5% ) with reversible
obstruction , and 28 patients ( 35% ) with nonreversible obstruction ( figure 1 ) .
younger patients ( p = 0.004 ) and patients who
had fewer pack years of exposure ( p = 0.013 ) were more likely to be in the no
obstruction group .
patients in the nonreversible obstruction group were more likely to be older and
had 1.7 times more tobacco exposure .
spirometry confirms copd diagnosis in the primary care setting.1315 however ,
a vermont survey found that only 66% of primary care respondents owned a spirometer and that
spirometry was only performed on half of copd patients.16 in our study , only 14 patients ( 17.5% ) reported a previous spirometry or pulmonary
function testing .
when spirometry is not used , practitioners use smoking history and clinical
features for copd diagnosis which lacks sensitivity.17 additionally , spirometry differentiates between asthma and copd which have different
therapy goals and treatment plans .
reliance on nonspecific symptoms of dyspnea , cough , and wheezing
has resulted in health practitioners responding to two physiologically different diseases in the
same manner.5,18,19 inappropriate
diagnosis leads to inappropriate symptom management and ignorance of underlying etiology .
unnecessary medications , poor management , and side effects associated with inappropriate medications
increase the overall cost while potentially harming the patient .
known side effects associated with
medications used by these patients may include angioedema , anaphylaxis , bronchospasm , arrhythmia ,
glaucoma , and adrenal suppression.20 the presence of over diagnosis in the current series of copd patients 42.5%
is consistent with reports outside of the us .
international studies have examined the prevalence of
copd misdiagnosis due to lack of spirometry .
copd misdiagnosis was 31% in australia,7 28.6% in belgium,9 25.8% in norway,21
27.2% in the uk,22 and 49.8% in greece.23 one study which combined patients from colorado and
scotland documented a 51.6% copd misdiagnosis ( table
2).10 studies vary in their definition of reversible obstructions .
due to literature inconsistency and
discrepant guidelines , we decided to only consider results that showed no obstruction
( fev1/fvc 0.7 ) as a clear copd misdiagnosis .
although being diagnosed with or
treated for copd , 42.5% of our patients showed no obstruction on spirometry .
given that some of the
patients who had reversible obstruction could have asthma and not copd , prevalence of copd
misdiagnosis could be higher in this series .
since the only study performed in the us included
patients from scotland and colorado,10 the
prevalence of copd misdiagnosis in the us remains unclear .
lower european copd misdiagnosis could be linked to emphasis on spirometry in ambulatory
settings.18 low utilization of spirometry in the
us may be attributed to factors such as unawareness of the value of spirometry , lack of access , busy
settings , and lack of device training.16 another
major challenge is provider perception of test uncertainty and the inability to interpret spirometry
data.24 incorporating training seminars to educate
health professionals has shown promise , increasing spirometry use by 59% in a 3 month period
following training.16 this study is the first to examine copd misdiagnosis and prevalence of spirometry in a group of
solely american patients .
study limitations include patient recall bias and data entry errors in the
medical record .
the current series was only referred for spirometry based on the individual
provider s preferences to manage uncontrolled copd or to confirm previously diagnosed copd .
therefore , current data may not be representative and should be viewed as a pilot investigation to
assess copd in an underserved population .
however , current data suggest that there is a problem with
copd over diagnosis in the underserved population and that problem could be significant due to the
lack of resources
. the underserved population may have more risk for fragmented care and lack of
access to health care resources which could make them more vulnerable to such issues .
justification of offering free or
discounted spirometry service could be explored based on anticipated cost avoidance of misdiagnosed
copd cases .
symptoms and smoking history are insufficient to diagnose copd . prevalence of copd over diagnosis
in uninsured patient populations may be higher than previously reported . confirming copd diagnosis
with spirometry is essential to avoid unnecessary and potentially harmful over treatment . | introductionwhile cross - national studies have documented rates of chronic obstructive pulmonary disease
( copd ) misdiagnosis among patients in primary care , us studies are scarce . studies investigating
diagnosis among uninsured patients are lacking.objectivethe purpose of this study is to identify patients who are over diagnosed and thus , mistreated ,
for copd in a federally qualified health center.methodsa descriptive study was conducted for a retrospective cohort from february 2011 to june 2012 .
spirometry was performed by trained personnel following american thoracic society recommendations .
patients were referred for spirometry to confirm previous copd diagnosis or to assess uncontrolled
copd symptoms .
airway obstruction was defined as a forced expiratory volume in the first second of
expiration ( fev1 ) to forced vital capacity ratio less than 0.7 .
reversibility was defined
as a postbronchodilator increase in fev1 greater than 200 ml and greater than 12%.resultseighty patients treated for a previous diagnosis of copd ( n = 72 ) or on anticholinergic inhalers
( n = 8) with no copd diagnosis were evaluated .
the average age was 52.9 years ; 71% were uninsured .
only 17.5% ( 14/80 ) of patients reported previous spirometry .
spirometry revealed that 42.5% had no
obstruction , 22.5% had reversible obstruction , and 35% had non - reversible obstruction.conclusionsymptoms and smoking history are insufficient to diagnose copd .
prevalence of copd over diagnosis
among uninsured patient populations may be higher than previously reported .
confirming previous copd
diagnosis with spirometry is essential to avoid unnecessary and potentially harmful treatment . |
osteoarthritis , characterized by the breakdown of articular cartilage in synovial joints , has long been viewed as the result of wear and tear1 .
although low - grade inflammation is detected in osteoarthritis , its role is unclear24 .
here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis . through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis
, we find that expression and activation of complement is abnormally high in human osteoarthritic joints .
using mice genetically deficient in c5 , c6 , or cd59a , we show that complement , and specifically the membrane attack complex ( mac)-mediated arm of complement , is critical to the development of arthritis in three different mouse models of osteoarthritis .
pharmacological modulation of complement in wild - type mice confirmed the results obtained with genetically deficient mice .
expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of c5-deficient mice than c5-sufficient mice , and mac induced production of these molecules in cultured chondrocytes .
furthermore , mac co - localized with matrix metalloprotease ( mmp)-13 and with activated extracellular signal - regulated kinase ( erk ) around chondrocytes in human osteoarthritic cartilage .
our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis . | |
the concept of a reference group and its potential influence on an individual has been know at least since the landmark publication of merton ( 1949 ) , but its beginnings can be traced to cooley ( 1902 ) . a brief definition of a reference group is given in park and lessig ( 1977 ) :
( ... ) a reference group is defined to be an actual or imaginary individual or group conceived of having significant relevance upon an individual s evaluations , aspirations , or behavior .
( ... ) a reference group is defined to be an actual or imaginary individual or group conceived of having significant relevance upon an individual s evaluations , aspirations , or behavior .
the present paper uses the concept of a reference group in a very broad sense as defined above .
the general discussion regarding the concept of a reference group is beyond the scope of this paper .
a brief discussion of the history of the concept of a reference group and its relevance can be found in dawson and chatman ( 2001 ) .
suppose that each individual in the population uses a product of one of two alternative brands .
once in a while the product breaks and needs to be replaced with a new one . at this time , an individual tries to determine which brand is used by the majority of a population .
he selects at random a group of \documentclass[12pt]{minimal }
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\begin{document}$k \in \mathbb { n}$\end{document}k individuals .
he chooses a brand used by the majority of those in the reference group with the probability (0,1 ) , referred to as the follow - up probability .
this procedure is repeated in time and leads to fluctuations of the share of each brand in the market .
the market share of the first brand at any given time t is denoted by x(t ) . the two parameters , the size of a reference group k and the follow - up probability , are exogenous .
the size k of the reference group governs the precision of information that an individual gets while choosing a brand.1 the follow - up probability is the measure of affect s strength of an individual for the group . the strength of the influence of the group on the individual thus measures how strongly an individual wants to be associated with the majority of a population .
the value = 1/2 is referred to as the neutral position larger values indicate the positive attitude to the information , while lower values indicate the negative attitude to the information .
the main issue investigated in the present paper is the isolated effect of the reference groups on the market share of a particular brand .
the second point concerns the dependence of equilibrium on the follow - up probability and the size of a reference group k. it is shown that , depending on the follow - up probability , there are two distinct types of model behavior , i.e. the model is not structurally stable for any k. the ( pitchfork ) bifurcation occurs for relatively high values of the follow - up probability provided that the size of a reference group is small , which may be interpreted as the negligible effect of the reference group in certain circumstances . the rest of the paper is organized in the following way . in section 2 ,
a large but finite population of a size \documentclass[12pt]{minimal }
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\begin{document}$ n \in \mathbb { n } $ \end{document}n of individuals is assumed .
at each period t = 0 , ,2 , , where >0 , there is a fraction of the population using the first brand .
the learning algorithm employed by members of the population is very simple . at each period t
, a single individual is selected at random.2 this individual randomly chooses a group of k persons from the population , referred to as the reference group.3 an individual in question determines which alternative is in the majority in a reference group and chooses this alternative with the probability (0,1 ) .
the probability of following the majority in a reference group is called the follow - up probability .
this process is repeated during every period . in the above construction , the time is assumed to be discrete , t = 0 , ,2 , , and the size of a population n is finite .
the state of a population can be described in several different ways , but because the model is concerned only with the market share of each brand , it is described by the share of a population x(t ) using the first brand .
given that the size of a population n is finite , the set of possible states of a population { 0,1/n,2/n, ,(n1)/n,1 } is also finite .
the procedure of brand selection defines the transition probabilities between the states of a population .
these probabilities are denoted by p
ij(x ) , that is , p
ij(x ) is the probability that a single individual changes the brand from the i - th alternative to the j - th alternative , i , j = 1,2 , given the state of a population x. the above model is , in fact , a finite markov chain .
this markov chain can be studied directly , but it is easier to consider a system of differential equations of the form
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\begin{document}$$ \left\ { \begin{array}{llll } \hspace*{1.5pc } \dot{x } & = & p_{21}(x ) - p_{12}(x ) , \\
.
system ( 1 ) approximates4 the average behavior of the markov chain ; cf .
( 1 ) is derived by taking the limit n with = 1/n but for the approximation to hold , it is enough to have the size n of a population finite and large enough .
the size k of a reference group defines , together with the follow - up probability , the transition probabilities and is not related in any way to the size of a population but only to the state of a population , that is , as long as the state of a population is x and both k and are fixed , then the size n of a population can be arbitrary .
it is assumed only that n is larger than k. in fact , in the context of the background story , it is better to think of k as a much smaller number than the size n of a population .
it is clear that , for two alternatives , one of the equations in eq .
1 can be dropped and from now on it is assumed that the second equation is dropped and only the following equation is used
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\begin{document}$$ \dot{x } = p_{21}(x ) - p_{12}(x ) .
is that the change in the share of a population supporting the first alternative is the difference between the rate of inflow into the first alternative and the outflow from it .
the approximation ( 2 ) is considered in continuous time and solutions take values in the interval . however , for large n , the grid of states of the markov chain is very dense and , with a slight abuse of notation , the same notation is used for both.5
for any given k , it is simple to calculate the probabilities p
ij(x ) .
however , for simplicity , it is assumed6 in this section that k = 3 . with this assumption
the probability p
12(x ) reads
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\begin{document}$$\begin{array}{@{}rcl@ { } } p_{12}(x ) & = & x \left ( \alpha \left((1-x)^{3 } + 3 ( 1-x)^{2 } x\right)\right.\\ & & \left .
+ ( 1-\alpha ) \left(x^{3 } + 3 ( 1-x ) x^{2}\right)\right ) \end{array } $ $ \end{document}p12(x)=x(1x)3 + 3(1x)2x+(1)x3 + 3(1x)x2and the probability p
21(x ) reads
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\begin{document}$$\begin{array}{@{}rcl@ { } } p_{21}(x ) & = & ( 1-x ) \left ( \alpha \left(x^{3 } + 3 x^{2 } ( 1-x)\right)\right.\\ & & \left .
+ ( 1-\alpha ) \left((1-x)^{3 } + 3 ( 1-x)^{2 } x\right)\right ) . \end{array } $ $ \end{document}p21(x)=(1x)x3 + 3x2(1x)+(1)(1x)3 + 3(1x)2x.the probability p
12 is the product of two probabilities .
they are the probability that the supporter of the first alternative is selected and the conditional probability that the second alternative is followed .
the probability that the supporter of the first alternative is selected , given the state x of a population , is simply x. the conditional probability that the second alternative is followed is the sum of probabilities of four events : every individual in the reference group uses the second alternative and the majority follows what happens with the probability (1x ) ; two individuals in the reference group use the second alternative and the majority follows with the probability 3(1x )
x ; only one individual in the reference group uses the second alternative and the minority follows with the probability ( 1)3(1x)x
; and finally all individuals in the reference group use the first alternative and the minority follows with the probability ( 1)(1x ) .
they are the probability that the supporter of the second alternative is selected and the conditional probability that the first alternative is followed . in the considered model
, it is assumed that the probability of following a given alternative is independent of the currently supported alternative .
thus , the conditional probability of following a given alternative is the same for both currently supported alternatives , i.e. the probabilities p
12(x ) and p
21(x ) differ only by the first term x and ( 1x ) , respectively , and switching every occurrence of to ( 1 ) and the other way around . ultimately , given the above assumptions , the differential equation describing the evolution of the share x(t ) reads
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\begin{document}$$ \dot{x } = f(x ) = ( 1 - 2x ) \left ( x^{2 } ( 2 \alpha -1 ) - x ( 2\alpha -1 ) + 1 - \alpha \right ) .
thus , for any initial conditions , there is a unique solution defined for t0 .
second , the interval , which is a projection of the two - dimensional simplex , is forward invariant because f(0)=1>0 and f(1)=1<0 . for any follow - up
probability , there is an equilibrium \documentclass[12pt]{minimal }
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\begin{document}$ \hat { x}^{a } = 1/2 $ \end{document}x^a=1/2 and because
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\begin{document}$$\frac{df(\hat{x}^{a})}{dx } = \frac{6}{2 } \left ( \alpha - \frac{5}{6 } \right ) $ $ \end{document}df(x^a)dx=6256this equilibrium is stable for < 5/6 and unstable for >5/6 . for >5/6 ,
there are two additional equilibria , namely
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\begin{document}$$\hat{x}^{b } = \frac{1}{2 } - \sqrt{\frac{5 - 6\alpha}{4 ( 1 - 2 \alpha ) } } \quad \text{and } \quad \hat{x}^{c } = \frac{1}{2 } + \sqrt{\frac{5 - 6\alpha}{4 ( 1 - 2 \alpha)}}. $ $ \end{document}x^b=12564(12)andx^c=12 + 564(12 ) .
both equilibria are stable for >5/6 because
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\begin{document}$$\frac{df(\hat{x}^{b})}{dx } = \frac{df(\hat{x}^{c})}{dx } = 5 - 6\alpha < 0 \quad \text{for}~~\alpha > \frac{5}{6 } .
0 = 5/6 a pitchfork bifurcation occurs.7 figure 1 shows the behavior of f for various values of the follow - up probability and the bifurcation diagram for ( 5 ) .
fig . 1
a behavior of f for various values of the follow - up probability . the dotted line is for = 9/12
, the solid bold line is for = 5/6 and the thin solid line is for = 11/12 .
solid lines are for the stable equilibriums , the dotted line is for the unstable equilibrium
a behavior of f for various values of the follow - up probability . the dotted line is for = 9/12 , the solid bold line is for = 5/6 and the thin solid line is for = 11/12 .
solid lines are for the stable equilibriums , the dotted line is for the unstable equilibrium the analysis in the previous section is done for a small reference group where k = 3 .
it is possible to derive exact formulas for p
ij for any fixed k. however , the polynomials describing the probabilities of transitions become large and unwieldy very quickly . therefore , it is easier to approximate these probabilities by the central limit theorem .
let l be the number of people supporting the first alternative in a randomly selected reference group of a size k ; l is a random variable following the binomial distribution with parameters k ( a number of trials ) ; and x is the probability of a success .
let w denote the probability that l > k/2 , that is , w is the probability that the majority of individuals in the selected reference group supports the first alternative given the state of a population x. for large values of k , the probability w is approximated by the standard normal cumulative distribution function as follows
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\begin{document}$$\begin{array}{@{}rcl@ { } } w & = & \text{pr}\left[{l > \frac{k}{2}}\right ] = 1 - \text{pr}\left[{l \leq \frac{k}{2}}\right]\\[-1pt ] & = & 1 - \text{pr}\left[{l - k x \leq \frac{k}{2 } - kx}\right]\\[-1pt ] & = & 1 - \text{pr}\left[{\frac{l - k x}{\sqrt{k x ( 1-x ) } } \leq \frac{k/2 - kx}{\sqrt{k x ( 1-x)}}}\right]\\[-1pt ] & \approx & 1 - { \phi}\left(\frac{k/2 - kx}{\sqrt{k x ( 1-x)}}\right)\\[-1pt ] & = & 1 - \frac{1}{2 } \left[1 + \text{erf}\left({\frac{k/2 - kx}{\sqrt{2 k x ( 1-x)}}}\right ) \right ] , \end{array } $ $ \end{document}w = prl > k2=1prlk2=1prlkxk2kx=1prlkxkx(1x)k/2kxkx(1x)1k/2kxkx(1x)=1121+erfk/2kx2kx(1x),where erf ( ) is the standard error function , given as
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\begin{document}$$\text{erf}({x } ) = \frac{2}{\sqrt{\pi } } { \int\limits_{0}^{x } } \mathrm{e}^{-\tau^{2 } } d\tau $ $ \end{document}erf(x)=20xe2dand the approximation follows by the central limit theorem .
the transition probabilities are
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\begin{document}$$\begin{array}{@{}rcl@ { } } p_{12}(x ) & = & x ( \alpha ( 1-w ) + ( 1-\alpha ) w)\\ p_{21}(x ) & = & ( 1-x ) ( \alpha w + ( 1-\alpha ) ( 1-w ) ) . \end{array } $ $ \end{document}p12(x)=x((1w)+(1)w)p21(x)=(1x)(w+(1)(1w)).taking into account ( 2 ) , ( 6 ) and ( 7 ) the equation analogous to eq .
5 reads
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\begin{document}$$ \dot{x } = g(x ) = \left(\frac{1}{2}-x\right ) + \left(\alpha - \frac{1}{2 } \right ) \text{erf}\left({\frac{\sqrt{k}}{2 \sqrt{2 } } \ ; \frac{2 x -1}{\sqrt{x ( 1-x)}}}\right ) .
however , the limits at these points are
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\begin{document}$$\lim\limits_{x \to 0^{+ } } g(x ) = 1-\alpha > 0 \qquad\text{and}\qquad \lim\limits_{x \to 1^{- } } g(x ) = \alpha-1 < 0 .
these limits are taken as the values of g(x ) for x = 0 and x = 1 . since g(0)>0 and g(1)<0 , the interval is forward invariant under g. also , g is a c
function .
thus , for any initial condition , there exists a unique solution defined for t0 . for any (0,1 )
, there is an equilibrium \documentclass[12pt]{minimal }
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\begin{document}$
the derivative at x = 1/2 reads
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\begin{document}$$\frac{d g(x)}{d x } = \frac{(2 \alpha -1 ) \sqrt{k } \mathrm{e}^{\frac{k ( 1 - 2 x)^{2}}{8 ( x-1 ) x}}}{4 \sqrt{2 \pi } ( -(x-1 ) x)^{3/2}}-1 \quad \text{and } \quad \frac{d g(1/2)}{d x } = \sqrt{\frac{2}{\pi } } ( 2 \alpha -1 ) \sqrt{k}-1 .
further , dg(1/2)/dx = 0 for
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\begin{document}$$\alpha_{0 } = \frac{1}{4 } \left(\frac{\sqrt{2 \pi } } { \sqrt{k}}+2\right ) .
<
0 , the equilibrium \documentclass[12pt]{minimal }
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\begin{document}$ \hat { x}^{a } $ \end{document}x^a is stable and , for >
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\begin{document}$ \hat { x}^{a }
$ \end{document}x^a is unstable . at
0 , the pitchfork bifurcation occurs.8
the behavior of a model for large reference groups is qualitatively identical to the simple model with k = 3 .
however , the particular value of the critical follow - up probability
0 depends crucially on the size of a reference group .
the larger the reference group , the smaller the critical follow - up probability
0 . eventually , for k , the critical follow - up probability would converge9 to 1/2 .
figure 2a shows the behavior of g for various values of and k = 20 .
figure 2b shows the dependence of
0 on the size of a reference group .
fig . 2
a behavior of g for various values of the follow - up probability .
the dotted line is for = 2/10 , the solid bold line is for =
0 and the thin solid line is for = 8/10 .
b behavior of the critical follow - up probability
0 with respect to the cardinality of the reference group k
a behavior of g for various values of the follow - up probability . the dotted line is for = 2/10 , the solid bold line is for =
0 and the thin solid line is for = 8/10 .
b behavior of the critical follow - up probability
0 with respect to the cardinality of the reference group k
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\begin{document}$ n \in \mathbb { n } $ \end{document}n of individuals is assumed .
at each period t = 0 , ,2 , , where >0 , there is a fraction of the population using the first brand .
the learning algorithm employed by members of the population is very simple . at each period t
, a single individual is selected at random.2 this individual randomly chooses a group of k persons from the population , referred to as the reference group.3 an individual in question determines which alternative is in the majority in a reference group and chooses this alternative with the probability (0,1 ) .
the probability of following the majority in a reference group is called the follow - up probability .
this process is repeated during every period . in the above construction , the time is assumed to be discrete , t = 0 , ,2 , , and the size of a population n is finite .
the state of a population can be described in several different ways , but because the model is concerned only with the market share of each brand , it is described by the share of a population x(t ) using the first brand .
given that the size of a population n is finite , the set of possible states of a population { 0,1/n,2/n, ,(n1)/n,1 } is also finite .
the procedure of brand selection defines the transition probabilities between the states of a population .
these probabilities are denoted by p
ij(x ) , that is , p
ij(x ) is the probability that a single individual changes the brand from the i - th alternative to the j - th alternative , i , j = 1,2 , given the state of a population x. the above model is , in fact , a finite markov chain .
this markov chain can be studied directly , but it is easier to consider a system of differential equations of the form
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\begin{document}$$ \left\ { \begin{array}{llll } \hspace*{1.5pc } \dot{x } & = & p_{21}(x ) - p_{12}(x ) , \\
.
system ( 1 ) approximates4 the average behavior of the markov chain ; cf .
( 1 ) is derived by taking the limit n with = 1/n but for the approximation to hold , it is enough to have the size n of a population finite and large enough .
the size k of a reference group defines , together with the follow - up probability , the transition probabilities and is not related in any way to the size of a population but only to the state of a population , that is , as long as the state of a population is x and both k and are fixed , then the size n of a population can be arbitrary .
it is assumed only that n is larger than k. in fact , in the context of the background story , it is better to think of k as a much smaller number than the size n of a population .
it is clear that , for two alternatives , one of the equations in eq .
1 can be dropped and from now on it is assumed that the second equation is dropped and only the following equation is used
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\begin{document}$$ \dot{x } = p_{21}(x ) - p_{12}(x ) .
is that the change in the share of a population supporting the first alternative is the difference between the rate of inflow into the first alternative and the outflow from it .
the approximation ( 2 ) is considered in continuous time and solutions take values in the interval . however , for large n , the grid of states of the markov chain is very dense and , with a slight abuse of notation , the same notation is used for both.5
for any given k , it is simple to calculate the probabilities p
ij(x ) .
however , for simplicity , it is assumed6 in this section that k = 3 . with this assumption
the probability p
12(x ) reads
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\begin{document}$$\begin{array}{@{}rcl@ { } } p_{12}(x ) & = & x \left ( \alpha \left((1-x)^{3 } + 3 ( 1-x)^{2 } x\right)\right.\\ & & \left .
+ ( 1-\alpha ) \left(x^{3 } + 3 ( 1-x ) x^{2}\right)\right ) \end{array } $ $ \end{document}p12(x)=x(1x)3 + 3(1x)2x+(1)x3 + 3(1x)x2and the probability p
21(x ) reads
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\begin{document}$$\begin{array}{@{}rcl@ { } } p_{21}(x ) & = & ( 1-x ) \left ( \alpha \left(x^{3 } + 3 x^{2 } ( 1-x)\right)\right.\\ & & \left .
+ ( 1-\alpha ) \left((1-x)^{3 } + 3 ( 1-x)^{2 } x\right)\right ) . \end{array } $ $ \end{document}p21(x)=(1x)x3 + 3x2(1x)+(1)(1x)3 + 3(1x)2x.the probability p
12 is the product of two probabilities .
they are the probability that the supporter of the first alternative is selected and the conditional probability that the second alternative is followed .
the probability that the supporter of the first alternative is selected , given the state x of a population , is simply x. the conditional probability that the second alternative is followed is the sum of probabilities of four events : every individual in the reference group uses the second alternative and the majority follows what happens with the probability (1x ) ; two individuals in the reference group use the second alternative and the majority follows with the probability 3(1x )
x ; only one individual in the reference group uses the second alternative and the minority follows with the probability ( 1)3(1x)x
; and finally all individuals in the reference group use the first alternative and the minority follows with the probability ( 1)(1x ) .
they are the probability that the supporter of the second alternative is selected and the conditional probability that the first alternative is followed . in the considered model
, it is assumed that the probability of following a given alternative is independent of the currently supported alternative .
thus , the conditional probability of following a given alternative is the same for both currently supported alternatives , i.e. the probabilities p
12(x ) and p
21(x ) differ only by the first term x and ( 1x ) , respectively , and switching every occurrence of to ( 1 ) and the other way around .
ultimately , given the above assumptions , the differential equation describing the evolution of the share x(t ) reads
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\begin{document}$$ \dot{x } = f(x ) = ( 1 - 2x ) \left ( x^{2 } ( 2 \alpha -1 ) - x ( 2\alpha -1 ) + 1 - \alpha \right ) .
thus , for any initial conditions , there is a unique solution defined for t0 .
second , the interval , which is a projection of the two - dimensional simplex , is forward invariant because f(0)=1>0 and f(1)=1<0 . for any follow - up
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\hat { x}^{a } = 1/2 $ \end{document}x^a=1/2 and because
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\begin{document}$$\frac{df(\hat{x}^{a})}{dx } = \frac{6}{2 } \left ( \alpha - \frac{5}{6 } \right ) $ $ \end{document}df(x^a)dx=6256this equilibrium is stable for < 5/6 and unstable for >5/6 . for >5/6 , there are two additional equilibria , namely
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\begin{document}$$\hat{x}^{b } = \frac{1}{2 } - \sqrt{\frac{5 - 6\alpha}{4 ( 1 - 2 \alpha ) } } \quad \text{and } \quad \hat{x}^{c } = \frac{1}{2 } + \sqrt{\frac{5 - 6\alpha}{4 ( 1 - 2 \alpha)}}. $ $ \end{document}x^b=12564(12)andx^c=12 + 564(12 ) .
both equilibria are stable for >5/6 because
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\begin{document}$$\frac{df(\hat{x}^{b})}{dx } = \frac{df(\hat{x}^{c})}{dx } = 5 - 6\alpha < 0 \quad \text{for}~~\alpha > \frac{5}{6 } .
0 = 5/6 a pitchfork bifurcation occurs.7 figure 1 shows the behavior of f for various values of the follow - up probability and the bifurcation diagram for ( 5 ) .
fig . 1
a behavior of f for various values of the follow - up probability . the dotted line is for = 9/12
, the solid bold line is for = 5/6 and the thin solid line is for = 11/12 .
solid lines are for the stable equilibriums , the dotted line is for the unstable equilibrium
a behavior of f for various values of the follow - up probability . the dotted line is for = 9/12 , the solid bold line is for = 5/6 and the thin solid line is for = 11/12 .
solid lines are for the stable equilibriums , the dotted line is for the unstable equilibrium
the analysis in the previous section is done for a small reference group where k = 3 .
it is possible to derive exact formulas for p
ij for any fixed k. however , the polynomials describing the probabilities of transitions become large and unwieldy very quickly . therefore , it is easier to approximate these probabilities by the central limit theorem .
let l be the number of people supporting the first alternative in a randomly selected reference group of a size k ; l is a random variable following the binomial distribution with parameters k ( a number of trials ) ; and x is the probability of a success .
let w denote the probability that l > k/2 , that is , w is the probability that the majority of individuals in the selected reference group supports the first alternative given the state of a population x. for large values of k , the probability w is approximated by the standard normal cumulative distribution function as follows
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\begin{document}$$\begin{array}{@{}rcl@ { } } w & = & \text{pr}\left[{l > \frac{k}{2}}\right ] = 1 - \text{pr}\left[{l \leq \frac{k}{2}}\right]\\[-1pt ] & = & 1 - \text{pr}\left[{l - k x \leq \frac{k}{2 } - kx}\right]\\[-1pt ] & = & 1 - \text{pr}\left[{\frac{l - k x}{\sqrt{k x ( 1-x ) } } \leq \frac{k/2 - kx}{\sqrt{k x ( 1-x)}}}\right]\\[-1pt ] & \approx & 1 - { \phi}\left(\frac{k/2 - kx}{\sqrt{k x ( 1-x)}}\right)\\[-1pt ] & = & 1 - \frac{1}{2 } \left[1 + \text{erf}\left({\frac{k/2 - kx}{\sqrt{2 k x ( 1-x)}}}\right ) \right ] , \end{array } $ $ \end{document}w = prl > k2=1prlk2=1prlkxk2kx=1prlkxkx(1x)k/2kxkx(1x)1k/2kxkx(1x)=1121+erfk/2kx2kx(1x),where erf ( ) is the standard error function , given as
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\begin{document}$$\text{erf}({x } ) = \frac{2}{\sqrt{\pi } } { \int\limits_{0}^{x } } \mathrm{e}^{-\tau^{2 } } d\tau $ $ \end{document}erf(x)=20xe2dand the approximation follows by the central limit theorem .
the transition probabilities are
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\begin{document}$$\begin{array}{@{}rcl@ { } } p_{12}(x ) & = & x ( \alpha ( 1-w ) + ( 1-\alpha ) w)\\ p_{21}(x ) & = & ( 1-x ) ( \alpha w + ( 1-\alpha ) ( 1-w ) ) . \end{array } $ $ \end{document}p12(x)=x((1w)+(1)w)p21(x)=(1x)(w+(1)(1w)).taking into account ( 2 ) , ( 6 ) and ( 7 ) the equation analogous to eq .
5 reads
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\begin{document}$$ \dot{x } = g(x ) = \left(\frac{1}{2}-x\right ) + \left(\alpha - \frac{1}{2 } \right ) \text{erf}\left({\frac{\sqrt{k}}{2 \sqrt{2 } } \ ; \frac{2 x -1}{\sqrt{x ( 1-x)}}}\right ) .
however , the limits at these points are
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\begin{document}$$\lim\limits_{x \to 0^{+ } } g(x ) = 1-\alpha > 0 \qquad\text{and}\qquad \lim\limits_{x \to 1^{- } } g(x ) = \alpha-1 < 0 .
these limits are taken as the values of g(x ) for x = 0 and x = 1 . since g(0)>0 and g(1)<0 , the interval is forward invariant under g. also , g is a c
function .
thus , for any initial condition , there exists a unique solution defined for t0 . for any (0,1 )
, there is an equilibrium \documentclass[12pt]{minimal }
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\begin{document}$
the derivative at x = 1/2 reads
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\begin{document}$$\frac{d g(x)}{d x } = \frac{(2 \alpha -1 ) \sqrt{k } \mathrm{e}^{\frac{k ( 1 - 2 x)^{2}}{8 ( x-1 ) x}}}{4 \sqrt{2 \pi } ( -(x-1 ) x)^{3/2}}-1 \quad \text{and } \quad \frac{d g(1/2)}{d x } = \sqrt{\frac{2}{\pi } } ( 2 \alpha -1 ) \sqrt{k}-1 .
further , dg(1/2)/dx = 0 for
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\begin{document}$$\alpha_{0 } = \frac{1}{4 } \left(\frac{\sqrt{2 \pi } } { \sqrt{k}}+2\right ) .
<
0 , the equilibrium \documentclass[12pt]{minimal }
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\begin{document}$ \hat { x}^{a } $ \end{document}x^a is stable and , for >
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\begin{document}$ \hat { x}^{a }
$ \end{document}x^a is unstable . at
0 , the pitchfork bifurcation occurs.8
the behavior of a model for large reference groups is qualitatively identical to the simple model with k = 3 .
however , the particular value of the critical follow - up probability
0 depends crucially on the size of a reference group .
the larger the reference group , the smaller the critical follow - up probability
0 . eventually , for k , the critical follow - up probability would converge9 to 1/2 .
figure 2a shows the behavior of g for various values of and k = 20 .
figure 2b shows the dependence of
0 on the size of a reference group .
fig . 2
a behavior of g for various values of the follow - up probability .
the dotted line is for = 2/10 , the solid bold line is for =
0 and the thin solid line is for = 8/10 .
b behavior of the critical follow - up probability
0 with respect to the cardinality of the reference group k
a behavior of g for various values of the follow - up probability . the dotted line is for = 2/10 , the solid bold line is for =
0 and the thin solid line is for = 8/10 .
b behavior of the critical follow - up probability
0 with respect to the cardinality of the reference group k
the presented model is concerned solely with the isolated effect of the assumed algorithm of choosing a brand . as a result ,
any potential characteristics differentiating the brands are not part of the model and , consequently , the results are symmetric about the equal split of a market between two brands .
the results of a model should be interpreted as the potential edge that one brand can have due only to the reference group influence and not the actual preferences of population s members , since those are absent from the model .
the main result of the model is that this potential edge should be relatively small if the size of a reference group is small and the follow - up probability is not too large .
this behavior results from the imprecise information from the reference group and is amplified if an individual is not certain how to act upon the information .
5 and 8 , for k = 3 and k>3 , respectively , are only approximations to the actual markov chain .
5 or 8 is ergodic for any (0,1 ) and so there is a unique stationary distribution depending on k and . this stationary distribution is symmetric about x = 1/2 .
<
0 , the stationary distribution is a single peak distribution . for >
0 , the stationary distribution becomes a bimodal distribution with peaks aligned with the stable equilibria of a differential equation .
as the value of a follow - up probability 1 , these peaks converge to 0 and 1 . for = 1
the market share of the first brand fluctuates around x = 1/2 as long as <
0 , with increasing variance for
0 .
any realization of the markov chain spends most of the time near the stable equilibria of a differential equation , with occasional switches between them .
the time spent near one of the equilibria depends on the size n of a population .
the larger is n , the longer the time10 spent near the equilibrium before switching to the other equilibrium .
the setup of the presented model is somewhat similar to other models , in particular to the models where the population is identified with the set of nodes of a graph and the reference group of a player ( node ) is then identified with the set of its neighbors , as in degroot ( 1974 ) , watts ( 2002 ) or acemoglu et al . (
the present model also can be fitted within this general setup , provided that the graph is the poisson graph11 with the average degree equal to the size of a reference group . in this
are usually referred to as the mean - field equations or the master equation ; cf .
this is the typical choice for evolutionary game theory , population learning theory and other similar models such as the ones mentioned before .
the compromise is the structure of a network of connections and the type of results ( average behavior ) that can be achieved .
the random choice procedure used in the model can be thought of as reflecting an individual observing other randomly selected people and choosing a brand accordingly .
there are other choices possible , e.g. the threshold rule used in watts ( 2002 ) , best - reply type rules or rules based on imitations .
all these rules have one thing in common : they do not allow for innovations in the sense that , if all members of a population use the same brand , then every individual making the switch decision chooses the brand used by all members of a population.12 in terms of the markov chain , it means that these two states ( everyone uses the first brand and everyone uses the second brand ) are absorbing ( and consequently asymptotically stable equilibria ) .
the concept of a reference group is used in the present paper in a very broad sense as introduced in section 1 .
the speculations on the precise meaning of the presented model for theoretical sociology and economics is left for future research .
it is , however , clear that , in the light of recent papers such as kramer et al .
( 2014 ) , it is important to try to understand if it is possible to construct and to provide a product that , through the manipulation of the follow - up probability , can swing the brand shares in favor of a particular brand . ultimately , the main insight from the presented model is the interplay between the precision of information and the strength of reaction to this information .
if the information is not precise ( small k ) , then , even for a relatively strong reaction ( large ) , the result for the market share is slim ( realizations of the markov chain wander about x = 1/2 ) .
it takes precise information and a strong reaction in a large population to influence the market share of a given brand for a longer time . whether this can be achieved in reality
consider the following differential equation
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\begin{document}$$\dot{x } = f(x , \alpha ) , \quad x \in \mathbb{r } , \alpha \in \mathbb{r}. $ $ \end{document}x=f(x,),x,. following ( wiggins 2003 , p.372 ) , in order to show that the above equation undergoes a pitchfork bifurcation at \documentclass[12pt]{minimal }
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\begin{document}$ ( x , \alpha ) = ( \hat { x}^{},\alpha _ { 0 } ) $ \end{document}(x,)=(x^,0 ) , it is sufficient to show that
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\begin{document}$$\begin{array}{@{}rcl@ { } } f(\hat{x}^{},\alpha_{0 } ) & = & 0 , \qquad \frac{\partial f}{\partial x } ( \hat{x}^{},\alpha_{0})= 0 , \\ \frac{\partial f}{\partial \alpha } ( \hat{x}^{},\alpha_{0 } ) & = & 0 , \qquad \frac{\partial^{2 } f}{\partial x^{2 } } ( \hat{x}^{},\alpha_{0 } ) = 0 , \\
\frac{\partial^{2 } f}{\partial x \partial \alpha } ( \hat{x}^{},\alpha_{0 } ) & \neq & 0 , \qquad \frac{\partial^{3 } f}{\partial x^{3 } } ( \hat{x}^{},\alpha_{0 } ) \neq 0 .
we have \documentclass[12pt]{minimal }
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\begin{document}$ ( \hat { x } , \alpha _ { 0 } ) = ( 1/2 , 5/6 ) $ \end{document}(x^,0)=(1/2,5/6 ) and the following calculations
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\begin{document}$$\begin{array}{@{}rcl@ { } } f(\hat{x}^{},\alpha_{0 } ) & = & 0,\\ \frac{\partial f}{\partial x } ( \hat{x}^{},\alpha_{0 } ) & = & ( 6 ( 1 - 2 \alpha ) ( x-1 ) x-1)|_{(\hat{x}^{},\alpha_{0})}=0 , \\ \frac{\partial f}{\partial \alpha } ( \hat{x}^{},\alpha_{0 } ) & = & ( -4 x^{3}+6 x^{2}-1)|_{(\hat{x}^{},\alpha_{0})}=0,\\ \frac{\partial^{2 } f}{\partial x^{2 } } ( \hat{x}^{},\alpha_{0 } ) & = & ( -6 ( 2 \alpha -1 ) ( 2 x-1))|_{(\hat{x}^{},\alpha_{0})}=0 , \\ \frac{\partial^{2 } f}{\partial x \partial \alpha } ( \hat{x}^{},\alpha_{0 } ) & = & ( -12 ( x-1 ) x ) |_{(\hat{x}^{},\alpha_{0})}=3 \neq 0 , \\
\frac{\partial^{3 } f}{\partial x^{3 } } ( \hat{x}^{},\alpha_{0 } ) & = & ( 12 - 24 \alpha ) |_{(\hat{x}^{},\alpha_{0})}=-8\neq 0 .
. 8 , we have
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\begin{document}$$(\hat{x } , \alpha_{0 } ) = \left(1/2,\frac{1}{4 } \left(\frac{\sqrt{2 \pi } } { \sqrt{k}}+2\right)\right ) $ $ \end{document}(x^,0)=1/2,142k+2 and the following calculations
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\begin{document}$$\begin{array}{@{}rcl@ { } } g(\hat{x}^{},\alpha_{0 } ) & = & 0,\\ \frac{\partial g}{\partial x } ( \hat{x}^{},\alpha_{0 } ) & = & \left(\frac{(2 \alpha -1 ) \sqrt{k } \mathrm{e}^{\frac{k ( 1 - 2 x)^{2}}{8 ( x-1 ) x}}}{4 \sqrt{2 \pi } ( -(x-1 ) x)^{3/2}}-1\right)|_{(\hat{x}^{},\alpha_{0})}=0 , \\
\frac{\partial g}{\partial \alpha } ( \hat{x}^{},\alpha_{0 } ) & = & \left(\text{erf}\left(\frac{\sqrt{k } ( 2 x-1)}{2 \sqrt{2 } \sqrt{-(x-1 ) x}}\right)\right)|_{(\hat{x}^{},\alpha_{0})}=0,\\ \frac{\partial^{2 } g}{\partial x^{2 } } ( \hat{x}^{},\alpha_{0 } ) & = & \left ( -\frac{(2 \alpha -1 ) \sqrt{k } ( 2 x-1 ) \mathrm{e}^{\frac{k ( 1 - 2 x)^{2}}{8 ( x-1 ) x } } ( k+12 ( x-1 ) x)}{32 \sqrt{2 \pi } ( -(x-1 ) x)^{7/2 } } \right)|_{(\hat{x}^{},\alpha_{0})}=0 , \\ \frac{\partial^{2 } g}{\partial x \partial \alpha } ( \hat{x}^{},\alpha_{0 } ) & = & \left(\frac{\sqrt{k } \mathrm{e}^{\frac{k ( 1 - 2 x)^{2}}{8 ( x-1 ) x}}}{2 \sqrt{2 \pi } ( -(x-1 ) x)^{3/2 } } \right)|_{(\hat{x}^{},\alpha_{0})}= 2 \sqrt{\frac{2}{\pi } } \sqrt{k } \neq 0 , \\
\frac{\partial^{3 } g}{\partial x^{3 } } ( \hat{x}^{},\alpha_{0 } ) & = & \left(\frac { ( 2 \alpha -1 ) \sqrt{k } \mathrm{e}^{\frac{k ( 1 - 2 x)^{2}}{8 ( x-1 ) x}}}{256 \sqrt{2 \pi } ( -(x-1 ) x)^{11/2 } } h(x , \alpha ) \right)|_{(\hat{x},\alpha_{0})}= -4 ( k-3 ) \neq 0 , \end{array } $ $ \end{document}g(x^,0)=0,gx(x^,0)=(21)kek(12x)28(x1)x42((x1)x)3/21|(x^,0)=0,g(x^,0)=erfk(2x1)22(x1)x|(x^,0)=0,2gx2(x^,0)=(21)k(2x1)ek(12x)28(x1)x(k+12(x1)x)322((x1)x)7/2|(x^,0)=0,2gx(x^,0)=kek(12x)28(x1)x22((x1)x)3/2|(x^,0)=22k0,3gx3(x^,0)=(21)kek(12x)28(x1)x2562((x1)x)11/2h(x,)|(x^,0)=4(k3)0 ,
where
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\begin{document}$$\begin{array}{@{}rcl@ { } } h(x,\alpha ) & = & \left(k^{2 } ( 1 - 2 x)^{2}+8 k ( x-1 ) ( 18 ( x-1 ) x+5 ) x \right.\\ & & \left .
+ 48 ( x-1)^{2 } ( 16 ( x-1 ) x+5 ) x^{2}\right ) . \end{array } $ $ \end{document}h(x,)=k2(12x)2 + 8k(x1)(18(x1)x+5)x+48(x1)2(16(x1)x+5)x2 . | the present paper tries to answer analytically how much the reference group influence can affect an actual market share of a particular brand or product . it is found that the increase of the size of a reference group and the probability of following the majority within the reference group may lead to the temporary modest prevalence of one brand .
this result requires a relatively large size of a reference group and a high probability of following the majority within the reference group . if these conditions are not satisfied the effect on the market share is negligible . |
hydroxyl radicals , activated from hydrogen peroxide and hydrogen superoxide anion under light radiation , are well known as potent mutagens that attack dna and convert them to many different kinds of base and ribose derivatives [ 1 , 2 ] .
every aerobic organism possesses several enzymes to remove such toxic oxides , as well as to recover the damaged dna .
however , when an excess amount of the radicals attacks dna , the thymine base is oxidized at the 5-methyl group to form 5-formyluracil base ( hereafter 2-deoxy-5-formyluridine residue is referred to as fu ) as a major product .
( the four characters , a , t , g , and c , represent the respective nucleotide residues in dna sequence .
the other abbreviations used are fu for 5-formyluracil or 2-deoxy-5-formyluridine residue , dfutp for 2-deoxy-5-formyluridine 5-triphosphate , hplc for high pressure liquid chromatography , fua for d(cgcgaatfucgcg ) , and fug for d(cgcggatfucgcg ) . )
it was demonstrated that 2-deoxy-5-formyluridine triphosphate ( dfutp ) was incorporated against both a and g templates , possibly forming fu : a and fu : g base pairs during in vitro dna replication [ 3 , 4 ] . on the other hand ,
it was reported that dfutp - induced pyrimidine transitions , g : ca : t and a : tg : c , as well as a gene transversion from g : c to t : a , could occur in vivo [ 5 , 6 ] .
these results suggest that fu can behave as c , a , and g in addition to its original property of t. in order to reveal the interaction geometry of the modified base fu , we performed x - ray analyses on fu - containing dna duplexes .
the fu residues were introduced into the self - complementary dickerson - drew - type dodecamer sequence , which is expected to be easy to crystallize .
the dnas used in this study are d(cgcgaatfucgcg ) and d(cgcggatfucgcg ) and will be referred to as fua and fug , respectively .
the fu base faces either an adenine or a guanine at the two sites in each duplex .
their crystal structures have been successfully determined at resolutions ranging from 1.5 to 3.0 . in the preliminary papers [ 7 , 8 ] ,
magnesium ion effects on crystallizations of fua were discussed , but the detailed structure of base - pair formations and its biological significance were not published . in this paper
we describe the structures of the base pairs formed between fu and g and between fu and a , based on which the pyrimidine transition induced by the oxidized thymine base will be discussed .
fua and fug were synthesized by the solid phase phosphoramidite method as described previously in and were purified by reverse - phase column chromatography and reverse - phase and anion - exchange hplcs .
after naio4 treatment , the oligonucleotides with fu were further purified by reverse - phase and anion - exchange hplcs .
initial screenings of crystallization conditions were performed using the hanging drop vapor diffusion method , equilibrating 2 l droplets against 1 ml of the reservoir solution .
the optimized conditions for growing the four different crystals of fua ( fua , fua , fua , and fua ) and three different crystals of fug are given in table 1 .
as the fug crystal was too small , two kinds of dyes , hoechst 33258 and dapi , were added to stabilize the duplex formation ( fug and fug ) .
crystals suitable for x - ray data collections were picked up from their mother liquors using a nylon loop ( hampton research ) and transferred into liquid nitrogen .
all x - ray experiments for the seven crystals were performed with synchrotron radiation at the photon factory in tsukuba .
diffraction patterns of the fua crystals , which were recorded on imaging plates , were processed subsequently using the programs denzo and scalepack and those of the fug crystals , recorded on quantum 4 ccd , with the program dps / mosflm .
low - resolution data around the 10 resolution shell were truncated because they did not fit well into the diffraction profile .
initial phases were derived by molecular replacement with the program amore using the atomic coordinates of the corresponding unmodified dna duplex ( pdb i d 355d , see ) as structural probes .
the molecular structures were constructed and modified on a graphic workstation with the program coot in the ccp4 program suite .
the atomic parameters were refined with the program refmac in ccp4 with maximum - likelihood techniques , followed by interpretation of an omit map at every nucleotide residue .
the program cns was used in the final refinements of fug and fug to stabilize the base pairs containing x with hydrogen bonds .
examples of the quality of the final electron density maps are depicted in figure 1 .
helical and local base - pair parameters , as well as the torsion angles and pseudorotation phase angles of sugar rings , were calculated using the program 3dna . some of them are shown in table 3 and figure 4 .
atomic coordinates and structure factors have been deposited in the protein data bank with accession codes 1g75 , 1g8n , 1g8u , 1g8v , 3ajj , 3ajk , and 3ajl for fua , fua , fua , fua , fug , fug , and fug , respectively .
crystallization of fua was easy as expected , and the crystals obtained diffracted within the 1.51.8 resolution range . in every crystal
, the formyl group of the fu8 residue on one of the two chains was disordered between the two alternative conformers , anti and syn , while that in the other chain adopted only the syn conformation .
relative occupancies of the disordered oxygen atoms , estimated from the electron densities , were assumed in the structure refinements .
( the residue numbering is 1 ~ 12 for one chain and 13 ~ 24 for another chain . ) on the other hand , fug was difficult to crystallize .
attempts to crystallize fug under conditions similar to those for fua , that is , at neutral or slightly acidic ph , were unsuccessful .
small single crystals of fug appeared at basic ph ( ph 8.1 ) but they poorly diffracted x - rays .
this approach led to the successful crystallization of fug and fug although the resolution of the fug crystal was still quite low .
hoechst 33258 and dapi were found to be bound in the central region of the minor grooves of fug and fug , respectively , in a manner similar to the other dna duplexes containing such dyes [ 2127 ] .
interestingly , during refinements of fug , it was found that the two fu bases moved to different directions , one toward the minor groove side and the other toward the major groove side from the canonical watson - crick - type pairing position , and the resulting electron density also showed the same movements . in the case of fug and fug , however , the two fu bases moved to the same direction toward the minor groove side , and the electron densities , though poor , also supported the movements .
the local helical parameters show that all the fua and the fug duplexes adopt the b - form conformation even in complex with dyes , as shown in figure 2 .
superimpositions of the present structures onto the unmodified duplex structure are shown in figure 3 and yield an average root mean square deviation of 1.4 .
closer inspection of the superimposed structures reveals no drastic differences between the modified and the unmodified duplexes .
however , the minor groove widths ( see figure 4 ) indicate that fug and fug are wider at the center compared with those of the other dna duplexes .
these changes in the dna conformation are presumably due to the binding of dapi and hoechst33258 rather than the fu introductions .
another feature is that , at residues 35 , the minor groove width is wider .
this is typical of dickerson - drew - type dna duplexes that are packed in the orthorhombic cell .
the widening occurs because the duplex accepts the end of a neighboring duplex along the c axis through two hydrogen bonds to form a c : g : g : c quartet .
the other end ( the residues 8 - 10 ) is not widened . however , the corresponding width of every fug crystal is wider , as discussed later . in every crystal
, the two dna strands are associated to each other through base - pair formations , and all the bases , except for the modified bases and their counter bases positioned on the opposite strands in the duplexes , form the canonical watson - crick base pairs .
all the pairing geometries of fu residues are shown in figure 5 . as depicted in figure 1(a )
, the electron densities clearly show that the fu residues in fua are paired with the opposite a residues in the watson - crick geometry .
these results indicate that the oxidized t residue ( fu ) still has an ability to form a watson - crick - type base pair with a. the formyl groups of fu could either adopt the anti or syn conformations depending on the surrounding water structure . on the other hand ,
the most interesting features can be seen in the interaction geometries between fu and g in the fug crystals . in the fug crystal ,
the fu20 residue forms a typical wobble pair with g5 through the nho and ohn hydrogen bonds , and the formyl group is disordered between syn and anti conformations .
however the fu8 residue forms a new type of pair with the opposite g17 residue through the two hydrogen bonds , ohn and nhn . in the pairing ,
the g base moves to the major groove side and the fu8 base moves to the minor groove side .
an atom was located on the electron density map ( see figure 1 ) , and it is within hydrogen distance of the o and o atoms of fu8 and the o atom of g17 . as the density of the atoms was of almost the same level as those of other water molecules , a water molecule was temporarily positioned at the peak for further structure refinements . in other words , it seems that a water molecule is trapped in the space surrounded by the three oxygen atoms to stabilize the pair formation . in the fug crystal
, the two fu residues at the different sites also form the respective pairs with the opposite g residues .
the geometries are , however , both in the new reversed wobble type , as shown in figure 1(c ) .
water molecules are also assigned at the positions similarly surrounded by the three oxygen atoms , as described above , though the corresponding electron density at the fu20 site is rather poor due perhaps to low resolution .
furthermore , in the fug crystal obtained at ph 8.1 without the help of dyes , the electron density , though again at low resolution , suggests that the two fu residues form pairs with the opposite g residues in a similar manner to those found in fug obtained with dapi .
therefore it is considered that the presence of dyes stabilizes duplex structure but does not affect the pairing modes .
figure 6 summarizes the pairing modes with their chemical structures , found in the fua and fug crystals .
it is noteworthy that an oxygen atom always exists at the center almost equidistant from the three surrounding oxygen atoms ( o and o of fu and o of g ) in the reversed wobble geometry . because of this pairing
, the n atom of fu should be deprotonated and the o atom of fu might be ionized so that the central pocket must accept a hydronium ion instead of a water molecule to stabilize the pair formation .
this is consistent with the fact that the fug crystal was obtained in alkaline state without the help of dyes .
the pka value decreases from 9.7 to 8.1 in response to the formylation by t oxidization . a sodium ion but not a potassium
a comparison of the local base - pair parameters at the modified pairs is given in table 3 .
as the sequence is basically palindromic , the duplex has a twofold axis at the centre of the duplex perpendicular to the helical axis .
therefore , the base pairs at fu8 and fu20 exhibit buckle angles and shear distances with signs ( + / ) different between the positions related by the twofold symmetry , but propeller twist angles with the same sign though their absolute values are affected by crystal packing .
( the shear values fluctuate around zero in the standard b - form duplex . )
the fug duplexes also adopt this geometrical rule , except for the shear parameter . in the fug duplex , the two fu : g pairs have the shear values with the same sign ( fu8 2.24 and fu20 2.08 ) .
this means that the g17 and g5 bases which are paired with fu8 and fu20 move in the different directions along the x - axis [ see the definition in ] , that is , g17 shifts up toward the major groove side and g5 shifts down toward the minor groove side ( or fu8 moves down toward the minor groove and fu20 moves up toward the major groove ) .
these movements are just ascribed to the reversed wobbling and the normal wobbling , respectively .
another feature of the fu : g reversed wobbling can be seen in the cc distance which is longer by 0.6 , as compared with those of the watson - crick pairing .
this expansion is also reflected in the wider minor groove widths found in every fu : g duplexes .
the present work has clarified a total of three interaction modes , a watson - crick type for fu : a pairing and two wobble types ( wobble and reversed wobble ) for fu : g pairing .
it has been believed that the dna polymerase accepts base pairs only with the watson - crick geometries .
the wobble pairing easily occurs between g and t , but the dna polymerase eliminates such a pair by sieving the shape of the pairs . in order to examine the possibility that a reversed wobble pair could also be accepted by the enzyme the pairing geometry was modeled into the enzyme in silico . as seen in figure 7 , the reversed wobble pair is reasonably accommodated without atomic collisions by slightly rotating the pair . on the contrary , a wobble pair , rotated in the opposite direction , would still collide with atoms of the enzyme .
thus , it could be concluded that g forms a pair with fu in the ionized form and a forms a watson - crick pair with fu and that the reversed wobble pair is allowed to be bound in the dna polymerase .
this tolerance explains the occurrence of mis - incorporation during replication and leads to pyrimidine transition mutagenesis .
n - methoxyl - adenine [ 31 , 32 ] and n - methoxyl - cytosine [ 3335 ] residues in the damaged dnas adopt the imino tautomer to form mismatched base pairs , which mimic the watson - crick pair geometries .
in contrast , fu derived from thymine oxidation forms a reversed wobble pair , which is also acceptable to the dna polymerase .
the focus of the present work is on pyrimidine transition mutation and not gene transversion .
the atomic mechanism of the latter will be revealed by similar x - ray studies . | hydroxyl radicals are potent mutagens that attack dna to form various base and ribose derivatives .
one of the major damaged thymine derivatives is 5-formyluracil ( fu ) , which induces pyrimidine transition during replication . in order to establish the structural basis for such mutagenesis ,
the crystal structures of two kinds of dna d(cgcgratfucgcg ) with r = a / g have been determined by x - ray crystallography .
the fu residues form a watson - crick - type pair with a and two types of pairs ( wobble and reversed wobble ) with g , the latter being a new type of base pair between ionized thymine base and guanine base . in silico
structural modeling suggests that the dna polymerase can accept the reversed wobble pair with g , as well as the watson - crick pair with a. |
although a rare , but an important cause of drug resistant seizures , the dyke davidoff masson syndrome ( ddms ) is not a difficult diagnosis to make in the era when neuroimaging has profoundly improved epilepsy care . acquired or congenital ( infantile )
cerebral hemiatrophy , also referred to as ddms , is a diagnostic constellation made up of hemiparesis , facial asymmetry , intellectual disability , and epilepsy . on brain imaging , the findings of cerebral hemiatrophy , calvarial thickening , and hyperpneumatization of the frontal sinuses provides radiographic support for the clinical diagnosis .
the significance of an early and accurate diagnosis is the prospect of improving the patient 's prognosis and improving their quality of life .
this case illustrates a unique epilepsy syndrome and the need for a heightened awareness among epilepsy care providers .
furthermore , it emphasizes the importance of early evaluation of patients with epilepsy in sub - saharan africa where the diagnostic gap is still close to 40% .
we report a case of ddms in a nigerian with childhood - onset epilepsy and review the literature .
a 27-year - old female presented to our clinic with her father because of recurrent seizures and poor scholastic performance .
she had normal development until she was 2 years when she had a febrile seizure which was described as focal clonic involving the left side of the body .
about 15 years prior to presentation , she developed recurrent generalized tonic - clonic seizures .
she demonstrated impaired cognition and poor scholastic performance which prevented her from completing her secondary education .
her general physical examination revealed a young female who was small for her age , with a weight of 43.7 kg and a height of 1.56 m ( body mass index , bmi = 17.9 kg / m ) .
her score on ravens standard progressive matrices ( rspm ) was 11/60 , indicating intellectual impairment ( below the 5th percentile for her age ) .
neurologically , she had left facial hemiatrophy , left hemibody atrophy , and spastic hemiparesis ( power of 4/5 in both left upper and lower limbs with a left extensor plantar response .
, we suspected rasmussen encephalitis and requested a cranial magnetic resonance imaging ( mri ) and an electroencephalogram ( eeg ) . her brain mri ( see fig
. 1 . ) revealed right cerebral hemiatrophy with features of encephalomalacia surrounded by gliosis ( figure ) .
furthermore , on mri , she had right calvarium thickening and hyperpneumatization of the frontal sinus .
her eeg revealed a low amplitude background over the right hemisphere with intermittent bursts of generalized sharp - and - slow wave complexes .
these features place her in the ilae category of epilepsy with structural metabolic causes .
her seizure frequency has been reduced from at least once a week to less than weekly but at least once a month .
the findings of cerebral hemiatrophy , calvarial thickening , and hyperpneumatization of the frontal sinuses on mri in the setting of drug - resistant seizure , intellectual impairment , and hemibody atrophy are the diagnostic criteria for ddms in this case . for epilepsy care givers ,
our case illustrates the fact that seizures associated with hemibody atrophy have a differential diagnosis beyond rasmussen 's syndrome .
acute focal weakness following a seizure in a child has many serious and treatable causes . although benign etiologies such as todd 's paralysis and metabolic etiologies such as glucose , sodium , potassium , calcium , and magnesium abnormalities are easily identifiable , structural lesions such as neoplasms , intracerebral abscesses , acute disseminated encephalomyelitis , developmental brain malformations , and signs of trauma and vascular disorders such as ischemic and hemorrhagic infarctions should be excluded .
hemiconvulsion - hemiplegia - epilepsy ( hhe ) syndrome should also be entertained . in hhe syndrome
, neuroradiological studies may show unilateral edematous swelling of the ipsilateral hemisphere at the time of initial status epilepticus ( se ) .
although neuroimaging studies were not conducted at the time of the initial insult in the presented case , the generalized tonic - clonic seizures after hemiconvulsions , which characterized this patient 's presentation , are less typical of hhe syndrome , which is characterized by hemiconvulsions alone .
this is in addition to the long latency ( 10 years ) between the acute encephalopathy and the onset of epilepsy ( typically 2 months to 2 years in hhe ) , though the two syndromes have been recognized synonymously .
other differential diagnoses should include sturge weber syndrome , basal ganglia germinoma , silver russel syndrome , fishman syndrome , and linear nevus sebaceous syndrome . in rasmussen 's syndrome , the calvarial changes are not found .
basal ganglia germinoma presents with similar clinical features , but brain imaging reveals cystic areas , focal hemorrhages , and mild surrounding edema along with calvarial changes . in sturge weber syndrome , brain imaging shows enhancing pial angiomas and cortical calcifications in addition to the clinical features of the phacomatosis .
russell syndrome is characterized by the classical facial phenotype ( triangular face ; small , pointed chin ; broad forehead ; and thin , wide mouth ) , poor growth with delayed bone age , clinodactyly , hemihypertrophy with normal head circumference , and normal intelligence .
fishman syndrome or encephalocraniocutaneous lipomatosis is a rare neurocutaneous syndrome including unilateral cranial lipoma with lipodermoid of the eye , which usually presents with seizures .
mims syndrome ) is characterized by a typical facial nevus , mental retardation , recurrent seizures , and hemimegaencephaly .
the etiology of ddms is due to a cerebral insult that may occur in utero or early in life .
the acquired type depends on the time of the insult and may be seen in adolescent or adults . in the congenital type , there is a midline shift towards the affected hemisphere , and the sulcal prominence replacing the gliotic tissue is absent .
pattern ii corresponds to diffuse cortical atrophy coupled with a porencephalic cyst , while pattern iii corresponds to previous infarction with gliosis in the middle cerebral artery ( mca ) territory .
the clinical profile and imaging features in our patient are in keeping with acquired ddms pattern iii .
the mechanism of cerebral atrophy and the related progressive neurological deficit are posited to be due to ischemic episodes which reduce the production of brain - derived neurotrophic factors , which , in turn , lead to cerebral atrophy . in some cases ,
ddms is accompanied by crossed cerebro - cerebellar atrophy , which is thought to be a continuum of cerebro - cerebellar diaschisis .
it has been postulated that frequent and excessive excitatory input during seizures via glutaminergic corticopontine
the 3 days of unconsciousness was due to either non - convulsive status epilepticus , a prolonged postictal state , or a coma of unknown etiology .
the possible etiological association between ddms , prolonged febrile seizures , and middle cerebral artery stroke has been described .
because of the high incidence ( 1118% ) of febrile seizures in our environment , with nearly 4% of patients going on to develop epilepsy , inappropriate management of febrile seizure should be highly discouraged to forestall untoward outcomes .
medical treatment of ddms includes rational use of antiseizure drugs . if seizures are drug - resistant , they can be treated surgically with hemispherectomy , which has an 85% chance of rendering the patient seizure free .
we could not offer hemispherectomy to our patient because epilepsy surgery and other tertiary care epilepsy management , including vagus nerve stimulation is still unavailable in our region of nigeria .
currently , management of epilepsy is still limited to rational use of the antiseizure drugs .
the relatively high cost of asds , on the background of high unemployment , poor remuneration , and out - of pocket payment for treatment , facilitates poor adherence to medications and widening of the treatment gap .
efforts at rapid resolution of acute symptomatic seizures should be advanced to prevent untoward outcome .
early diagnosis by an appropriate imaging study , optimization of medical management of the seizures , and enhancement of neurorehabilitation are equally important .
to the best of our knowledge , this is the first case of ddms described in our environment in nigeria , although it has been described in other lower - middle income countries like iran , india , tunisia , and turkey .
the paucity of neuroimaging facilities in our region may deter early recognition , and , therefore , a high clinical index of suspicion is required .
we advocate prompt referral of patients with suspected ddms and refractory epilepsy to regional specialty care facilities in underserved countries to facilitate an early diagnosis and to provide optimal regional treatment .
| dyke davidoff masson syndrome ( ddms ) is a rare , but important cause of drug - resistant seizures .
dyke davidoff
masson syndrome is a constellation of clinical features that consists of hemiparesis , seizure , facial asymmetry , and intellectual disability with distinct neuroimaging features .
a 27-year - old lady presented to us with drug - resistant epilepsy , hemiparesis , and intellectual disability that necessitated her withdrawal from school .
her brain magnetic resonance imaging ( mri ) showed cerebral hemiatrophy , calvarial thickening , and hyperpneumatization of the frontal sinuses consistent with ddms .
we discuss the diagnostic and therapeutic implications of ddms and advocate early referral and evaluation of people with epilepsy in sub - saharan african settings . |
microglia , the only immunocompetent cells in the cns , originate from progenitors derived from the yolk sac ( 1 , 2 ) . studies have shown that , after birth , microglia maintain their numbers under normal conditions by self - renewal without the recruitment of monocyte - derived microglia precursors ( 2 , 3 ) .
although there is currently no specific marker for distinguishing resident microglia from monocyte - derived macrophages unequivocally , some studies have shown that microglia and peripheral blood cell
derived macrophages can be distinguished by the presence of surface markers , such as ccr2 and cx3cr1 , even under inflammatory conditions ( 4 , 5 ) .
moreover , a study has shown that human y chromosome bearing male cells can not differentiate into microglia in the cns of a female host after bone marrow transplantation ( 6 ) .
by contrast , bone marrow stem cells and peripheral monocytes can differentiate into microglia - like cells in vitro ( 7 , 8) , suggesting that one of the most important barriers to the recruitment of microglial precursors from peripheral blood is the blood - brain barrier ( bbb ) .
indeed , cranial irradiation or administration of the alkylating agent busulfan can disrupt the bbb , enabling bone marrow derived cells to invade the brain parenchyma and remain there ( 9 ) .
stress was found to induce the recruitment of bone marrow derived monocytes to the brain parenchyma in mice ( 10 ) , but the results of many other studies did not clarify the origin of microglia in human adult brains . here ,
we report the findings of a human autopsy case in which umbilical cord blood stem cell derived cells differentiated into ramified microglia in the recipient brain parenchyma .
a 51-year - old japanese woman noticed swelling of her nasal floor at age 48 years ; she was diagnosed as having natural killer / t - cell lymphoma at our hospital .
she received focal radiation therapy targeting the paranasal , maxillary , and ethmoid sinuses , the parotid glands , and the right and left orbits ( 40 gy ) , and novalis shaped beam surgery ( 12 gy ) .
after radiation therapy , she received 3 courses of devic ( dexamethasone , etoposide , ifosfamide , and carboplatin ) chemotherapy .
after treatment , she achieved complete remission ; 2 months after chemotherapy , she received a cord blood stem cell transplant ( cbsct ) from a male donor with whom she had 2 human leukocyte antigen ( hla ) mismatches ( donor : a*31:01 , a*33:03 , b*51:01 , b*40:03 , drb1 * 08:02 , drb1 * 14:05 ; recipient : a*31:01 , a*02:01 , b*51:01 , b*40:02 , drb1 * 08:02 , drb1 * 14:05 ) .
acute graft - versus - host disease was mild and well controlled by oral administration of tacrolimus .
four months after cbsct , the patient presented with shingles of the right 12th thoracic segment and was treated with acyclovir ; however , herpes zoster developed into cervical myelitis . combined therapy with steroid pulse treatment and intravenous administration of gamma globulin was effective .
two years after cbsct , she experienced muscle weakness and sensory disturbance of the lower extremities .
in addition , she was given a single intrathecal administration of methotrexate ( 15 mg ) 2 weeks before her death .
she died 29 months after cbsct . a general autopsy was performed 3 hours after death .
neuropathologic examination demonstrated the presence of lymphoma in the spinal cord ( including a lesion at c5 ) and inflammation of the left medulla , with no evidence of tumor in the cerebrum and cerebellum .
multiple tissue blocks were embedded in paraffin , and 6-m sections were cut and immunostained using a liquid dab substrate chromogen system kit ( dako , tokyo , japan ) and a tmb peroxidase substrate kit ( vector laboratories , burlingame , ca ) .
primary antibodies included rabbit polyclonal anti - iba1 ( 1:8000 ; wako , osaka , japan ) , anti glial fibrillary acidic protein ( 1:10000 ; sigma , tokyo , japan ) , anti - cx3cr1 ( 1:100 ; abcam , tokyo , japan ) , rabbit monoclonal anti - ccr2 ( 1:4000 ; abcam ) , and mouse monoclonal anti
briefly , 5-m sections from formalin - fixed paraffin - embedded blocks were deparaffinized , rehydrated , and predigested with 1 drop of pepsin solution .
the sections were incubated with hybridization solution containing zytodot cen yq12 probe ( zytovision , bremerhaven , germany ) overnight at 37c . the zytodot cish implementation kit ( zytovision )
a 51-year - old japanese woman noticed swelling of her nasal floor at age 48 years ; she was diagnosed as having natural killer / t - cell lymphoma at our hospital .
she received focal radiation therapy targeting the paranasal , maxillary , and ethmoid sinuses , the parotid glands , and the right and left orbits ( 40 gy ) , and novalis shaped beam surgery ( 12 gy ) .
after radiation therapy , she received 3 courses of devic ( dexamethasone , etoposide , ifosfamide , and carboplatin ) chemotherapy .
after treatment , she achieved complete remission ; 2 months after chemotherapy , she received a cord blood stem cell transplant ( cbsct ) from a male donor with whom she had 2 human leukocyte antigen ( hla ) mismatches ( donor : a*31:01 , a*33:03 , b*51:01 , b*40:03 , drb1 * 08:02 , drb1 * 14:05 ; recipient : a*31:01 , a*02:01 , b*51:01 , b*40:02 , drb1 * 08:02 , drb1 * 14:05 ) .
acute graft - versus - host disease was mild and well controlled by oral administration of tacrolimus .
four months after cbsct , the patient presented with shingles of the right 12th thoracic segment and was treated with acyclovir ; however , herpes zoster developed into cervical myelitis . combined therapy with steroid pulse treatment and intravenous administration of gamma globulin was effective .
two years after cbsct , she experienced muscle weakness and sensory disturbance of the lower extremities .
in addition , she was given a single intrathecal administration of methotrexate ( 15 mg ) 2 weeks before her death .
she died 29 months after cbsct . a general autopsy was performed 3 hours after death .
neuropathologic examination demonstrated the presence of lymphoma in the spinal cord ( including a lesion at c5 ) and inflammation of the left medulla , with no evidence of tumor in the cerebrum and cerebellum .
multiple tissue blocks were embedded in paraffin , and 6-m sections were cut and immunostained using a liquid dab substrate chromogen system kit ( dako , tokyo , japan ) and a tmb peroxidase substrate kit ( vector laboratories , burlingame , ca ) .
primary antibodies included rabbit polyclonal anti - iba1 ( 1:8000 ; wako , osaka , japan ) , anti glial fibrillary acidic protein ( 1:10000 ; sigma , tokyo , japan ) , anti - cx3cr1 ( 1:100 ; abcam , tokyo , japan ) , rabbit monoclonal anti - ccr2 ( 1:4000 ; abcam ) , and mouse monoclonal anti
we used a commercial kit to identify the y chromosome . briefly , 5-m sections from formalin - fixed paraffin - embedded blocks were deparaffinized , rehydrated , and predigested with 1 drop of pepsin solution .
the sections were incubated with hybridization solution containing zytodot cen yq12 probe ( zytovision , bremerhaven , germany ) overnight at 37c . the zytodot cish implementation kit ( zytovision )
to study the migration of donor - derived cells into the patient s brain , we performed immunohistochemistry with anti hla - a2 antibody , which could distinguish donor - derived cells from host cells .
interestingly , donor - derived hla - a2positive cells were found in the cortex and around vessels ( figs .
invading hla - a2positive cells in the cortical region had a ramified morphology similar to that of microglia ( fig .
by contrast , immunohistochemical staining of the c5 lesion with anti hla - a2 antibodies showed more hla - a2positive cells in the parenchyma ( figs .
hla - a2positive cells in that area were round or ring - shaped but did not have a ramified morphology ( figs .
( a ) hla - a2positive cells accumulate in the cerebral cortex ( brown cells within the circle ) .
( c ) a round hla - a2positive cell around a vessel ( arrow ) .
hla - a2positive cells with a round or ring morphology in the c5 lesion of the spinal cord at lower magnification ( d ) and at higher magnification ( e ) .
we next performed double staining to investigate the differentiation of graft cells into microglia or astrocytes .
hla - a2positive cells stained positive for iba1 , which is representative of microglia ( fig .
2a ) , but not for glial fibrillary acidic protein , which is specific for astrocytes ( fig .
2b ) . because these results suggested that graft cells differentiated into microglia , we also performed in situ hybridization with anti - yq12 probes . although most of the cells in the male positive control sample showed a brown dot in their nuclei , whereas cells in the female negative control sample had no brown dot in their nuclei , only a few cells in the transplant recipient sample had a brown dot in their nuclei , indicating y chromosome positive cells from the male donor ( figs . 2c e ) .
moreover , in situ hybridization with anti - yq12 probes after immunohistochemistry with anti - iba1 antibodies showed that y chromosome positive cells from the male donor were iba1-positive ( fig .
double staining of donor - derived cells by immunohistochemistry ( a , b ) and in situ hybridization ( c f ) .
( a ) double staining of donor - derived cells with anti hla - a2 antibody ( brown ) and anti - iba1 antibody ( blue ) .
( b ) double staining of donor - derived cells with anti hla - a2 antibody ( brown ) and anti
( c ) in situ hybridization of the patient sample using anti - yq12 probes ( brown dot ) with counterstaining of nuclei by mayer hematoxylin solution .
( f ) double staining of donor - derived cells using anti - yq12 probes ( brown dot ) and anti - iba antibody ( blue ) .
arrows show double - positive cells indicating donor origin ; arrowheads show iba1-positive and yq12-negative cells indicating host origin .
scale bar = 100 m . because ccr2 and cx3cr1 are very important for distinguishing resident microglia from monocyte - originating microglia - like cells , we next explored the expression of ccr2 and cx3cr1 in hla - a2positive cells .
approximately 60% of hla - a2positive cells were ccr2-negative , and most of the invading hla - a2positive cells were cx3cr1-positive ( figs .
( a ) double staining of donor - derived cells with anti hla - a2 antibody ( brown ) and anti - ccr2 antibody ( blue ) .
arrows indicate ccr2-negative and hla - a2positive cells ; arrowheads indicate double - positive cells .
( b ) migrated hla - a2positive cells were counted in 3 independent sections ; the frequency of ccr2-positive cells and ccr2-negative cells is shown in the bar graph .
( c ) double staining of donor - derived cells with anti hla - a2 antibody ( brown ) and anti - cx3cr1 antibody ( blue ) .
arrow indicates a double - positive cell indicating donor origin ; arrowhead indicates a cx3cr1-positive and hla - a2negative cell indicating host origin .
this case shows that umbilical cord blood stem cell derived precursors can differentiate into cx3cr1-positive , ccr2-negative , and iba1-positive ramified microglia even without brain irradiation , whereas previous studies did not find that donor - derived cells entering the cns parenchyma can transform into microglia ( 6 ) .
first , our patient had herpes zoster myelitis and relapse in the spinal cord , perhaps including meningitis of the whole brain .
moreover , a lesion of the cortex in the left midfrontal gyrus ( data not shown ) may indicate that there had been a trace of herpes zoster virus induced encephalitis .
indeed , donor - derived cells can enter the recipient brain and differentiate into microglia after severe streptococcal meningitis ( 11 ) .
bacterial meningitis often destroys the bbb and pial membrane , thereby allowing donor - derived cells to enter the cns parenchyma . even after meningitis
massengale et al ( 12 ) found that no donor - derived cells enter the cns parenchyma even with brain injury .
ajami et al ( 13 ) reported that infiltrating monocytes at an inflammatory site have a short life span and do not contribute to the resident microglia pool . although it is possible that diffuse leptomeningeal involvement by lymphoma may have occurred at the time of clinical relapse in the spinal cord , our case showed no trace of meningitis at autopsy .
our finding that donor - derived cells had not invaded the deep white matter of the recipient is consistent with the possibility that there was no brain irradiation , as has been shown in head - shielded mouse models ( 14 ) .
indeed , we rarely observed donor - derived microglia in the deep white matter , thalamus , and basal ganglia , although we found donor - derived perivascular cells ; however , inflammation of the left medulla ( as reported in the final diagnosis ) might have been caused by the direct effects of focal irradiation . because the alkylating agent busulfan can cause bone marrow derived cells to penetrate the brain parenchyma ( 9 ) , devic chemotherapy
may also disrupt the bbb . however , donor - derived ramified cells were rare in the deep region of the brain , suggesting that the bbb in our patient was sufficiently intact to prevent invasion of donor - derived cells .
although unger et al ( 6 ) did not provide information pertaining to irradiation and chemotherapy , all patients likely received aggressive chemotherapy as pretreatment before bone marrow transplantation for leukemia or lymphoma . nevertheless , no donor - derived cells were found to have differentiated into ramified microglia .
because donor - derived cells did not invade the deep white matter of the recipient in our case , we consider the possibility that chemotherapy had minimal effects .
moreover , even if a single intrathecal administration of methotrexate had disrupted the bbb , we consider it highly improbable that invading cells differentiated into cx3cr1-positive , ccr2-negative , and iba1-positive ramified microglia ( i.e. indicating the resting form ) within 2 weeks . in conclusion , we report an autopsy case in which cells derived from transplanted umbilical cord blood differentiated into ramified microglia in the host brain parenchyma .
although the bbb and glia limitans seem to have prevented the invasion of these donor - derived cells , most of the invading donor - derived ramified cells remained in the cortical zone .
this result suggests that the invasion of donor - derived cells occurs through the pial membrane . | abstractrecent studies have indicated that microglia originate from immature progenitors in the yolk sac . after birth ,
microglial populations are maintained under normal conditions via self - renewal without the need to recruit monocyte - derived microglial precursors .
peripheral cell invasion of the brain parenchyma can only occur with disruption of the blood - brain barrier . here
, we report an autopsy case of an umbilical cord blood transplant recipient in whom cells derived from the donor blood differentiated into ramified microglia in the recipient brain parenchyma .
although the blood - brain barrier and glia limitans seemed to prevent invasion of these donor - derived cells , most of the invading donor - derived ramified cells were maintained in the cerebral cortex .
this result suggests that invasion of donor - derived cells occurs through the pial membrane . |
most of the time teeth with periapical lesions heal satisfactorily after non - surgical endodontic intervention .
however , we do come across cases with persisting symptoms and infection that require periradicular surgery in order to remove the pathological tissues and to eliminate the source of irritation , and promote healing .
abramovitz et al . , have discussed the case selection for apical surgery and non - surgical re - treatment .
they have reported that treatment of 24.5% of the cases was impossible without surgical therapy .
the key to tissue regeneration is to simulate a cascade of healing events that , if coordinated , can result in the completion of integrated tissue formation .
this is possible only with the use of growth factors , extracellular matrix , and the use of bone morphogenetic proteins instead of routinely used synthetic bone grafts , because synthetic bone grafts induce regeneration by osteoconduction , whereas , these biological modulators induce regeneration by osteoinduction .
the need for these biological modulators resulted in the development of platelet - rich plasma [ prp ] by whitmen et al .
platelet - rich plasma is the component of blood with concentrated platelets in a limited volume of plasma .
this autologous plasma is a rich source of growth factors and its application is thought to be an effective way of inducing tissue repair and regeneration .
marx et al . , in 1998 , reported the first clinical dental results with prp and they suggested that adding prp to bone grafts accelerated the rate and degree of bone formation .
platelet membranes had been shown to stimulate the mitogenic activity of human trabecular bone cells .
the proliferation rate of human osteoblast - like cells was increased , up to a certain level , by adding thrombocytes .
recent studies have indicated the stimulator influence of prp in collagen biosynthesis and proliferation of osteoblastic cells .
these in vitro results have supported the current assumption that the clinical use of prp might enhance bone regeneration.[914 ] the aim of this in vivo study was to evaluate the bone regeneration in endodontically induced periapical lesions using hydroxyapatite , platelet - rich plasma , and a combination of both , for a period of one year .
twenty systemically healthy patients of both genders , between the ages 20 and 40 years , were included .
the procedure was explained to the patients in their own language and informed consent was obtained from them .
to qualify , the patient had to have a tooth where non - surgical root canal therapy had failed , periapical radiolucency was present , and periapical root - end surgery was required .
the bony defect had to be confined to the apical area with the bone covering the entire root surface coronally and had an intact lingual cortical plate .
the patients were randomly divided into four groups of five patients each : group i replacement with hydroxyapatite ( periobone g , top notch health care product , india .
batch no : pb01040006 ) , group ii replacement with prp , group iii replacement with prp and hydroxyapatite , and group iv control group with no substitutes .
ten milliliters of venous blood was drawn from the patient and transferred into two test tubes containing ethylene diamine tetra acetic acid ( edta ) as an anticoagulant .
the plasma was then aspirated with a micropipette and transferred to a sterile test tube without an anticoagulant .
the samples of plasma were subjected to a second centrifugation at 2000 rpm for 10 minutes , which allowed the precipitation of the platelets ( 0.8 to 1.2 ml ) to fall onto the bottom , while the surface platelet poor plasma was discarded using a micropipette .
the prp was transferred to a dappen dish and stored at room temperature until further use . just before placing the prp into the bony cavity
, a small amount of bovine thrombin ( dade behring test thrombin reagent , germany ) and a few drops of 10% calcium chloride [ obtained by mixing 1 g of calcium chloride in 10 ml of distilled water ] were added to the prp to form a gel in a few seconds [ figure 1 ] .
this preparation of the prp gel was conducted by another operator during the surgical procedure .
platelet - rich plasma gel the first step of the treatment plan was to complete the root canal therapy in cases where dry canals existed and in others obturation was done on the day of surgery .
the surgical procedure included reflection of a full thickness mucoperiosteal flap by sulcular incision and two relieving vertical incisions .
debridement of tissues at the defect site was followed by irrigation with sterile saline solution .
root planing was performed on the root surface under the apical portion of the root .
for group i patients , the hydroxyapatite crystals were mixed with the patients own blood and thoroughly compacted into the bony defect until it reached the level of the periphery of the bone margins .
for group ii patients , the prp gel was carefully placed into the cavity till the entire cavity was filled [ figure 2 ] . for group iii patients ,
then hydroxyapatite crystals were mixed with the remaining prp and were packed into the defect as a second layer .
bony cavity filled with platelet - rich plasma gel wound closure was obtained with a 3 - 0 black silk suture .
analgesics ( combiflam [ aristo ] { ibuprofen 400 mg + paracetamol 325 mg } twice a day ) , antibiotics ( amoxycillin 500 mg [ alembic pharma ] twice a day ) and 0.2% chlorhexidine mouthwash ( hexidine , icpa health products ) , was prescribed for five days post surgically .
the patients were reviewed at regular intervals of one week , and one , three , six , nine , and twelve months . during the review
, radiographs were taken using the paralleling cone technique ( rwt rontgenfilmhalter mit langtubus , kkd , d-7090 ellwangen ) .
all these cases were evaluated clinically and radiographically . clinically , the patients were assessed for edema , postoperative pain , signs of infection , untoward reaction , and wound dehiscence .
ten milliliters of venous blood was drawn from the patient and transferred into two test tubes containing ethylene diamine tetra acetic acid ( edta ) as an anticoagulant .
the plasma was then aspirated with a micropipette and transferred to a sterile test tube without an anticoagulant .
the samples of plasma were subjected to a second centrifugation at 2000 rpm for 10 minutes , which allowed the precipitation of the platelets ( 0.8 to 1.2 ml ) to fall onto the bottom , while the surface platelet poor plasma was discarded using a micropipette .
the prp was transferred to a dappen dish and stored at room temperature until further use . just before placing the prp into the bony cavity
, a small amount of bovine thrombin ( dade behring test thrombin reagent , germany ) and a few drops of 10% calcium chloride [ obtained by mixing 1 g of calcium chloride in 10 ml of distilled water ] were added to the prp to form a gel in a few seconds [ figure 1 ] .
this preparation of the prp gel was conducted by another operator during the surgical procedure .
platelet - rich plasma gel the first step of the treatment plan was to complete the root canal therapy in cases where dry canals existed and in others obturation was done on the day of surgery .
the surgical procedure included reflection of a full thickness mucoperiosteal flap by sulcular incision and two relieving vertical incisions .
debridement of tissues at the defect site was followed by irrigation with sterile saline solution .
root planing was performed on the root surface under the apical portion of the root .
for group i patients , the hydroxyapatite crystals were mixed with the patients own blood and thoroughly compacted into the bony defect until it reached the level of the periphery of the bone margins .
for group ii patients , the prp gel was carefully placed into the cavity till the entire cavity was filled [ figure 2 ] . for group iii patients ,
then hydroxyapatite crystals were mixed with the remaining prp and were packed into the defect as a second layer .
bony cavity filled with platelet - rich plasma gel wound closure was obtained with a 3 - 0 black silk suture .
analgesics ( combiflam [ aristo ] { ibuprofen 400 mg + paracetamol 325 mg } twice a day ) , antibiotics ( amoxycillin 500 mg [ alembic pharma ] twice a day ) and 0.2% chlorhexidine mouthwash ( hexidine , icpa health products ) , was prescribed for five days post surgically .
the patients were reviewed at regular intervals of one week , and one , three , six , nine , and twelve months . during the review
, radiographs were taken using the paralleling cone technique ( rwt rontgenfilmhalter mit langtubus , kkd , d-7090 ellwangen ) .
clinically , the patients were assessed for edema , postoperative pain , signs of infection , untoward reaction , and wound dehiscence .
clinically , all cases showed moderate edematous swelling 24 hours after the surgery , which gradually subsided over a period of five days .
there were no signs of infection , untoward reaction , wound dehiscence or extrusion of material in any of the patients .
radiographically , group i patients showed complete bone regeneration , with evidence of a trabecular pattern at the end of one year , group ii patients showed complete bone regeneration at the end of nine months , group iii patients showed complete bone regeneration at the end of six months and group iv patients showed bone regeneration , which was not satisfactory even after one year [ figures 310 ] .
group i follow - up radiographs , hydroxyapatite group i follow - up radiographs group ii follow - up radiographs , platelet - rich plasma group ii follow - up radiographs group iii follow - up radiographs , combination of platelet - rich plasma & ha group iii follow - up radiographs group iv follow - up radiographs , control group iv follow - up radiographs
today , while non - surgical endodontics is considered as the primary mode of treatment , the decision to intervene surgically is open to deliberations .
the success of an endodontic procedure depends upon complete healing of the lost periapical tissues .
however , some may require surgical intervention to remove the pathological tissue and simultaneously eliminate the source of irritation that could not be removed by the orthograde root canal treatment .
hence , to enhance these processes a number of bone substitutes are being tried out .
the objective of using a bone graft is to achieve successful and complete healing of the bone .
a variety of materials are available for bone regeneration , which are highly osteoconductive or osteoinductive like , freeze dried bone graft , bioactive glass , emdogain , ptr polymer , mta , tricalcium phosphate , and octacalcium phosphate .
blood and blood products have been used for over a century for various purposes , including the use of serum and individual components .
since the early twentieth century , various functions of platelets have been known , such as , hemostasis and platelet - plug formation .
research regarding the active use of growth factors present within the platelets started in the middle of twentieth century .
these studies began to explore the fact that platelets could be used to modulate regeneration , and to repair and heal the tissues . in 1997 , whitman et al .
, showed prp as an autologous source of platelet derived growth factor ( pdgf ) and transforming growth factor - b ( tgf ) that was obtained by sequestering and concentrating platelets by gradient density centrifugation .
platelet gel was prepared by blood collection preoperatively , and was prepared by another operator during surgery .
platelet rich plasma contain various growth factors namely pdgf , transforming growth factor , insulin - like growth factor , and epidermal growth factor .
these factors play a prime role in bone regeneration the various advantages of prp are :
improved support of tissue healingmore rapid mineralization of collagen in bone repairearlier stability of grafts because of the sticky consistency of fibrin componentcytokines and growth factors brought to the siteformation of a firm nonfriable clotclevatage of fibrinopeptides a and b from a molecule caused by thrombin , resulting in the formation of fibrin monomer , thrombin activated factor xiii that in turn allowed for a linkage in the presence of ionized calciumno potential for disease transmissionless thrombogenic than untreated graft materialminimal adhesion formation resolving in four to six monthsenhanced osteoconduction : osteoblasts were normally non - motile cells , in that , they would not move across a distance greater than 0.4 mm ( 400 microns ) , prp enhanced the fibrin network , and the movement of osteoblasts along the fibrin network was enhanced resulting in a woven bone formation , much earlier than normal .
this phase would last for three monthscellular activity : bone morphogenetic protein ( bmp ) acted as a
switch through which the reception as well as bone formation occurred and acted as a modulatorstem cells in the graft started the cascade of osteogenesis
improved support of tissue healing more rapid mineralization of collagen in bone repair earlier stability of grafts because of the sticky consistency of fibrin component cytokines and growth factors brought to the site formation of a firm nonfriable clot clevatage of fibrinopeptides a and b from a molecule caused by thrombin , resulting in the formation of fibrin monomer , thrombin activated factor xiii that in turn allowed for a linkage in the presence of ionized calcium no potential for disease transmission less thrombogenic than untreated graft material minimal adhesion formation resolving in four to six months enhanced osteoconduction : osteoblasts were normally non - motile cells , in that , they would not move across a distance greater than 0.4 mm ( 400 microns ) , prp enhanced the fibrin network , and the movement of osteoblasts along the fibrin network was enhanced resulting in a woven bone formation , much earlier than normal .
this phase would last for three months cellular activity : bone morphogenetic protein ( bmp ) acted as a
switch through which the reception as well as bone formation occurred and acted as a modulator stem cells in the graft started the cascade of osteogenesis hydroxyapatite is used , as it helps in osteoconduction .
osteoconduction is a process wherein the bone is formed by the in - growth of capillaries and osteoprogenitor cells from the recipient bed into , around , and through a graft .
these hydroxyapatite crystals when used act as a scaffold upon which new bone is deposited , which is then followed by a slow resorption of the graft .
prp is used , as it helps in better healing and faster regeneration , because of the biological modulators and also helps in osteoinduction .
prp acts as an osteoinductive material because osteoinduction is a process whereby new bone is produced in an area where there was no bone earlier , where one tissue or its derivative causes another undifferentiated tissue to differentiate into a new bone .
calcium chloride is added to nullify the effect of the anticoagulant edta , and bovine thrombin is added to gellate the plasma . as only small grains of thrombin
are added , it does not produce any antigenic response , as proved by neil winter bottom et al . , in the year 2002 .
a number of methods have been used for the procurement of prp , such as :
electromedic 500 gradient density cell separator ( medtronics ) by marx et al . , in 1998smart prep by bhanot and alex , in 2002elmd 500 transfusion system by whitman and green , in 2002.automated systems like placon 7 and hemonetics mcs
electromedic 500 gradient density cell separator ( medtronics ) by marx et al .
, in 1998 smart prep by bhanot and alex , in 2002 elmd 500 transfusion system by whitman and green , in 2002 .
automated systems like placon 7 and hemonetics mcs the method used in this study to procure prp and prp gel was based on the test - tube or manual isolation techniques ( vacutainer system ) by sonnenleit and sullivan et al .
radiographic evaluation in group i ( periobone g ) showed increased radio - opacity immediately , due to the compaction of the bone graft into the defect . in the first month
there was reduction in volume of the material due to initiation of resorption of the bone graft . in the third month
there was diffuse radio - opacity due to increased calcification of the graft . in the sixth and ninth months
, bone regeneration was seen , but not complete , due to partial resorption of the graft . in the first year
there was complete bone regeneration due to osteoconduction as explained earlier . in group ii ( prp ) the immediate ,
first week and first month postoperative radiographs showed a radiolucency similar to that of the preoperative radiograph , as prp was a radiolucent material . in the third and sixth months bone regeneration was evident , but not complete , due to the presence of biological modulators as discussed earlier . in the ninth month bone regeneration was complete . in group iii ( prp and periobone g )
there was an increase in radio - opacity immediately , due to the compaction of the bone graft into the defect .
in the first month there was reduction in volume of the material due to initiation of resorption of the bone graft . in the third month
there was evidence of bone regeneration , but not complete , due to partial resorption of the graft . in the sixth month bone regeneration was complete because of the combined effect of periobone g and prp . in group iv ( control )
there was incomplete bone regeneration , even at the one - year follow - up , due to nonavailability of bone substitutes to induce osteoconduction . in this
study the grafts did not produce any adverse reactions . wound healing and primary closure of the surgical site were good .
today 's understanding of the bone science recognizes the pivotal role of the growth factor in the clinical bone grafting success .
this study elucidates the mechanism of action and the points of influence that the fundamental growth factors of pdgf and tgf- exert on bone regeneration .
the amplification of pdgf and tgf- through the technique of platelet sequestration and concentration into prp is seen as an available and practical tool for enhancing the rate of bone formation and the final quality of the bone formed .
under the conditions of this in vivo study , it can be concluded that :
the use of bone substitutes helps in rapid and qualitative bone regeneration when compared to natural healinghydroxyapatite crystals facilitated bone regeneration , but not to the extent of the other two tested groups ( prp and combination)plasma - rich platelets with biological modulators showed better results , but a combination of prp and hydroxyapatite showed the best resultsin a clinical situation the use of bone substitutes will definitely enhance periapical regeneration
the use of bone substitutes helps in rapid and qualitative bone regeneration when compared to natural healing hydroxyapatite crystals facilitated bone regeneration , but not to the extent of the other two tested groups ( prp and combination ) plasma - rich platelets with biological modulators showed better results , but a combination of prp and hydroxyapatite showed the best results in a clinical situation the use of bone substitutes will definitely enhance periapical regeneration | aim and objectives : to evaluate bone regeneration in endodontically induced periapical lesions using hydroxyapatite , platelet - rich plasma ( prp ) , and a combination of hydroxyapatite and platelet - rich plasma for a period of one year.materials and methods : twenty systemically healthy patients of both genders between the ages 20 and 40 years were included .
to qualify , the patient had to have a tooth where non - surgical root canal therapy had failed , periapical radiolucency was present , and periapical root end surgery was required .
the bony defect had to be confined to the apical area , with the bone covering the entire root surface coronally , with an intact lingual cortical plate .
patients were randomly divided into four groups , with five patients each , as follows : group i replacement with hydroxyapatite , group ii replacement with prp , group iii replacement with prp and hydroxyapatite , and group iv control group with no substitutes .
the patients were evaluated both clinically and radiographically.results:the radiographic evaluation revealed that group i patients showed complete bone regeneration with evidence of a trabecular pattern , at the end of one year , group ii patients showed complete bone regeneration at the end of nine months , group iii patients showed complete bone regeneration at the end of six months , and group iv patients showed bone regeneration , which was not satisfactory even after one year.conclusions:the prp and hydroxyapatite combination facilitated better and faster bone regeneration when compared to prp alone . |
more than 4 million children in the united states have a chronic condition that results in some degree of limitation in their ability to participate in school or play or to engage independently in activities of daily living , such as eating or walking .
these children are more likely than their healthier peers to obtain their health insurance coverage through medicaid . in 1994
roughly 1.5 million children with limitation of activity were covered by medicaid . among poor children ,
70 percent of those with an activity limitation had medicaid coverage , compared with only 59 percent of those with no limitation . among non - poor children , medicaid covered 15 percent of those with a limitation , but only 6 percent of those without limitation ( newacheck , 1997 ) .
the medicaid program guarantees eligible children a comprehensive package of health insurance benefits , one that is far more extensive than those typically offered through commercial plans . under the program children have access to benefits that include routine preventive visits , medically necessary diagnostic and evaluative services , and medically necessary treatment services that range from office - based medical services to various mental health and developmental therapies in non - traditional settings , to home health and other long - term care services .
medicaid children 's entitlement to these services was established early in 1990 when the omnibus budget reconciliation act of 1989 's amendments to medicaid 's epsdt provision took effect . since
that time states have been statutorily required to pay not only for comprehensive pediatric screening services , including those additional to the screens prescribed under the state 's periodicity schedule , but also for all federally allowable health care , diagnostic , and treatment services determined as a result of a screen to be necessary to correct or ameliorate a defect or physical or mental condition .
these services must be reimbursed for children regardless of whether they are covered for other medicaid beneficiaries . although states retain some discretion in defining and authorizing epsdt expanded benefits , the statutory language of epsdt constitutes a fairly uniform standard of coverage for all medicaid - eligible children and one that is particularly important to those with disabling conditions . at the same time that states have been struggling to structure epsdt coverage policies , however ,
many have also been accelerating their use of capitated managed care , in effect shifting decisionmaking authority for children 's services from medicaid agencies to plans . the plans with which they are contracting often are commercial entities new to serving low - income children or furnishing a pediatric benefit package as comprehensive as medicaid 's .
unless otherwise directed these plans are likely to draw upon their commercial experiences , where definitive coverage limits usually apply and services are considered medically necessary only when they are needed to treat an accident or illness ( fox and mcmanus , 1996a ) .
enrollment into capitated managed care arrangements marks a major shift in the way that medicaid children with disabling conditions receive their care ; it creates new provider relationships and establishes new rules for accessing services . just as little has been known about the effectiveness of medicaid 's fee - for - service arrangements on children with disabilities , the evidence regarding managed care 's impact is also limited .
research suggests , however , that these children are likely to confront difficulties in obtaining necessary specialty services in capitated arrangements .
findings from a survey of pediatricians revealed that a significant proportion had referral requests for pediatric subspecialists denied , and that one - third of these pediatricians believed their patients ' care was compromised as a result ( cartland and yudkowsky , 1992 ) .
similarly , a recent study of medicaid demonstration waiver programs in four states found that prior authorization policies imposed at the plan level were often structured to prevent children from obtaining ancillary therapies , mental health services , durable medical equipment , and pediatric subspecialty services , particularly if those services were available only from out - of - network providers ( fox and mcmanus , 1996b ) .
specialty care access problems have also been identified in the managed care literature for medicaid children generally .
much of this literature , which refers only to children eligible for medicaid because of their aid to families with dependent children ( afdc ) status , is dated and does not reflect the utilization control methods employed by today 's fully capitated plans . nor
does it always take sufficient account of selection bias in enrollment or the generosity of benefits available under comparative fee - for - service arrangements .
nevertheless the studies generally report a reduction in specialty physician ( freund et al . , 1989 ; hurley , freund , and gage , 1991 ) and emergency room use ( bonham and barber , 1987 ; hurley , freund , and paul , 1993 ; hurley , freund , and taylor , 1989 ) , but show little or no change in the rate of inpatient hospital stays ( hurley , freund , and paul , 1993 ; bonham and barber , 1987 ; leibowitz , buchanon , and mann , 1992 ) .
with respect to primary care services , the findings suggest that utilization of preventive services by children may be the same or higher ( balaban , mccall , and bauer , 1994 ; hohlen et al . , 1990 ;
mauldon et al . , 1994 ) , while acute care services may be the same or lower ( hurley , freund , and gage , 1991 ; mauldon et al , 1994 ) . freund and lewit ( 1993 ) conclude that children in medicaid managed care arrangements appear to experience decreased access to specialty care without a commensurate increase in primary care services . in this article
we present new information on state medicaid managed care policies affecting children with disabilities for 1995 and 1996 .
we provide information on enrollment policies and on pediatric benefits , including epsdt and medical - necessity guidance for health maintenance organizations ( hmos ) and other fully capitated managed care arrangements .
although medicaid managed care policies have been discussed by other authors ( horvath and kaye , 1995 , 1997 ; johnson , 1997 ; rawlings - sekunda , 1997 ; saucier and mitchell , 1995 ) , they have not included multiyear analyses focused on enrollment and benefit issues of particular relevance to children with special health care needs .
this study is based on a combination of telephone interviews with state medicaid agency staff and reviews of state managed care contracts in 1995 and 1996 . in 1995 , 29 states using hmos and other fully capitated plans to serve children were studied ; in 1996 the number of states rose to 39 .
we focused this analysis on state policies regarding general managed care plans and excluded policies related to specialized plans that serve disabled populations exclusively .
our telephone interviews consisted of a series of detailed questions concerning enrollment and benefit policies under fully capitated plans .
we asked about enrollment policies for categorical and special child population groups and whether enrollment was voluntary or mandatory .
we also asked about whether 13 medicaid - allowable services relevant to children with disabilities were included in the capitated contract or separately reimbursed , whether plans were expected to provide all epsdt expanded benefits , and what guidance was furnished to plans regarding medical necessity .
the interviews were conducted with the director of managed care in each state medicaid office during the spring of 1995 and again , approximately 18 months later , during the late fall of 1996 .
the telephone surveys were supplemented by reviews of state contracts for fully capitated plans that were in effect at the time of our survey .
we examined those sections of the contract related to enrollment , service responsibilities , epsdt , medical necessity , and definitions of terms . in cases where the contracts referenced other relevant documents , such as a provider manual or the administrative code , we obtained copies of these documents and incorporated them in our analysis .
epsdt language was assessed according to whether it included core elements federally required under the epsdt benefit .
medical - necessity language was assessed according to the extent to which it would permit coverage for habilitative and other therapeutic interventions for children with disabling chronic conditions . to verify our enrollment and coverage policy findings
, summary confirmation was sent back to each state medicaid managed care office for review , along with state - specific questions we needed to have answered . through this process
we were able to clarify any discrepancies between what was reported to us and what appeared in the contract documents and to address any gaps in our information .
in some instances we learned that state managed care policies actually differed from those specified in the contract because of delays in implementation .
in other instances we determined that medicaid managed care staff were not fully aware of all of the plan features , particularly with respect to medicaid payments for public program services , and we made additional telephone calls to public program or other medicaid staff to gain clarification . it should be noted that in classifying a state 's enrollment policy as voluntary or mandatory , we chose the policy applicable to the largest composite geographic area of the state , that is , the largest number of managed care counties .
geographic scope was selected as the best measurement because of the difficulties in obtaining estimates of the number of medicaid child beneficiaries in specific counties or even the population size of specific counties .
in addition we considered a state to have a voluntary enrollment policy with respect to fully capitated plans if the state required enrollment in either a primary care case management system or a fully capitated plan .
at the end of 1996 , more than three - quarters of states were serving medicaid children through fully capitated plans , either on a statewide basis or in limited geographic areas .
state policies regarding the enrollment of different categorical groups of children , the extent of pediatric benefits included in capitation contracts , and the guidance given to plans regarding epsdt expanded benefits and medical - necessity standards are discussed in the next section .
states ' enrollment of children in fully capitated plans has focused primarily on those eligible for medicaid by virtue of their afdc or afdc - related status , as shown in table 1 . among the 39 states that were using fully capitated plans to serve children in 1996 , all states enrolled children for whom
afdc payments are made and children in afdc - related categories , including those who qualified for medicaid because of their poverty - level status and , where section 1115 demonstration waivers were in effect , those who qualified by meeting higher income criteria . in about one - half of the states ,
by contrast states were more reluctant to enroll medicaid children who are receiving ssi disability benefits or are in subsidized foster care , citing concerns that they did not have the requisite information to set capitation rates or monitor quality and that the plans themselves might not be structured to deliver the range and intensity of services that these children require . just under two - thirds of the states
enrolled foster care children , only slightly more than one - half enrolled ssi children , and only slightly more than one - third enrolled both populations .
states were also significantly less likely to require mandatory enrollment of these children . less than one - third of the states that enrolled the ssi or foster care populations in fully capitated plans mandated their enrollment .
not only did nearly two - thirds of states exclude either ssi or foster care children or both from participation in fully capitated managed care arrangements in 1996 , but more than one - third also had policies to exempt certain other groups of children with special needs , many of whom may be eligible for medicaid because of their afdc or afdc - related status .
such policies have been established for children served by the federal title v program for children with special health care needs ( two states ) , children with serious emotional disturbances ( three states ) , children served by the early intervention program ( one state ) , and children with developmental delay ( one state ) .
other states allowed for exemptions on an individual basis , with evidence of a disability or severe medical problem that could not be served adequately by participating plans . in fact , six states , most with mandatory enrollment , excluded one or more of these groups and also excluded ssi children .
a comparison to state policies in 1995 reveals rapid growth in medicaid managed care enrollment . in just 18 months ( from the time of our first round of interviews in the spring of 1995 to our second in the fall of 1996 ) , the number of states using fully capitated plans to serve children grew by just over one - third , from 29 states in 1995 to 39 states in 1996 . during the same period , the proportion enrolling the ssi population in fully capitated plans grew from 45 to 54 percent , while the proportion of states enrolling children in subsidized foster care grew from 41 to 64 percent . yet
the increase in mandatory enrollment of the ssi and foster care populations between 1995 and 1996 was even more striking . for ssi children
the portion of states mandating their enrollment almost doubled , up from only 10 percent in 1995 to 18 percent in 1996 .
for children in foster care , it tripled , growing from 7 percent in 1995 to 21 percent in 1996 .
although states showed no overall tendency to increase their use of child - enrollment exemptions based on public program eligibility or individual criteria , they did allow for more individual exemptions where plan enrollment of ssi children was mandatory .
children enrolled in fully capitated arrangements in 1996 received most medicaid services from their plan , as shown in table 2 .
state contracts with plans typically required the provision of all mandatory medicaid services , including inpatient and outpatient hospital services , physician services , and laboratory and x - ray services .
most state contracts also required plans to furnish the majority of the optional medicaid services covered by the state , including most of the treatment services required under epsdt .
however , all but 2 of the 39 states that used fully capitated plans to serve children elected to exclude at least some medicaid services for children from their capitated contract and reimburse these services separately .
many states excluded certain pediatric services so that the medicaid benefit package provided by the plan would resemble the type and amount of services offered to commercial enrollees , presuming that plans did not have the capacity or inclination to furnish these services .
in some instances services were excluded only above specified coverage limits or under certain situations .
services that were carved out from state contracts for these reasons included : dental services ( in 26 states ) , personal care services ( in 19 states ) , prescription drugs ( in 10 states ) , vision services ( in 9 states ) , ancillary therapies ( in 5 states ) , and durable medical equipment ( in 4 states ) . states sometimes used a separate capitated arrangement to pay for excluded services : four states capitated dental services under another contract , for example . in some instances , plans were given the option of furnishing certain otherwise excluded services if they had the capacity and desire to furnish them . equally often , however , states excluded services from their contracts to protect certain populations and to ensure continuity of their care , or to preserve medicaid financing for service providers that have historically relied on public funds and provided the state 's share of the medicaid service cost .
about three - quarters of the states ( 29 ) excluded some or all mental health services from their contract for these reasons .
of these , 17 excluded all mental health services , sometimes paying for these services under a separate capitated contract .
the other states limited plans ' responsibilities for mental health services in essentially one of two ways : either the states created a separate payment arrangement for services provided primarily or exclusively to children with serious emotional disturbances ( 10 states ) , or they paid on a fee - for - service basis for services beyond an established benefit limit ( 5 states ) .
, the carve - out of mental health services usually was intended to ensure that community mental health centers and other providers within the children 's mental health system would continue to receive medicaid reimbursement for services provided to medicaid - eligible children .
other types of public program providers were also frequently protected by contract exclusions of the services they provide .
two - thirds of states ( 26 ) carved out health - related special education services provided by schools to children with disabilities , just over 45 percent ( 18 ) carved out services furnished under the early intervention program to developmentally delayed children under age three , and nearly 40 percent carved out both services .
five states excluded special assessment , counseling , and court - ordered services for foster care children ( although , as previously mentioned , a larger number of states elected to exclude the children themselves from enrollment ) .
finally , three states omitted the medical and other specialty services provided by the title v program for children with special health care needs .
specialty services furnished by title v program providers reportedly were less likely to be excluded from managed care contracting , because , unlike the other public program services , title v services have historically been included in the state medicaid budget and would presumably be perceived by plans as medically necessary . considering the extent to which children with disabilities require speciality services , many of which are non - traditional , and the difficulties of establishing reasonable capitation rates for them
, it was perhaps surprising that the enrollment of ssi children in fully capitated plans did not appear to have any significant overall effect on states ' carve - out policies for services particularly important to this population .
on average states that enrolled ssi children were only slightly more likely to exclude public program services and other specialized services such as personal care , prescription drugs , durable medical equipment , private duty nursing , and ancillary therapies , from their contracts as were states that exempted these children from enrollment . when states that mandatorily enrolled ssi children in fully capitated plans were compared with states in which these children were exempt or allowed to enroll on a voluntary basis , carve - out policies for public program and other specialized services still were not dramatically different . if anything
, states with mandatory ssi enrollment established somewhat fewer service carve - out protections than the other states .
this was most notably the case for carve - out policies regarding mental health services primarily or exclusively for children with serious emotional disturbances and private duty nursing .
overall states did not make substantial changes between 1995 and 1996 in the number of services excluded from their contracts . in
both years states excluded an average of 4 of the 13 pediatric services we examined .
states did , however , make some significant changes in the types of services they excluded . for example , states moved away from carving out expanded epsdt benefits from their contracts ; by 1996 no state carved out this set of services , whereas in 1995 four states did . looking specifically at state contract policies regarding public program services for special - needs children in 1995 and 1996 , we found a slight increase in the average number of services that states carved out .
the proportion that excluded health - related special education services increased somewhat , and the proportion that excluded services furnished through the children 's mental health program decreased .
looking at other specialized services of particular importance to children with disabling conditions in 1995 and 1996 , the average number of service carve - outs remained about the same .
however , states became less likely to exclude durable medical equipment and somewhat more likely to exclude ancillary therapies .
state shifts in mental health service carve - out policies over the 18-month period of our study deserve special notice . although the proportion of states with mental health carve - outs remained at three - quarters , there were notable changes in state policy .
states showed a greater tendency to carve out all mental health services from their general managed care contract and were less inclined to exclude services for children with serious emotional disturbances or mental health services beyond certain limits .
except for specifically excluded services , children in fully capitated plans depend on their plan for all of the diagnostic and treatment services to which they are entitled under medicaid 's epsdt provision .
yet our review of 1996 contracts shows substantial variation in the extent of guidance states gave plans about epsdt coverage and how it differs from children 's private health insurance coverage and medicaid coverage for adults , as shown in table 3 . among the 38 states that included epsdt in their capitated contracts ( oregon has a waiver to eliminate the epsdt benefit ) , all communicated the preventive focus of epsdt and described the screening component of the benefit , and about 85 percent referenced federal regulations pertaining to epsdt or , at a minimum , indicated that the benefit is federally mandated .
however , just under 40 percent ( 15 ) of the states used contract language regarding the diagnosis and treatment components of the benefit that is substantially similar to federal law , stipulating ( 1 ) the requirement to provide services to correct or ameliorate a problem detected by a screen , ( 2 ) the requirement to provide services for both physical and mental health problems , and ( 3 ) the requirement to provide all federally allowable diagnostic , treatment , and other health care services for a problem identified by an epsdt screen . of the remaining 23 states
, 6 did not include in their contract any of the core elements of the epsdt expanded benefit requirement , and 2 did not even make reference to federal ( or state ) law pertaining to epsdt .
thirteen of the states failed to specify that epsdt allows for coverage to correct or ameliorate
nine did not specify that epsdt encompasses coverage of mental as well as physical health problems , although five of these were states that carved out all mental health services .
further , 18 states failed to describe children 's entitlement to all federally allowable medicaid services , although 5 had very extensive carve - out policies for children 's public program and other specialized services .
although epsdt guidance can affect access to care by children with chronic or disabling conditions , a state 's medical - necessity policy can have even more significance , because it establishes the circumstances under which a service is presumed to be covered . in 1996 about 80 percent ( 31 ) of the 39 states enrolling
children in fully capitated plans included a medical - necessity definition in their contract , as shown in table 4 .
of these , 20 states required plans to follow a medical - necessity standard that included preventive , diagnostic , and treatment services for a condition or disability as well as an illness or injury .
( the medical - necessity standard in three of these states was child - specific . ) in three instances , however , the preventive focus was limited to cases in which the problem is significant , severe , or life - threatening .
also in four instances , qualifying criteria were included that could restrict coverage by directing plans either to use the least costly alternative treatment ( three states ) or to provide only services with evidence of effectiveness or proven medical value ( three states ) . among the remaining 11 states with a medical - necessity definition that did not incorporate the prevention and treatment of conditions and disabilities ,
only 1 state offered a definition equivalent to a commercial standard , defining as medically necessary only those services that are required to diagnose or treat an illness or injury .
three states , however , included qualifying criteria that restricted medically necessary services to those that were the least costly ( three states ) , of proven effectiveness ( two states ) , or required only for conditions that were severe or significant ( one state ) .
notwithstanding the many problems we identified in state guidance concerning epsdt and medical necessity , a comparison to contracts in effect in the spring of 1995 shows that states have begun to make noticeable improvements in this area in just 18 months . by the fall of 1996 , all states included in their contract a description of the epsdt diagnostic and treatment benefit , whereas one state in 1995 had failed to explain epsdt service requirements at all .
moreover by 1996 the number of states that provided contract language on epsdt that is substantially similar to federal law increased from 32 to 39 percent in particular , states became more likely to include language on mental health treatment in their descriptions of epsdt .
similarly states in 1996 were more likely to include a definition of medical necessity in their contracts in 1996 . in 1995 almost 40 percent of state contracts were silent on the issue of medical necessity , but by 1996 the proportion dropped by one - half . also in 1996 two additional states moved to establish a child - specific definition of medical necessity tailored specifically to the health , mental health , and developmental needs of children . beyond this , however , trends in medical - necessity guidance were mixed .
states were less likely to restrict treatment for a handicap or disability to situations where the condition is severe or significant .
on the other hand , they were more likely to require that interventions show evidence of medical effectiveness or proven medical value in order to be considered medically necessary and also somewhat less likely to reference standards of good medical practice .
states ' enrollment of children in fully capitated plans has focused primarily on those eligible for medicaid by virtue of their afdc or afdc - related status , as shown in table 1 . among the 39 states that were using fully capitated plans to serve children in 1996 ,
all states enrolled children for whom afdc payments are made and children in afdc - related categories , including those who qualified for medicaid because of their poverty - level status and , where section 1115 demonstration waivers were in effect , those who qualified by meeting higher income criteria . in about one - half of the states , enrollment for these children was mandatory .
by contrast states were more reluctant to enroll medicaid children who are receiving ssi disability benefits or are in subsidized foster care , citing concerns that they did not have the requisite information to set capitation rates or monitor quality and that the plans themselves might not be structured to deliver the range and intensity of services that these children require . just under two - thirds of the states
enrolled foster care children , only slightly more than one - half enrolled ssi children , and only slightly more than one - third enrolled both populations .
less than one - third of the states that enrolled the ssi or foster care populations in fully capitated plans mandated their enrollment .
not only did nearly two - thirds of states exclude either ssi or foster care children or both from participation in fully capitated managed care arrangements in 1996 , but more than one - third also had policies to exempt certain other groups of children with special needs , many of whom may be eligible for medicaid because of their afdc or afdc - related status .
such policies have been established for children served by the federal title v program for children with special health care needs ( two states ) , children with serious emotional disturbances ( three states ) , children served by the early intervention program ( one state ) , and children with developmental delay ( one state ) .
other states allowed for exemptions on an individual basis , with evidence of a disability or severe medical problem that could not be served adequately by participating plans . in fact , six states , most with mandatory enrollment , excluded one or more of these groups and also excluded ssi children .
a comparison to state policies in 1995 reveals rapid growth in medicaid managed care enrollment . in just 18 months ( from the time of our first round of interviews in the spring of 1995 to our second in the fall of 1996 ) , the number of states using fully capitated plans to serve children grew by just over one - third , from 29 states in 1995 to 39 states in 1996 . during the same period , the proportion enrolling the ssi population in fully capitated plans grew from 45 to 54 percent , while the proportion of states enrolling children in subsidized foster care grew from 41 to 64 percent .
yet the increase in mandatory enrollment of the ssi and foster care populations between 1995 and 1996 was even more striking . for ssi children
the portion of states mandating their enrollment almost doubled , up from only 10 percent in 1995 to 18 percent in 1996 .
for children in foster care , it tripled , growing from 7 percent in 1995 to 21 percent in 1996 .
although states showed no overall tendency to increase their use of child - enrollment exemptions based on public program eligibility or individual criteria , they did allow for more individual exemptions where plan enrollment of ssi children was mandatory .
children enrolled in fully capitated arrangements in 1996 received most medicaid services from their plan , as shown in table 2 .
state contracts with plans typically required the provision of all mandatory medicaid services , including inpatient and outpatient hospital services , physician services , and laboratory and x - ray services .
most state contracts also required plans to furnish the majority of the optional medicaid services covered by the state , including most of the treatment services required under epsdt .
however , all but 2 of the 39 states that used fully capitated plans to serve children elected to exclude at least some medicaid services for children from their capitated contract and reimburse these services separately .
many states excluded certain pediatric services so that the medicaid benefit package provided by the plan would resemble the type and amount of services offered to commercial enrollees , presuming that plans did not have the capacity or inclination to furnish these services . in some instances services
services that were carved out from state contracts for these reasons included : dental services ( in 26 states ) , personal care services ( in 19 states ) , prescription drugs ( in 10 states ) , vision services ( in 9 states ) , ancillary therapies ( in 5 states ) , and durable medical equipment ( in 4 states ) .
states sometimes used a separate capitated arrangement to pay for excluded services : four states capitated dental services under another contract , for example . in some instances , plans were given the option of furnishing certain otherwise excluded services if they had the capacity and desire to furnish them . equally often , however , states excluded services from their contracts to protect certain populations and to ensure continuity of their care , or to preserve medicaid financing for service providers that have historically relied on public funds and provided the state 's share of the medicaid service cost .
about three - quarters of the states ( 29 ) excluded some or all mental health services from their contract for these reasons . of these ,
17 excluded all mental health services , sometimes paying for these services under a separate capitated contract .
the other states limited plans ' responsibilities for mental health services in essentially one of two ways : either the states created a separate payment arrangement for services provided primarily or exclusively to children with serious emotional disturbances ( 10 states ) , or they paid on a fee - for - service basis for services beyond an established benefit limit ( 5 states ) .
regardless of the specific policy , the carve - out of mental health services usually was intended to ensure that community mental health centers and other providers within the children 's mental health system would continue to receive medicaid reimbursement for services provided to medicaid - eligible children .
other types of public program providers were also frequently protected by contract exclusions of the services they provide .
two - thirds of states ( 26 ) carved out health - related special education services provided by schools to children with disabilities , just over 45 percent ( 18 ) carved out services furnished under the early intervention program to developmentally delayed children under age three , and nearly 40 percent carved out both services .
five states excluded special assessment , counseling , and court - ordered services for foster care children ( although , as previously mentioned , a larger number of states elected to exclude the children themselves from enrollment ) .
finally , three states omitted the medical and other specialty services provided by the title v program for children with special health care needs .
specialty services furnished by title v program providers reportedly were less likely to be excluded from managed care contracting , because , unlike the other public program services , title v services have historically been included in the state medicaid budget and would presumably be perceived by plans as medically necessary . considering the extent to which children with disabilities require speciality services , many of which are non - traditional , and the difficulties of establishing reasonable capitation rates for them
, it was perhaps surprising that the enrollment of ssi children in fully capitated plans did not appear to have any significant overall effect on states ' carve - out policies for services particularly important to this population . on average states that enrolled ssi children were only slightly more likely to exclude public program services and other specialized services such as personal care , prescription drugs , durable medical equipment , private duty nursing , and ancillary therapies , from their contracts as were states that exempted these children from enrollment .
when states that mandatorily enrolled ssi children in fully capitated plans were compared with states in which these children were exempt or allowed to enroll on a voluntary basis , carve - out policies for public program and other specialized services still were not dramatically different .
if anything , states with mandatory ssi enrollment established somewhat fewer service carve - out protections than the other states .
this was most notably the case for carve - out policies regarding mental health services primarily or exclusively for children with serious emotional disturbances and private duty nursing .
overall states did not make substantial changes between 1995 and 1996 in the number of services excluded from their contracts . in both years
states did , however , make some significant changes in the types of services they excluded . for example , states moved away from carving out expanded epsdt benefits from their contracts ; by 1996 no state carved out this set of services , whereas in 1995 four states did . looking specifically at state contract policies regarding public program services for special - needs children in 1995 and 1996
, we found a slight increase in the average number of services that states carved out .
the proportion that excluded health - related special education services increased somewhat , and the proportion that excluded services furnished through the children 's mental health program decreased .
looking at other specialized services of particular importance to children with disabling conditions in 1995 and 1996 , the average number of service carve - outs remained about the same .
however , states became less likely to exclude durable medical equipment and somewhat more likely to exclude ancillary therapies .
state shifts in mental health service carve - out policies over the 18-month period of our study deserve special notice . although the proportion of states with mental health carve - outs remained at three - quarters , there were notable changes in state policy .
states showed a greater tendency to carve out all mental health services from their general managed care contract and were less inclined to exclude services for children with serious emotional disturbances or mental health services beyond certain limits .
except for specifically excluded services , children in fully capitated plans depend on their plan for all of the diagnostic and treatment services to which they are entitled under medicaid 's epsdt provision .
yet our review of 1996 contracts shows substantial variation in the extent of guidance states gave plans about epsdt coverage and how it differs from children 's private health insurance coverage and medicaid coverage for adults , as shown in table 3 . among the 38 states that included epsdt in their capitated contracts ( oregon has a waiver to eliminate the epsdt benefit ) , all communicated the preventive focus of epsdt and described the screening component of the benefit , and about 85 percent referenced federal regulations pertaining to epsdt or , at a minimum , indicated that the benefit is federally mandated .
however , just under 40 percent ( 15 ) of the states used contract language regarding the diagnosis and treatment components of the benefit that is substantially similar to federal law , stipulating ( 1 ) the requirement to provide services to correct or ameliorate a problem detected by a screen , ( 2 ) the requirement to provide services for both physical and mental health problems , and ( 3 ) the requirement to provide all federally allowable diagnostic , treatment , and other health care services for a problem identified by an epsdt screen . of the remaining 23 states , 6 did not include in their contract any of the core elements of the epsdt expanded benefit requirement , and 2 did not even make reference to federal ( or state ) law pertaining to epsdt .
thirteen of the states failed to specify that epsdt allows for coverage to correct or ameliorate
nine did not specify that epsdt encompasses coverage of mental as well as physical health problems , although five of these were states that carved out all mental health services .
further , 18 states failed to describe children 's entitlement to all federally allowable medicaid services , although 5 had very extensive carve - out policies for children 's public program and other specialized services .
although epsdt guidance can affect access to care by children with chronic or disabling conditions , a state 's medical - necessity policy can have even more significance , because it establishes the circumstances under which a service is presumed to be covered . in 1996 about 80 percent ( 31 ) of the 39 states enrolling children in fully capitated plans
included a medical - necessity definition in their contract , as shown in table 4 .
of these , 20 states required plans to follow a medical - necessity standard that included preventive , diagnostic , and treatment services for a condition or disability as well as an illness or injury .
( the medical - necessity standard in three of these states was child - specific . ) in three instances , however , the preventive focus was limited to cases in which the problem is significant , severe , or life - threatening .
also in four instances , qualifying criteria were included that could restrict coverage by directing plans either to use the least costly alternative treatment ( three states ) or to provide only services with evidence of effectiveness or proven medical value ( three states ) . among the remaining 11 states with a medical - necessity definition that did not incorporate the prevention and treatment of conditions and disabilities , only 1 state offered a definition equivalent to a commercial standard , defining as medically necessary only those services that are required to diagnose or treat an illness or injury .
three states , however , included qualifying criteria that restricted medically necessary services to those that were the least costly ( three states ) , of proven effectiveness ( two states ) , or required only for conditions that were severe or significant ( one state ) .
notwithstanding the many problems we identified in state guidance concerning epsdt and medical necessity , a comparison to contracts in effect in the spring of 1995 shows that states have begun to make noticeable improvements in this area in just 18 months . by the fall of 1996 , all states included in their contract a description of the epsdt diagnostic and treatment benefit , whereas one state in 1995 had failed to explain epsdt service requirements at all . moreover by 1996 the number of states that provided contract language on epsdt that is substantially similar to federal law increased from 32 to 39 percent in particular , states became more likely to include language on mental health treatment in their descriptions of epsdt . similarly states in 1996 were more likely to include a definition of medical necessity in their contracts in 1996 . in 1995 almost 40 percent of state contracts were silent on the issue of medical necessity , but by 1996 the proportion dropped by one - half .
also in 1996 two additional states moved to establish a child - specific definition of medical necessity tailored specifically to the health , mental health , and developmental needs of children . beyond this , however , trends in medical - necessity guidance were mixed .
states were less likely to restrict treatment for a handicap or disability to situations where the condition is severe or significant .
on the other hand , they were more likely to require that interventions show evidence of medical effectiveness or proven medical value in order to be considered medically necessary and also somewhat less likely to reference standards of good medical practice .
states are proceeding cautiously with the enrollment of ssi and foster care children into fully capitated managed care plans , in contrast to the enrollment of children in afdc and afdc - related categories .
still , by the fall of 1996 more than one - third of the states using fully capitated plans did enroll ssi and foster care children , and nearly one - third of these states enrolled these children on a mandatory basis .
indeed , notwithstanding certain exemptions based on program eligibility or individual criteria , most children with disabling chronic conditions were included in states ' fully capitated arrangements . between the spring of 1995 and the fall of 1996 , more categories of children
were enrolled into fully capitated plans and more often on a mandatory basis . where medicaid children are enrolled in fully capitated plans , they receive most of their preventive , primary , and chronic care services from the plan . in 1996 responsibility for many of the services of particular importance to children with disabilities epsdt expanded benefits , ancillary therapies , durable medical equipment , foster care services , private duty nursing , prescription drugs , and title v services for children with special health needs was left to the plans in about three - quarters of states . in fact personal care , early intervention , and health - related special education were the only specialized services for this population that were omitted from contracts and reimbursed separately in about one - half the states .
overall , between 1995 and 1996 , states became more likely to exclude certain public program services and less likely to exclude other specialized services . on average states carved out about four medicaid benefits for children in 1995 and 1996 , irrespective of ssi and foster care enrollment policies .
given the extent of pediatric services included in states ' fully capitated contracts , the specification of the epsdt benefit and the content of medical - necessity guidance in state contracts is of critical concern for children with disabilities .
our study found that all states provide managed care plans with at least some information about epsdt and are particularly expansive regarding the preventive component of the benefit .
yet about 60 percent of states failed to specify epsdt diagnostic and treatment services required in a manner that is consistent with federal medicaid law .
our study also found that medical necessity was defined in about 80 percent of states ' contracts and usually in a somewhat more liberal manner than in the commercial sector .
however , 35 percent of these states used a standard that did not require preventive , diagnostic , and treatment services for conditions and disabilities as well as accidents or injuries .
significant improvements occurred in 1996 with respect to epsdt language , but there was more variability in the direction states were taking with respect to medical - necessity guidance .
our results indicate that although states are proceeding cautiously with managed care enrollment of foster care children and ssi children , they are rapidly enrolling afdc and afdc - related children with little attention to the special service requirements of those with disabilities .
one reason for this appears to be the common presumption that afdc and afdc - related children are generally healthy .
an urban institute study found that between 11 and 16 percent of afdc children have a disability ( loprest and acs , 1996 ) .
another reason may be the difficulties that states face in identifying children with chronic or disabling conditions within the broader afdc population .
unfortunately there is no widely accepted approach for defining or identifying children with disabilities in managed care organizations , leaving states without practical guidance or strategies about whether to use public program eligibility , diagnostic criteria , functional criteria , or some combination ( division of services for children with special health care needs , 1996 ) .
relying solely on ssi and foster care eligibility status as indicators for childhood disability is not only problematic for enrollment purposes but also for determining pediatric capitation rates that avoid adverse risk selection . a large number of ssi children , for example ,
yet , states have established ssi capitation rates that are in many instances at least twice the afdc rate , with stop - loss protections typically set only for extreme outliers .
recent research based on colorado 's medicaid claims data shows , however , that afdc and ssi children had virtually the same distribution of expenditures ( kronick et al . , 1996 ) . considering the prevalence of disability and the diversity of service needs among all medicaid - eligible groups , states probably need to consider a broader strategy for identifying children with disabilities , obtaining reliable actuarial data , and setting appropriate risk - adjusted capitation rates .
state medicaid agencies have been struggling with how to structure managed care contracts with respect to service and population carve - outs .
most states favor carving out specific services and not children , given that their long - term strategy is to enroll as many eligible children as possible into fully capitated managed care arrangements .
service carve - outs , though relatively few in number , are common and have been designed in large part to protect public program services .
states appear to be considering several factors including the capacity of plans , continuity of care , pressure from public programs and their constituents , as well as existing agreements regarding medicaid revenues ( fox et al . , 1996 ) .
by maintaining certain service carve - outs , states are purchasing from general managed care plans the benefit package that they are best able to deliver , while at the same time ensuring that children have access to services that are not likely to be available or perceived as medically necessary in the commercial sector .
mental health , health - related special education , and early intervention services are the most common examples of this . in each case
states recognize the difficulties in transferring the broad service responsibilities performed by these programs and in ensuring that their public program providers continue to obtain medicaid reimbursement for their services .
still , by retaining these carve - outs particularly for mental health services the ability of plans and pediatric providers to integrate physical health , mental health , and developmental services for children may be comprised .
considering the nature and extent of the health care problems presented by medicaid - eligible children and the array of medicaid benefits for which managed care plans are now responsible , contractual requirements concerning medicaid services , particularly in the area of developmental , habilitative , and mental health interventions , have become critically important .
our survey findings reveal that states have made some improvements in defining medicaid 's epsdt provision
. this may be the result of increased awareness on the part of states resulting from health care financing administration ( hcfa ) guidance on this subject ( office of managed care and medicaid bureau , 1996 ) . among those states that have not yet defined the epsdt expanded benefit requirement
, however , there is a significant potential for plans that have served privately insured children to reduce diagnostic and treatment services critical to children with disabilities .
although more states are including medical - necessity definitions in their managed care contracts , some have begun to impose requirements for services to meet medical effectiveness criteria . moreover , where states have been silent on the medical - necessity issue
there are eight such states plans themselves are free to require that services be of proven effectiveness in order to be considered medically necessary . for children
this requirement to prove effectiveness is particularly problematic , because little has been published about the effectiveness of many of the interventions required by children , particularly those with disabilities .
if states omit from their contract a particular benefit category , such as personal care services , the state 's responsibility is clear .
however , if states transfer to plans responsibility for a particular benefit , then families , providers , plans , and even the state presume that decisionmaking responsibility for that benefit rests with the plan . a state 's residual responsibility for types or amounts of services denied may not become clear in the absence of legal procedures .
it appears inevitable that many medicaid children with disabilities will continue to be enrolled in some form of capitated managed care in the next 5 to 10 years .
how effectively this move takes place will depend on many factors , including how well states are able to identify and count medicaid children with chronic or disabling conditions , the specification of the epsdt benefit and a medical - necessity standard for children , and the partnerships that can be formed between plans and the various public program providers that have historically served this population .
this is a critical period for medicaid children with disabilities , and a great deal is at stake in the transition to managed care .
unfortunately no comprehensive evaluation studies have been conducted on the effects of current managed care arrangements for this population .
in addition few examples of special pediatric managed care arrangements exist . a much greater level of investment is urgently needed by both the public and private sectors in program design and implementation , quality performance measurement , federal and state oversight , and evaluation . | the authors present findings from a study of state medicaid managed care enrollment and benefit policies in 1995 and 1996 for children with disabilities . during this time
the number of states serving children through fully capitated plans grew by more than one - third , and enrollment of children receiving supplemental security income ( ssi ) payments and children in subsidized foster care increased .
most states required plans to provide all mandatory and most optional medicaid services . although states have begun to make noticeable improvements in their contract language concerning medical necessity and the early and periodic screening , diagnosis , and treatment ( epsdt ) benefit , overall state guidance in these areas remains weak . |
elevated liver function tests ( lfts ) in obese patients either with or without diabetes are a common clinical problem .
the most frequent cause is non - alcoholic fatty liver disease ( nafld ) , which represents both a diagnostic and therapeutic challenge .
the diagnostic criteria also include absence of significant alcohol intake ( women : > 20 g per day ; men : > 30 g per day ) and use of steatogenic drugs ( e.g. amiodarone and glucocorticoids ) .
nafld is associated with an increase in mortality due to cardiovascular disease , irrespective of diabetes .
the prevalence is closely associated with obesity and type 2 diabetes . up to 70% of patients with type 2 diabetes
the underlying pathophysiology of the development from simple steatosis to non - alcoholic steatohepatitis ( nash ) is not fully understood .
excess hepatic fat infiltration seems to cause lipid - induced mitochondrial dysfunction and oxidative stress ( lipotoxicity ) , which result in inflammation and fibrosis .
studies have shown that nash is associated with cirrhosis and hepatocellular carcinoma and is predicted to be the leading cause of liver transplantation by 2020 in the us .
weight loss is an effective treatment of nash , but is difficult to maintain for a majority of patients .
current medical interventions are limited and seem to be associated with risk of side - effects .
we present a case of severe liver disease in a young woman with type 2 diabetes .
she was treated with the glucagon - like peptide-1 receptor ( glp-1r ) agonist liraglutide with remarkable results on glycemic control , lfts and liver histology .
a 25-year - old female nurse was referred to our diabetes outpatient clinic with poorly controlled type 2 diabetes , obesity and elevated lfts .
her general practitioner had initiated treatment with metformin ( 1,000 mg twice - daily ) and simvastatin ( 40 mg once - daily ) , but compliance was limited . at her first visit to our outpatient clinic her body weight was 90 kg and her body mass index 32.6 kg / m .
she was asymptomatic and clinical examination , fundus photography , filament testing and albumin / creatinine ratio revealed no signs of complications related to type 2 diabetes .
blood samples showed a glycated hemoglobin a1c ( hba1c ) of 8.9% ( 74 mmol / mol ) and a fasting plasma glucose of 7.3 mmol / l .
lfts showed an alanine aminotransferase ( alt ) of 196 u / l ( reference range 1045 u / l ) , an aspartate aminotransferase ( ast ) of 132 u / l ( reference range 1535 u / l ) and an alkaline phosphatase ( alp ) of 127 u / l ( reference range 35105 u / l ) .
lipids were elevated : total cholesterol 4.5 mmol / l , high - density lipoprotein 0.84 mmol / l , low - density lipoprotein 2.4 mmol / l and triglycerides 2.86 mmol / l .
the patient had no history of alcohol abuse nor did she take any herbal drug .
abdominal ultrasound revealed increased echogenicity and poor visualization of the intrahepatic vessel walls , suggesting diffuse hepatic steatosis .
statins were discontinued and the patient was strongly encouraged to be compliant with her metformin treatment ( 1,000 mg twice - daily ) .
after 8 weeks on metformin and insulin , hba1c had markedly improved to 6.3% , but lfts except for alp remained elevated ( alt 133 u / l , ast 76 u / l , alp 69
the patient was then scheduled for a liver biopsy , which showed hepatic fat infiltration involving more than 66% of hepatocytes , ballooned hepatocytes , lobular inflammation as well as pericellular and periportal fibrosis .
the histological diagnosis was nash with a nafld activity score of 5 ( score range 08 ) and a fibrosis score of 2 ( score range 04 ) ( fig .
the dose was increased with weekly increments of 0.61.8 mg once daily during the following weeks .
after 46 weeks of treatment with liraglutide , total weight loss was 16 kg , lfts were in the lower normal range ( alt 29 u / l , ast 25 u / l , alp 67 u / l ) , glycemic control was excellent ( hba1c 5.6% ) and the lipid profile was normalized without statin treatment .
a repeated liver biopsy confirmed decreased hepatic fat infiltration ( involving 4050% of hepatocytes ) , no ballooned hepatocytes and only distinct lobular inflammation ( fig .
the histology was no longer consistent with nash , but pericellular and periportal fibrosis were still present ( nafld activity score 3 , fibrosis score 2 ) . to investigate the expression of hepatic glp-1rs in the present case ,
the baseline and the post - treatment liver biopsy were compared to liver tissue from another patient with type 2 diabetes but without steatosis , using quantitative polymerase chain reaction .
the prevalence of obesity and type 2 diabetes is increasing , and obese patients with type 2 diabetes and elevated lfts are regularly referred to outpatient clinics .
this case illustrates the necessity for both diabetologists and hepatologist to be aware , and together take part in the diagnosis and treatment of nafld .
abdominal ultrasound is recommended as a first - line evaluation if lfts are elevated , but has high inter - observer variability and hepatic fat infiltration fat must exceed 2030% to be detected . magnetic resonance spectroscopy is expensive and not widely available , but can identify > 5.5% hepatic fat infiltration .
transient elastography enhanced with controlled attenuation parameter can quantify liver steatosis , but has not been validated in large trials .
the nafld fibrosis score can help identify patients with a high risk of nash and fibrosis and , thus , eligible for liver biopsy .
the score is based on body mass index , age , presence of diabetes and blood levels of alt , ast , platelets and albumin .
if coexisting liver diseases to nash are suspected , liver biopsy should always be considered .
the natural history of simple steatosis is benign from a liver standpoint and should be managed by treating comorbidities such as obesity , hyperlipidemia and insulin resistance . on the other hand ,
nash per se increases the risk of liver - related death and management should include liver specific treatment(s ) . in patients with nash
the primary goal is to reverse oxidative stress and reduce hepatic fat infiltration , thereby reducing insulin resistance and further reverse hepatic inflammation .
weight loss and glycemic control are effective measures in nash , but can be difficult to achieve and maintain for a majority of patients .
nevertheless , if weight loss can be obtained it improves insulin sensitivity and reduces lfts .
a weight loss of 35% improves steatosis and a 10% decrease in body weight reduces hepatic inflammation . in line with these considerations , bariatric surgery should be considered in patients who are overweight and have type 2 diabetes as well as nash .
metformin increases hepatic and muscular insulin sensitivity , but does not improve lfts or liver histology in nash .
pioglitazones reduce hepatic steatosis but are rarely used due to adverse events including heart failure , bladder cancer and loss of bone density .
one trial suggests that vitamin e 800 iu per day improves lfts , steatosis , ballooning and inflammation , and vitamin e is recommended for nash in non - diabetic subjects .
however , emerging evidence suggests that vitamin e may increase mortality . the use of glp-1r agonists for patients with type 2 diabetes is increasing . these drugs are based on the incretin hormone glp-1 , which is released from enteroendocrine cells in response to food ingestion .
native glp-1 acts via the glp-1r ( a g protein - coupled receptor expressed in several tissues ) and plays an essential role in the maintenance of normal glucose homeostasis and regulation of appetite and food intake .
the effects of the native hormone have been exploited through the development of stable glp-1r agonists .
these drugs have sustained effects on glucose levels , increase insulin secretion and reduce glucagon secretion , satiety , food intake and body weight .
furthermore , a recent post hoc analysis concluded that liraglutide was well tolerated and safe to use in patients with type 2 diabetes and elevated lfts . in rodents ,
glp-1r agonists reduce hepatic steatosis by suppressing enzymes involved in hepatic lipogenesis through activation of the 5 adenosine monophosphate - activated protein kinase , and have been suggested to reduce hepatic expression of pro - inflammatory mediators
. nevertheless , initial observations of the glp-1r on human hepatocytes have not been confirmed in subsequent studies [ 12 , 13 ] . as previously described , no liver biopsies from this case had expression of glp-1r .
the patient in this report showed remarkable results after 46 weeks of treatment with a glp-1r agonist .
she lost 16 kg of body weight ( from a baseline of 90 kg ) and achieved normalized lfts and lipid profile .
her hepatic fat infiltration was reduced by 30% , a decreased number of inflammatory cells were observed and , thus , she no longer fulfilled the criteria for nash . since no glp-1rs were found in the liver tissue ,
these improvements may be explained by indirect results of glucose metabolic improvement and weight loss induced by treatment with the glp-1r agonist . in spite of the fact that we were unable to identify glp-1rs in liver tissue in the present case
, some evidence suggests that glp-1r agonists may have direct effects on hepatic steatosis ( in vitro models ) [ 10 , 11 ] .
furthermore , one other paper has investigated the effect of glp-1r agonist on liver histology in nafld .
kenny et al . presented a case series with eight biopsy - proven nafld patients who received exenatide 10 mg twice daily for 26 weeks .
repeat liver biopsies showed improved nafld activity scores of 12 in four patients , but no changed in fibrosis score .
cuthbertson et al . treated 19 patients with exenatide 10 mg twice - daily and 6 patients with liraglutide 1.2 mg once daily for 6 month .
patients had a 42% relative reduction in intrahepatic lipid content assessed by magnetic resonance spectroscopy , independent of body weight loss but in correlation with a decrease in hba1c .
however , the study was open - labeled , without a control group , and did not asses liver histology . thus , there is some evidence suggesting that there may be a direct effect of glp-1r agonists on hepatocytes , but this case does not allow for any conclusion regarding the potential direct or indirect effect of glp-1 on the liver .
options for the treatment of nash are limited and hold risk of severe side effects . in this case , the glp-1r agonist liraglutide was well tolerated and markedly improved lfts and histology in this patient with type 2 diabetes and nash .
randomized controlled trials are needed to evaluate whether the present findings hold promise for the treatment of nash .
the authors declare no conflict of interest regarding this paper . there were no funding sources . | a 25-year - old female nurse was referred to our diabetes outpatient clinic with poorly controlled type 2 diabetes , obesity and elevated liver function tests ( lfts ) . following a liver biopsy
she was diagnosed with non - alcoholic steatohepatitis ( nash ) and liver fibrosis .
treatment with subcutaneous injections of the glucagon - like peptide-1 receptor ( glp-1r ) agonist liraglutide was initiated .
after 46 weeks of treatment the patient had lost 16 kg , glycemic control was excellent and lfts had normalized .
repeat liver biopsy and ultrasound showed reduction in hepatic fat content and inflammatory cells .
the biopsy no longer fulfilled the criteria for nash .
the liver biopsies did not express hepatic glp-1rs using quantitative polymerase chain reaction .
our case suggests that liraglutide may benefit patients with nash . |
carcinoma primarily developing in the cystic duct is uncommon , compared to other locations in the biliary ducts .
they may have better survival than malignancies located in other biliary ducts [ 2 , 3 ] .
we report a 66-year - old male who is still doing well 13 years after surgical resection for ccd .
a 66-year - old male was admitted to the digestive surgery service of the teaching hospital of trousseau for recurrent right upper quadrant pain in january 1995 .
laparoscopic surgery was performed and peroperative cholangiography made opaque only 1 cm of the cystic duct because of an intraluminal obstruction . at laparotomy
a tumor was found in the distal portion of the cystic duct suggesting a ccd .
the patient underwent en - bloc resection of the gallbladder , the cystic duct with its tumor , the common bile duct and the choledochus . an extending lymph node dissection in the hepatoduodenal ligament and a roux - en - y hepaticojejunal anastomosis
final histological examination of the resected specimen showed a moderately differentiated excretobiliopancreatic adenocarcinoma of the cystic duct ( fig .
2 ) there was no invasion of the gallbladder or the common bile duct and the choledochus .
the patient is doing well 13 years after the resection without any sign of recurrence or metastasis .
ccd is rare according to the literature [ 1 , 3 , 4 , 5 ] .
however its incidence would be higher than is believed now if all the advanced stage cases were added .
it is proved that inflammation of the biliary duct epithelium potentially leads to malignancy sooner or later , in consideration to the frequently associated carcinomas with pancreaticobiliary maljunction . indeed , because of its narrow lumen , the cystic duct is more often affected by gallstones during their migration from the gallbladder to the choledochus than other extrahepatic biliary ducts .
therefore this carcinogenesis factor should increase the incidence of carcinoma in the cystic compared to the other biliary ducts .
clinically , ccd may be completely asymptomatic or be revealed by signs suggesting other pathologies .
it sometimes presents with mirrizi syndrome [ 3 , 4 , 5 ] and is diagnosed preoperatively .
peroperative diagnosis is common when performing operation for acute cholecystitis such as in our patient . in consequence ,
any relapsing cholecystitis , particularly without gallstones , associated with uniform distended gallbladder should suggest the possibility of cystic duct tumor [ 1 , 2 ] . for us ,
the impossibility to catheterize the cystic duct for cholangiography or to opacify the hepaticocholedochus , such as in this case , should lead to the search for cystic duct tumor . on the treatment level , most authors recommend radical surgery [ 2 , 3 , 4 ] though , according to the definition , advanced stages of ccd are excluded .
these authors propose en bloc resection of gallbladder , cystic duct , common bile duct , choledochus and an extending lymph node dissection of the hepaticoduodenal ligament .
we think this attitude is too aggressive and may not be necessary even if only early - stage ccd are concerned .
biliary duct resection and extending lymph node dissection should be sufficient . according to the literature , among all patients with carcinoma of the extrahepatic trees , those fulfilling the definition criteria of ccd should have better and prolonged survival than those with other locations .
this better survival is justified by advanced stage exclusion . in spite of these restricted criteria , the overall published survival to
our knowledge varies from 14 months to 125 months [ 1 , 2 , 3 , 4 ] with a median survival of 20 months .
thirteen years 156 months ) after surgery our patient is still doing well without any recurrence or metastasis . to our knowledge this is the second case with a survival over ten years and the first one with a disease - free survival over thirteen years . beside this prolonged survival , this case report may suggest possibly curability when large en bloc resection of the biliary trees with extending lymph node dissection is performed .
the incidence of ccd might be increased if advanced stage ccd were taken into account .
disease - free survival over thirteen years is possible , but large resection and extending lymph node dissection are necessary . | cystic duct carcinoma is known to have a better prognosis compared to its location in other biliary ducts .
only one case with a survival over ten years has been previously published .
the authors report a case of survival over 13 years without recurrence or metastasis .
preoperative diagnosis of cystic duct tumor was carried in a 66-year - old male . under the diagnosis of carcinoma of the cystic duct , the patient underwent en bloc resection of the gallbladder , cystic duct , hepaticocholedochus and lymph node dissection . a roux - en - y hepaticojejunostomy was performed .
histological examination revealed a moderately differentiated adenocarcinoma of the cystic duct .
five months later the patient underwent second look surgery for benign obstruction of the hepaticojejunal anastomosis .
the patient is still doing well 13 years later without any local recurrence or metastasis . to our knowledge , this is the longest disease - free survival ever published in the literature .
this case sustains that better and longer survival is possible with a real chance of potential cure if radical surgery is performed . |
smoking cessation requires commitment shared by individuals and national authorities . along with an individual approach
, a supportive environment is needed to encourage tobacco consumers in their attempts to quit ( 1 ) . according to who data , about 87% of countries in the world health organization ( who ) european region allow nicotine replacement therapies ( nrt ) to be sold , but only about 40% offer toll - free quit lines .
( 2 ) the evidence shows that a pharmaceutical treatment ( nrt and/or bupropion ) approximately doubles success rates of smoking cessation . nevertheless
, only three countries fully cover and four partially cover the cost of nrts through national / federal health insurance or the national health service ( 3,4,5 ) .
recent surveys reveal great opportunity for smoking cessation among both young people as well in adults .
survey findings in over 30 countries in the who european region shows that about 7 out of 10 smokers aged 1315 years expressed interest in quitting smoking .
disadvantaged people are likely to start smoking at a younger age and have less access to cessation services .
recent study of 10 partner countries in the european union shows , the development of smoking - cessation interventions for young people has been neglected ( 6,7,8,9,10 ) .
analysis of current cigarette smoking and attitude of school children related to smoking cessation , as a significant indicator for recommendations of tobacco control policy and strategy measures in the federation of bih .
the analysis involves data collected through two surveys conducted in the federation of bih , during period 2008 -2013 under support of the federal ministry of health and federal ministry of education who authorized the survey .
survey is conducted by the institute for public health of the federation of bih as the implementing agency ( 11 ) . for the analysis purpose , current cigarette smoking and motivation for cessation in school children assessed as a part of multiple - choice questionnaire adapted from a global youth tobacco standard core questionnaire .
surveys involves two - stage cluster sample design aimed to enroll students in grades 7 , 8 and 9 of primary and 1 grade of secondary school , with focus on representative sample of children age 13 - 15 . in 2013 , from 6,972-sampled school children , total 6,415 completed usable questionnaires , of which 5,060 were aged 13 - 15 years with overall response rate of schoolchildren 92.0% .
data entry and statistical analysis completed with sudaan , a software package used to calculate weighted prevalence estimates and standard errors ( se ) of the estimates ( 95% confidence intervals [ ci ] were calculated from the ses ) ( 11,12 ) .
data analysis is based on comparative trends of selected indicators collected through two surveys performed in the federation of bosnia and herzegovina in 2008 and 2013 .
every cigarettes smokers decreases from 45.9% in 2008 to 43.2% in 2013 , both in boys from 52.9% to 47.8% in 2013 as well in girls from 39.0% in 2008 to 38.3% in 2013 .
a survey data shows that overall current cigarette smokers in school children decreased from 14.3% in 2008 to 12.7% in 2013 , both boys and girls ( table 1 ) .
tobacco use in school children age 13 - 15 , the federation of bosnia and herzegovina 2008 - 2013 a survey data indicate early initiation of smoking among school children in the federation of bih , with some changes in period 2008 - 2013 .
a over third of school children ( 36.6% ) in 2008 who initiated to smoke in age before ten , decreases to 32.8% in 2013 , both in boys from 41.3% in 2008 to 34.3% in 2013 as well in girls from 30.9% in 2008 to 28.9% in 2013 a percentage of never smokers in school children who are thinking about possible initiation to start smoking in near future are important data for susceptibility of tobacco use among youth .
a survey findings show that never tobacco users susceptible to tobacco use in the future decrease from 26.1% in 2008 to 12.3% in 2013 both in boys decreases from 24.6%in 2008 to 11.5% in 2013 as well in girls from 27.1% in 2008 to 12.9% in 2013 ( figure 2 ) ever smokers initiated smoking before age of ten , the federation of bosnia and herzegovina 2008 - 2013 never tobacco users susceptible to tobacco use in the future , the federation of bosnia and herzegovina 2008 - 2013 from two third or 68.1% school children in 2008 who confirmed that tried to stop smoking in the past 12 months , over half of schoolchildren or 57.7% in 2013 confirmed attempting to stop smoking .
a survey data shows decrease of current smoking students who want to stop smoking now from 50.5% in 2008 to 45.8% in 2013 , more likely boys than girls . from over half or 53.2% of school children in 2008 who thought they would be able to stop smoking if they wanted to , significantly increases to 80.3% of school children in 2013 .
however , slightly 1 in 10 school children or 12.3% in 2013 have ever received help / advice from a program or professional to stop smoking ( table 2 ) .
smoking cessation in school children age 13 - 15 , the federation of bosnia and herzegovina 2008 - 2013
survey findings collected a significant data for monitoring of tobacco as health risk factor in school children and youth in the federation of bih . every cigarettes smoker s decreases from 45.9% in 2008 to 43.2% in 2013 , as well overall current cigarette smokers from 14.3% in 2008 to 12.7% in 2013 , both boys and girls . in compare with
recently performed regional surveys , current cigarette smokers in the federation of bih data from is lower in compare to croatia ( 27.2% ) , slovenia ( 17.8% ) , hungary ( 23.2% ) , czech republic ( 30.6% ) and higher compare to current cigarette smokers among school children in serbia ( 9.3% ) , montenegro ( 5.1% ) and italy ( 7.3% ) ( 11,12,13 ) .
a survey data indicate early initiation of smoking among school children in the federation of bih , from 36.6% in 2008 who initiated to smoke in age before ten , decreases to 32.8% in 2013 which is similar to survey data in serbia ( 36.8% ) and montenegro ( 39.6% ) and higher compare to surveys performed in croatia ( 23.3% ) , slovenia ( 19.6% ) czech republic ( 21.4% ) , hungary ( 18.0% ) and italy ( 7.4% ) ( 11,12,13 ) .
a survey finding indicates evidence of decrease of attempting to stop smoking among school children in the federation of bih , from 68.1% in 2008 to 57.7% in 2013 , which is lower in line with trends of cross - country surveys and data of attempting to stop smoking in croatia ( 65.3% ) , serbia ( 52.5% ) , montenegro ( 75.6% ) , slovenia ( 61.6% ) , italy ( 66.2% ) , hungary ( 66.3% ) and czech republic ( 70.3% ) ( 11,12,13 ) .
also , a survey data shows decrease of current smoking students who want to stop smoking now from 50.5% in 2008 to 45.8% in 2013 , which is higher compare to surveys results in croatia ( 36,2% ) , slovenia ( 33.2% ) , montenegro ( 41.2% ) , italy ( 28.0% ) , hungary ( 41.0% ) , and czech republic ( 44.9% ) and lower compare to serbia ( 47.5% ) ( 11,12,13 ) .
it is significant that only of 12.3% school children in 2013 have ever received help / advice from a program or professional to stop smoking , which is significantly lower compare to surveys results in croatia ( 51,3% ) , serbia ( 81.2% ) , slovenia ( 43.1% ) , montenegro ( 65,0% ) , italy ( 57.3% ) , hungary ( 48.1% ) , czech republic ( 46.1% ) ( 11,12,13 ) .
a survey findings indicates that almost half of school children ever smoked cigarettes more likely boys than girls as well almost a third of school children confirmed to start smoking in age before ten , as a sign of early initiation and evidence of significant nicotine addiction . however , collected data confirmed decreases of current cigarette smoking in school children in the federation of bih during period 2008 - 2013 , both in boys and girls which is an indicator for further enforcement of initiated tobacco control measures . for prevention and decrease of smoking among school children
it is very important to provide available cessation services and quit lines . particularly as survey data shows that current cigarette smokers among school children who tried to stop smoking in past year as well those who want to stop smoking now decreases in period 20082013 . in line with good international practice of youth cessation services , more efforts should be focus to promote awareness and knowledge of school children related to smoking cessation
there is need for more comprehensive public campaigns as well enforcement of friendly use quitting services and help lines developed in collaboration of health and education system in the federation of bih ( 14,15,16,17,18,19 ) | introduction : quitting tobacco use has many benefits for both individuals and society . for individuals , quitting of smoking provides immediate and long - term benefits to health .
higher levels of nicotine addiction and start smoking at a younger age are factors consider as strongly influence on quit smoking .
access to youth friendly quit lines increases a smoking cessation of school children.goal:monitoring of trends of age of initiation of smoking and current smoking cigarettes as good motivation for smoking cessation among school children in the federation of bih.methods:analysis of survey s findings performed in the federation of bih in period 2008 - 2013 , with particular focus on indicators of every smokers , currents smokers , start to smoke before age of ten , signs of smoking dependence and motivation for quitting smoking in school children .
surveys involves two - stage cluster sample design aimed to enroll students in grades 7 , 8 and 9th of primary and 1th grade of secondary school.results:a survey findings indicates ever cigarettes smokers decreases from 45.9% in 2008 to 43.2% in 2013 , as well decreases of current cigarette smoking from 14.3% in 2008 to 12.7% in 2013 , both boys and girls . however , survey data shows decrease of current smoking students who want to stop smoking now from 50.5% in 2008 to 45.8% in 2013.conclusions:collected data confirmed decreases of current cigarette smoking in school children in the federation of bih in period 2008 - 2013 .
access to available smoking cessation services and quit lines for school children and youth considers should be a significant part of tobacco control measure in the federation of bih . |
computerized techniques for the evaluation of o'farrell two - dimensional electrophoretic gels have been applied to proteins derived from asbestos bearing macrophages .
preliminary results indicate definite changes in the protein content of cells depending on fiber phagocytosis.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 7.figure 8.figure 9.figure 10.figure 11.figure 12.figure 13.figure 14.figure 15.figure 22 .
afigure 22 .
bfigure 22 .
cfigure 22 .
d | |
1 ) is a perennial herb with a eurasian - mediterranean distribution area ( kretzschmar et al .
the species is confined to calcareous soils and occurs in sunny or partially shaded sites .
it ranges from sea level to 2,200 m and it can grow in chalk grasslands , sand dunes , wet meadows , abandoned cultivated lands , scrub communities or open woodlands .
o. militaris appears as a moderately long stem , generally varying from 20 to 45 cm , with a single inflorescence bearing 1040 hermaphroditic flowers ( farrell 1985 , delforge 2012 ) .
o. militaris is assumed to be self - compatible ( neiland and wilcock 1998 , metsare et al .
2015 ) and non - autogamous ( delforge 2012 ) , and it depends on the food deception strategy to attract pollinators ( claessens and kleynen 2011 ) .
low levels of fruit set ( < 30% ) are common ( tremblay et al . 2005 ,
militaris visited by a. mellifera ( wijre - akkers , netherlands , 1 june 2010 , photographer : jean claessens ) .
o. militaris visited by a. mellifera ( wijre - akkers , netherlands , 1 june 2010 , photographer : jean claessens ) .
the study was carried out in 2013 in five belgian populations ( table 1 ) . according to alexandersson and gren ( 1996 )
, a population was defined as a group of inflorescences separated from their closest conspecific by at least 100 m. all populations were at least 5 km apart , except those of vis , which were 800 m apart ( but separated by a canal 140 m wide ) .
the biotopes were various : recolonized slag heaps , semi - dry calcareous grassland , mesic grassland , thickets and deciduous forestry plantations .
table 1.characteristics of o. militaris populationssite no.municipalitysite namecoordinatesaltitude ( m)population sizedate of insect collectionsavisfriche du canal albert50 4616 n 5 4104 e5510,00045 june 2013bvisoseraie de lanaye50 4645 n 5 4059 e651,50067 june 2013cvirtoncontournement de virton49 3438 n 5 3058 e2501501617 june 2013dmussoncrassier de musson49 3309 n 5 4304 e2902302728 may 2013erouvroynature reserve raymond mayn49 3034 n 5 2853 e295801112 june
characteristics of o. militaris populations the population size is given in terms of number of inflorescences .
field work took place during sunny and warm ( at least 18 c ) days with little or no wind , between 8.00 a.m. and 8.00 p.m. between 27 may 27 and 17 june ( table 1 ) .
we made around 30 total hours of captures ( 180 10-min censuses ) , thus 6 h ( 36 10-min censuses ) per population .
each population was studied during 2 consecutive days , and the 6 h were regularly distributed throughout the 2 days . during 10-min censuses , taking a standard route ( 100 m ) across the population , we used a butterfly net to catch all insects that landed on o. militaris inflorescences .
we put them in individual pill - boxes containing a piece of cotton wool soaked with ethyl acetate .
pill - boxes were conserved in a cool box ( eda plastiques , oyonnax , france ) with cold accumulators ( kern frio s.a .
, lhospitalet de llobregat , spain ) and then in a freezer at 18 c . when insects bore pollinia , we used morphological characters in order to distinguish the pollinia of o. militaris from those of other orchids found in the study sites and their surroundings [ anacamptis pyramidalis ( l. ) rich .
, o ophrys insectifera ( l. ) , orchis anthropophora ( l. ) all . , and platanthera chlorantha ( custer ) rchb . ] .
most of the specimens are kept at the biodiversity research centre ( universit catholique de louvain ) ; some dipterans are stored at the royal belgian institute of natural sciences .
1 ) is a perennial herb with a eurasian - mediterranean distribution area ( kretzschmar et al .
the species is confined to calcareous soils and occurs in sunny or partially shaded sites .
it ranges from sea level to 2,200 m and it can grow in chalk grasslands , sand dunes , wet meadows , abandoned cultivated lands , scrub communities or open woodlands .
o. militaris appears as a moderately long stem , generally varying from 20 to 45 cm , with a single inflorescence bearing 1040 hermaphroditic flowers ( farrell 1985 , delforge 2012 ) .
o. militaris is assumed to be self - compatible ( neiland and wilcock 1998 , metsare et al .
2015 ) and non - autogamous ( delforge 2012 ) , and it depends on the food deception strategy to attract pollinators ( claessens and kleynen 2011 ) .
low levels of fruit set ( < 30% ) are common ( tremblay et al . 2005 ,
militaris visited by a. mellifera ( wijre - akkers , netherlands , 1 june 2010 , photographer : jean claessens ) .
o. militaris visited by a. mellifera ( wijre - akkers , netherlands , 1 june 2010 , photographer : jean claessens ) .
the study was carried out in 2013 in five belgian populations ( table 1 ) . according to alexandersson and gren ( 1996 )
, a population was defined as a group of inflorescences separated from their closest conspecific by at least 100 m. all populations were at least 5 km apart , except those of vis , which were 800 m apart ( but separated by a canal 140 m wide ) .
the biotopes were various : recolonized slag heaps , semi - dry calcareous grassland , mesic grassland , thickets and deciduous forestry plantations .
table 1.characteristics of o. militaris populationssite no.municipalitysite namecoordinatesaltitude ( m)population sizedate of insect collectionsavisfriche du canal albert50 4616 n 5 4104 e5510,00045 june 2013bvisoseraie de lanaye50 4645 n 5 4059 e651,50067 june 2013cvirtoncontournement de virton49 3438 n 5 3058 e2501501617 june 2013dmussoncrassier de musson49 3309 n 5 4304 e2902302728 may 2013erouvroynature reserve
raymond mayn49 3034 n 5 2853 e295801112 june 2013the population size is given in terms of number of inflorescences .
characteristics of o. militaris populations the population size is given in terms of number of inflorescences .
field work took place during sunny and warm ( at least 18 c ) days with little or no wind , between 8.00 a.m. and 8.00 p.m. between 27 may 27 and 17 june ( table 1 ) .
we made around 30 total hours of captures ( 180 10-min censuses ) , thus 6 h ( 36 10-min censuses ) per population .
each population was studied during 2 consecutive days , and the 6 h were regularly distributed throughout the 2 days . during 10-min censuses , taking a standard route ( 100 m ) across the population , we used a butterfly net to catch all insects that landed on o. militaris inflorescences .
we put them in individual pill - boxes containing a piece of cotton wool soaked with ethyl acetate .
pill - boxes were conserved in a cool box ( eda plastiques , oyonnax , france ) with cold accumulators ( kern frio s.a .
, lhospitalet de llobregat , spain ) and then in a freezer at 18 c . when insects bore pollinia , we used morphological characters in order to distinguish the pollinia of o. militaris from those of other orchids found in the study sites and their surroundings [ anacamptis pyramidalis ( l. ) rich .
, o ophrys insectifera ( l. ) , orchis anthropophora ( l. ) all . , and platanthera chlorantha ( custer ) rchb .
most of the specimens are kept at the biodiversity research centre ( universit catholique de louvain ) ; some dipterans are stored at the royal belgian institute of natural sciences .
we caught a total of 104 insects belonging to 49 species , 21 families and four orders ( table 2 ) .
rhingia campestris meigen ( 14 individuals ) and bombylius venosus mikan ( 10 individuals ) were the most abundant dipterans ; apis mellifera ( l. ) ( 8 individuals ) and bombus lapidarius ( l. ) ( 6 individuals ) were the most abundant hymenopterans . some lepidopterans and coleopterans
pollinia ( only from o. militaris ) were only found in hymenopterans : four honeybee workers ( a. mellifera ) bore respectively 13 , 12 , 3 ( attached to the clypeus ) and 2 pollinia , and one bumblebee worker ( b. lapidarius ) bore 1 pollinium .
a. mellifera was caught in three sites and b. lapidarius in four of the five sites .
because ethyl acetate fumes rapidly dissolve the viscidium glue ( peter and johnson 2013 ) , we failed to determine the position of pollinia on four of the five insects .
a number of insects were also observed but not caught on o. militaris inflorescences : clytus arietis ( l. ) ( coleoptera , cerambycidae ) , harmonia axyridis pallas ( coleoptera , coccinellidae ) , eristalis sp .
( diptera , sarcophagidae ) , pyrausta aurata scopoli ( lepidoptera , crambidae ) , polyommatus icarus rottemburg ( lepidoptera , lycaenidae ) , vanessa sp .
( lepidoptera , nymphalidae ) , and anthocharis cardamines ( l. ) ( lepidoptera , pieridae ) .
table 2.identity and number of insects caught on o. militaris inflorescencesorderfamilytaxonabcdecoleopteraelateridaeindet.1scarabaeidaephyllopertha horticola ( l. 1758)22valgus hemipterus ( l. 1758)2dipteraagromyzidaeindet.1asilidaeneoitamus cf . socius ( loew 1871)1bombyliidaebombylius major ( l. 1758)3b
. venosus ( mikan 1796)316empididaeempis discolor ( loew 1856)1empis femorata ( f. 1798)1empis livida ( l. 1758)1hybotidaehybos sp.1scathophagidaescathophaga stercoraria ( l. 1758)1syrphidaechrysotoxum cautum ( harris 1776)31episyrphus balteatus ( de geer 1776)1eupeodes nitens ( zetterstedt 1843)1helophilus pendulus ( l. 1758)1merodon equestris ( f. 1794)1platycheirus albimanus ( f. 1781)11platycheirus peltatus ( meigen 1822)1orderfamilytaxonabcdeplatycheirus scutatus ( meigen 1822)1r .
campestris ( meigen 1822)437sphaerophoria scripta ( l. 1758)11syritta pipiens ( l. 1758)111volucella bombylans ( l. 1758)1hymenopteraandrenidaeandrena carantonica ( perez 1902)1()andrena flavipes ( panzer 1799)1()andrena nigroaenaea ( kirby 1802)1()apidaea .
pascuorum ( scopoli 1763)1()1()bombus pratorum ( l. 1761)4(3 , 1 )bombus terrestris ( l. 1758)1()colletidaehylaeus confusus ( nylander 1852)1()halictidaelasioglossum fulvicorne ( kirby 1802)1()lasioglossum malachurum ( kirby 1802)1()lasioglossum parvulum ( schenck 1853)1()sphecodes sp.1()megachilidaeo . bicolor ( schrank 1781)1()lepidopteracrambidaepyrausta sp.1geometridaesiona lineata ( scopoli 1763)1hesperiidaeerynnis tages ( l. 1758)1carterocephalus palaemon ( pallas 1771)2nymphalidaeaglais io ( l. 1758)1lasiommata megera ( l. 1767)1melitaea cinxia ( l. 1758)1noctuidaeeuclidia glyphica ( l. 1758)11pieridaepieris napi ( l. 1758)2formicidae were present in all the populations but were nt caught .
= female ; = male ; = worker .
identity and number of insects caught on o. militaris inflorescences formicidae were present in all the populations but were nt caught .
= female ; = male ; = worker .
in this study , o. militaris attracted a variety of visitors , mainly hoverflies ( 32 individuals ) , bumblebees ( 16 individuals ) and bee flies ( 13 individuals ) , probably through the display of general floral signals ( typical for rewarding plants ) such as showy flower colors , presence of a spur , etc .
rewarding plants attract insects and non - rewarding o. militaris individuals flowering nearby benefit from chance visits ( jerskov et al .
2009 ) . all caught dipterans and lepidopterans have to be considered as visitors ( sensu
schatz et al .
to our knowledge , dipterans and butterflies are never cited as pollinators in the literature .
the former visit various orchid species for nectar ( absent in deceptive species like o. militaris , which exploit the food - seeking behavior of insects and lure visitors jerskov et al .
2006 ) , substances secreted by the stigma , pollen or occasionally to find a shelter ; lepidopterans seek nectar essentially in species with a long spur ( berger 2003 ) , like gymnadenia spp . or platanthera spp . caught and observed coleopterans were simple visitors
( sensu
berger 2003 ) ; they apparently landed randomly on inflorescences and never actively visited flowers .
a. mellifera was already known as a potential pollinator ( i.e. insects collected in an inflorescence while carrying one or more pollinia after schatz et al .
2005 ) of o. militaris but it was apparently not the case of b. lapidarius , although this species was previously observed on o. militaris in different countries ( table 3 ) .
the high number of pollinia found on two of the four caught honeybees ( 13 and 12 , respectively ) indicates repeated visits to o. militaris .
table 3.non-exhaustive list of insects observed on o. militaris inflorescences in different countriesorderfamilytaxoncountry ( iso code)referencescoleopteracantharidaecantharis lateralis ( l. 1758)ru12cantharis rustica ( fallen 1807)gb9cerambycidaepachyta quadrimaculata ( l. 1758)ru10rutpela maculata ( poda 1761)gb9cetoniidaet .
hirta ( poda 1761)ru , ru , at10 * , 12 * , 15*chrysomelidaecassida viridis ( l. 1758)gb9smaragdina salicina ( scopoli 1763)ru12malachiidaemalachius bipustulatus ( l. 1758)ru12malachius coccineus ( waltl 1838)ru12oedemeridaeoedemera nobilis ( scopoli 1763)ru10 , 12dipterabibionidaebibio marci ( l. 1758)gb9bombyliidaeb .
venosus ( mikan 1796)at13calliphoridaeonesia sp.gb7empididaeempis tessellata ( f. 1794)fr , gb3 , 9muscidaemusca sp.at13thricops semicinereus ( wiedemann 1817)gb7opomyzidaeopomyza germinationis ( l. 1758)gb7scathophagidaes .
balteatus ( de geer 1776)gb , at9 , 13eristalis pertinax ( scopoli 1763)gb9leucozona lucorum ( l. 1758)gb7melanostoma mellinum ( l. 1758)gb7melanostoma scalare ( f. 1794)gb9platycheirus ambiguus ( fallen 1817)at13p .
flavipes ( panzer 1799)at13andrena hattorfiana ( f. 1775)at13 * , 14*andrena helvola ( l. 1758)at13andrena minutula ( kirby 1802)at13andrena subopaca ( nylander 1848)at13andrena taraxaci ( giraud 1861)at13andrena tarsata ( nylander 1848)at13apidaeanthophora aestivalis ( panzer 1801)at13*anthophora borealis ( morawitz 1864)ru10 * , 12*a .
mellifera ( l. 1758)fr , fr , nl , gb , at , at2 * , 3 * , 5 * , 9 * , 13 * , 14*b .
lapidarius ( l. 1758)fr , gb , fr , gb , at3 , 7 , 8 , 9 , 13b . lucorum ( l. 1761)gb7 , 9b .
terrestris ( l. 1758)gb , at9 * , 13bombus vestalis ( geoffroy 1785)gb1*ceratina callosa ( f. 1794)at13ceratina cyanea ( kirby 1802)at13eucera sp.fr3 , 4n .
eurygnathus ( bluethgen 1931)at13 * , 14*halictus simplex ( bluethgen 1923)at13 * , 14*lasioglossum albipes ( f. 1781)at13lasioglossum calceatum ( scopoli 1763)at13lasioglossum morio ( f. 1793)at13lasioglossum nigripes ( lepeletier 1841)at13lasioglossum pauxillum ( schenck 1853)at13lasioglossum xanthopum ( kirby 1802)at13sphecodes ferruginatus ( von hagens 1882)at14*megachilidaeh .
adunca ( panzer 1798)be11*osmia aurulenta ( panzer 1799)at13 * , 14*o . bicolor ( schrank 1781)at13*osmia rufa ( l. 1758)be11*sphecidaeammophila sabulosa ( l. 1758)gb9sphex funerarius ( gussakovskij 1934)gb9orderfamilytaxoncountry ( iso code)referencevespidaevespula vulgaris ( l. 1758)gb9lepidopterageometridaepseudopanthera macularia ( l. 1758)at13hesperiidaec .
cardamines ( l. 1758)de6colias hyale ( l. 1758)de6gonepteryx rhamni ( l. 1758)de6leptidea sinapis ( l. 1758)de6pieris brassicae ( l. 1758)de , gb , gb6 , 7 , 9p .
napi ( l. 1758)de , gb , gb , at6 , 7 , 9 , 13pieris rapae ( l. 1758)gb91 , bateman and rudall ( 2014 ) ; 2 , berger ( 2003 ) ; 3 , berger ( 2004 ) ; 4 , berger ( 2010 ) ; 5 , claessens and kleynen ( 2011 ) ; 6 , ebert and rennwald ( 1993 ) ; 7 , farrell ( 1985 ) ; 8 , godfery ( 1933 ) ; 9 , harding ( 1996 ) ; 10 , krivosheev et al . ( 2009 ) ; 11 , petit ( 1998 ) ; 12 , shamigulova ( 2012 ) ; 13 , vth ( 1987 ) ; 14 , vth ( 1999 ) ; 15 , vth ( 2003 ) .
non - exhaustive list of insects observed on o. militaris inflorescences in different countries 1 , bateman and rudall ( 2014 ) ; 2 , berger ( 2003 ) ; 3 , berger ( 2004 ) ; 4 , berger ( 2010 ) ; 5 , claessens and kleynen ( 2011 ) ; 6 , ebert and rennwald ( 1993 ) ; 7 , farrell ( 1985 ) ; 8 , godfery ( 1933 ) ; 9 , harding ( 1996 ) ; 10 , krivosheev et al .
( 2009 ) ; 11 , petit ( 1998 ) ; 12 , shamigulova ( 2012 ) ; 13 , vth ( 1987 ) ; 14 , vth ( 1999 ) ; 15 , vth ( 2003 ) .
several factors can explain why an insect is only a visitor and not a pollinator of a plant species ( pellmyr 2002 ; shivanna and tandon 2014 ) .
in the case of o. militaris , the behavior and or the morphology of visitors on the orchid flowers normally prevents these insects from picking up pollinia .
. then the bursicle s membrane breaks and the viscidia are released and glued to the insect s head ( claessens and kleynen 2011 ) . as long as individuals from a given insect species are not documented as having collected at least one pollinium ,
this categorization could be temporary or permanent ; for example , some insect groups , like orthopterans ( but see micheneau et al . 2010 ) , usually are never considered as pollinators .
caught insects bearing pollinia are potential pollinators and could be either incidental ( transporting pollinia by chance ) or confirmed ones .
a potential pollinator becomes confirmed when the observer has seen the removal of one or more pollinia from a flower and the deposit of pollen on the stigma of another flower from the same species ( schatz et al .
2005 ; but see berger ( 2006 ) who details the limits of this definition ) .
more than 90 insect species ( half are hymenopterans ) are known to visit the flowers of o. militaris ( table 3 ) .
one - fourth ( almost exclusively hymenopterans , with the exception of the coleopteran tropinota hirta poda ) have to be considered as potential pollinators . among them , some species were caught in this study but bore no pollinia ( apidae : bombus pascuorum scopoli , b. pratorum ( l. ) , and b. terrestris ( l. ) ; megachilidae : osmia bicolor schrank ) . the low sample size per species ( in average , two specimens )
moreover , a few potential pollinators from table 3 are not present or extremely rare in belgium : andrena enslinella stoeckhert ( andrenidae : rasmont et al .
2013 ) , anthophora aestivalis panzer , a. borealis morawitz ( apidae : rasmont 2014 ) , and halictus eurygnathus bluethgen ( halictidae : pauly et al .
2016 ) ; their role in o. militaris pollination is probably very low in this country .
oseraie de lanaye ( table 1 ) , petit ( 1998 ) found specimens from three species of nomada scopoli ( apidae : nomada fabriciana ( l. ) , nomada ruficornis ( l. ) , and nomada succincta panzer ) and hoplitis adunca panzer ( megachilidae ) with o. militaris pollinia . however , none of these species was observed in our study .
finally , as mentioned earlier , a. mellifera was the only species bearing pollinia in common in this study ( table 2 ) and in previous ones ( table 3 ) . according to vth ( 1987 ) ,
confirmed pollinators ( at least in lower austria ) are short - tongued hymenopterans from the genera andrena ( f. ) ( andrenidae : a. enslinella and a. hattorfiana f. ) and halictus latreille ( halictidae : h. eurygnathus and h. simplex bluethgen ) .
a. hattorfiana ( rasmont et al . 2013 ) , h. eurygnathus , and h. simplex ( pauly et al .
2016 ) are found in belgium , but these species were not observed on o. militaris during the censuses . as mentioned in the previous paragraph , a. enslinella is absent from the belgian territory ( rasmont et al .
vth s conclusion is based upon the presence of pollinia on insects and the similarity between spur length ( and lip length ) and proboscis length ( and body length ) . according to this interpretation
, pollinia adhesion and subsequent transport would be unadapted to the morphology of long - tongued bees ( in vth s study , apidae : a. mellifera , b. lapidarius , and b. terrestris ) because they reach the end of the spur before the viscidium adheres to the insect ( vth 1987 , van der cingel 1995 ) .
however , real pollination events are not described by vth ( 1987 ) and his conclusion is thus mostly putative .
bateman and rudall ( 2014 ) hypothesized that bumblebees ( b. pratorum and b. vestalis geoffroy ) could be confirmed pollinators in a british population . in vth
s study , the two mentioned bumblebee species ( b. lapidarius and b. terrestris ) were nt pollinators because no specimen bore pollinia ( but see harding 1996 ) and their proboscis was longer than the spur .
b. lapidarius and b. terrestris are long - tongued bees but this is not the case of the short - tongued b. pratorum and b. vestalis ( goulson 2010 , bateman and rudall 2014 ) .
the results of vth ( 1987 ) and those of bateman and rudall ( 2014 ) suggest the importance of short - tongued ( non-)apidae bees as confirmed pollinators of o. militaris . in general , long - tongued bees , like a. mellifera and b. lapidarius in this study , probably remove pollinia accidentally during their visits . in certain cases ,
the number of removing events can be high but specimens bearing numerous pollinia should be rare , although we mostly observed honeybees bearing many pollinia .
the function of short - tongued bees should be examined in more details through direct observation of pollination events and study of pollination efficiency .
the status of incidental pollinators attributed to long - tongued bees could also be confirmed through experiments involving a. mellifera or b. terrestris ( e.g. hives provided by biobest , westerlo , belgium ) . direct field observations and
captures also remain of the greatest importance to determine the role of insect visitors and study the visitor spectrum of a plant species . | as part of a research project on the food deception strategy in orchis militaris ( l. ) , the objective of this study was to identify insect visitors and potential pollinators of this orchid species in belgium . in 2013 ,
insects were collected over 2 d per site in five localities distributed in southern belgium ( wallonia ) .
a total of 104 insects belonging to 49 species were caught .
dipterans were the most abundant visitors ( 50% of total specimens ) , followed by hymenopterans ( 32% ) .
rhingia campestris meigen , bombylius venosus mikan , apis mellifera ( l. ) , and bombus lapidarius ( l. ) were the most abundant species .
only five specimens bore one to more than 10 pollinia : four honeybees ( a. mellifera ) and one bumblebee worker ( b. lapidarius ) .
these two species should be considered as potential pollinators in the study area , but probably not confirmed ones . |
radiographic diagnosis of impacted third molars precedes their surgical removal . the factors such as the position of tooth , the number , and morphology of roots , in particular , the relationship between the roots and mandibular canal , require assessment for an atraumatic untoward transalveolar extraction of these impacted mandibular molars .
the bucco - lingual relation between the tooth and other anatomical structures , is better asssessed by the tube shift or buccal object rule which is traditionally employed .
orthopantamograph has diversified use in both general diagnosis and varied specialized clinical diagnosis , and is commonly used to assess third molars prior to transalveolar extraction . during the last decade , different techniques have been developed for digital , and in recent years , the solid - state digital x - ray units and photostimulable phosphor plate systems which are used with conventional opg units are available in the market .
the diagnostic outcome of panoramic images is the same as that of intraoral periapical radiograph ( iopa ) , however , they have been evaluated only sporadically ; such comparison should be done until a new diagnostic procedure is introduced for better diagnosis .
the third molar follicle first becomes apparent at an age of 67 years and can be diagnosed by 89 years . at the age of 1416 years , the third molar follicle is apparent in radiographs .
girls appear to be slightly ahead of boys up to the age of root formation , however , this difference disappears in the final stage of root development .
third molar agenesis is very common and is considered to have an incidence of 533% . when the cervical part of the root complex is formed , the tooth germ starts to move away from the mandibular canal , assuming a distally curved eruption pathway .
if resistance is met during eruption , intrusive growth occurs in the proximity of the mandibular canal , resulting in indentations in the apical part of the root , apical deflections , or circumferential growth around the mandibular canal , resulting in curving of the roots and a change in the eruption pathway .
these changes that occur during the developmental and the eruption stage of the mandibular third molar make meticulous preoperative radiographic evaluation an important step .
this would facilitate comprehensive evaluation of the anatomic disposition of the tooth in relation to the vital structures in the surrounding .
these types of studies have been conducted in the past comparing computed tomography and panoramic radiographs by pecker et al . in turkey .
though various diagnostic modalities have come up in the recent past including the use of computed tomography , iopa and panoramic radiographs ( opg ) ought to be considered the gold standards for planning transalveolar extraction of impacted third molar of mandible . however , there is sparse literature on comparison between iopa and opg .
the aim of our study is to correlate the clinical findings with these standard radiographs and compare the efficacy of iopa with opg for determining the status of the anatomical factors that dictate the third molar surgery .
the aims of our study are
to confirm the clinical parameters of impacted mandibular third molars with opg and iopa and to assess the efficacy between the two.to corroborate the opg and iopa findings of the root apex of the impacted mandibular third to the inferior alveolar nerve canal .
to confirm the clinical parameters of impacted mandibular third molars with opg and iopa and to assess the efficacy between the two . to corroborate the opg and iopa findings of the root apex of the impacted mandibular third to the inferior alveolar nerve canal .
the aims of our study are
to confirm the clinical parameters of impacted mandibular third molars with opg and iopa and to assess the efficacy between the two.to corroborate the opg and iopa findings of the root apex of the impacted mandibular third to the inferior alveolar nerve canal .
to confirm the clinical parameters of impacted mandibular third molars with opg and iopa and to assess the efficacy between the two . to corroborate the opg and iopa findings of the root apex of the impacted mandibular third to the inferior alveolar nerve canal
two hundred patients diagnosed with moderate - to - severe pericoronitis electing to undergo surgical extraction were choosen , out of which 50 patients were selected for the study by simple random sampling .
the sample size of 50 was selected based on similar comparative studies done previously by ishak et al .
we enrolled individuals who voluntarily signed an informed consent after obtaining institutional ethical committee clearance .
all the patients were referred to the department of oral medicine and radiology for digital opg and iopa .
the digital opg was obtained using sirona ortho phos xg5 unit along with automatic processing of the film .
iopa was obtained using villa india and blue x unit in a paralleling cone technique , and manual processing was done .
the clinical and radiographic assessments were done under standard conditions by a trained oral surgeon experienced in interpreting iopa and opg , as well as one observer beginning his training in oral and maxillofacial surgery .
clinically , the anteroposterior width and depth of the crown exposed was measured with a divider and a scale [ figures 1 and 2 ] .
a trace paper was attached to the opg [ figure 3 ] and iopa [ figure 4 ] , and then the ramus , second , third molars , and inferior alveolar canal was traced on it .
a horizontal line was drawn from the anterior border of the ramus to the posterior extremity of the distal cusp of second molar to determine the amount of bone that has to be removed anteroposteriorly ; a vertical line was drawn from the tip of the distal cusp of mandibular second molar to the crown of the impacted mandibular third molar to determine the depth of impaction .
the proximity of the nerve canal to the root apex of mandibular third molar appreciated in the radiographs was compared clinically by visualizing the socket after the extraction for nerve exposure .
vertical position in relation to occlusal surface of second molar antero - posterior width of crown exposure radiological evaluation of panoramic radiograph and intraoral periapical radiograph yellow line in fig 3 and 4 indicate vertical and antero posterior relation of impacted tooth with ramus of mandible ( horizontal ) and second molar ( vertical ) anatomic relationship
partial bony impactioncomplete bony impaction
partial bony impaction complete bony impaction external oblique ridge
anterior to third molarposterior to third molar
anterior to third molar posterior to third molar type of impaction or space relationship ( pell and gregory classification ) :
mesioangular ( 1070 degrees)distoangular ( 1070 degrees)vertical ( 10 degrees)horizontal ( > 70 degrees )
mesioangular ( 1070 degrees ) distoangular ( 1070 degrees ) vertical ( 10 degrees ) horizontal ( > 70 degrees ) space available or ramus relationship ( pell and gregory classification ) :
sufficient space ( class i)reduced space ( class ii)no space ( class iii )
sufficient space ( class i ) reduced space ( class ii ) depth of impaction or position in relation to second molar ( pell and gregory classification ) :
high occlusal ( level a)medium occlusal ( level b)deep occlusal ( level c )
high occlusal ( level a ) medium occlusal ( level b ) deep occlusal ( level c ) before the operation , the iopa and opg were assessed for the following factors :
type of impactionexternal oblique ridgevertical relationship in relation to second molarspace available or ramus relationshipnumber of rootsroot curvatureproximity of nerve canal
external oblique ridge vertical relationship in relation to second molar space available or ramus relationship proximity of nerve canal
the study revealed that iopa was more accurate in determining majority of the factors affecting the third molar surgery , which includes the relationship of the external oblique ridge to the third molars , where 96% showed that the iopa was superior to the opg .
( iopa vs opg = 96%:90% ) ; p = 0.436(two - sided significance by chi - square test [ table 1 ] . the anteroposterior relation of the impacted tooth with the ramus was best determined by iopa with 70% justifying the decision ( iopa vs. opg = 70%:66% ) ; p = 0.265 ( two - sided significance by chi - square test ) [ table 1 ; graph 1 ] . the vertical depth of impaction in relation to the occlusal aspect of the second molar was better visualized with an iopa .
( iopa vs. opg = 72%:68% ) ; p = 0.828 ( two - sided significance by chi - square test ) [ table 1 ; graph 1 ] .
this was statistically significant with p = 0.013 ( two - sided significance by chi - square test ) [ table 1 ; graph 2 ] .
morphology of roots was also better visualized with an iopa ( iopa vs. opg = 96%:90% ) ; p = 0.436 ( two - sided significance by chi - square test ) [ table 1 ; graph 2 ] .
the comparison of iopa radiograph with opg in evaluating the various parameters of third molar impaction , with clinical correlation graph comparing the accuracy between iopa and opg in the type of impaction , vertical position of impacted molar and antero posterior position of impacted molar graph comparing the accuracy between iopa and opg in the number and morphology of roots of impacted third molar however , panoramic radiograph was found to be more accurate in evaluating the type of impaction ( iopa vs. opg = 88%:94% ) [ p = 0.487 ( two - sided significance by chi - square test ) ] [ table 1 ; graph 1 ] , as well as the canal relationship to the apex of the root of the impacted molar ( iopa vs. opg = 74%:86% ) ; p = 0.211 ( two - sided significance by chi - square test ) [ table 1 ; graph 3 ] .
though in most cases panoramic was more accurate in determining the exact type of impaction and the canal relationship to the tip of the impacted molar , these values were not statistically significant on analysis .
graph comparing the accuracy between iopa and opg in showing the relationship of impacted mandibular third molar root tip with inferior alveolar nerve canal
conventional iopa has diversified use in both general diagnosis and specialized clinical diagnosis , especially to assess third molars prior to transalveolar extraction . with the invention of digital opg during the last decade , many clinicians have opted for this modality of investigation to assess third molars prior to surgical removal . though several comparative studies focusing on the image quality between the conventional and digital opg have been conducted with a significant difference in quality between the two , there had been been no study so for comparing the efficacy of iopa and digital opg , which are most popular among the practitioners .
the primary outcome of the study is that the iopa is more accurate in determining the external oblique ridge relation , space availability , number of roots , and morphology of roots , whereas the type of impaction and the relationship of inferior alveolar canal to the root apex of impacted mandibular third molars are more obvious in opg .
the transalveolar extraction of mandibular third molar necessitate thorough radiographic evaluation of tooth and its related structures to facilitate its removal and eliminate or reduce the risk of nerve damage .
therefore , the most important factor to be considered for surgeons to recognize may be the proximity of the mandibular canal , the number and configuration of the roots , and the degree of mechanical impediment caused by the overlying bone .
hence , the difference in diagnostic accuracy between the conventional iopa and the digital opg were evaluated to assess the efficacy between the two in the study .
several studies have been performed earlier to correlate the different radiographic modalities used in the diagnosis of impacted mandibular third molar .
suggested that opg and/or iopa suffice the presurgical imaging needs in the majority of cases where there is no overlaping between the mandibular canal and the impacted molars of mandible .
a posteroanterior open mouth projection is a supplementary imaging technique in most of the remaining cases . in cases where the mandibular canal and the wisdom tooth are intimately placed ,
hassan concluded that , in clinical dentistry setup , the conventional radiographs which are used mainly include iopa and to a lesser extent opg . according to their study ,
in contrast , a survey shows that up to 95% of dental surgeons want their patients to undergo only opg before implant placement and relatively small number of patients are referred to ct scan .
though the basic investigation preferred by many practitioners are opg and iopa , no study been conducted to compare the efficacy between the two , especially with the most frequent minor oral surgical pathosis of impacted mandibular third molar .
because the anatomical structures and allingment of teeth are always not in a straight line and the tooth root and the inferior alveolar canal in a radiographic image are not in a straight line , intimate care is taken during observation and to obtain a conclusion .
iopa and opg are the two conventional imaging techniques of choice for maxillofacial surgeons for thorough evaluation of the lower third molar position and its associated structures .
several radiographic markers in opg are identified for their reliability in predicting the risk of inferior alveolar nerve damage or exposure .
performance of the two conventional radiographic methods ( opg and iopa ) is high for identification of the intimate relation of the mandibular third molar and the inferior alveolar canal ; however , they are not accurate and do not predict the exact root apex and canal relationship in three dimensions . when the panoramic images show darkening of the roots and interruption of the white line , we should assume that there may be contact between the impacted mandibular third molar and the inferior alveolar canal in such a scenario cbct is considered .
rios - santos in their study on the assessment of periapical status using film - based iopa and digital panoramic images found that opg allows the assessment of the periapical status of 87.4% of teeth , including third molars .
digital panoramic images allow the assessment of the periapical status of only 58.0% of the teeth .
hassan suggested that there is a better correlation between cbct scans and iopa scores than between cbct scans and opg images scores .
this is due to the paralleling technique used for iopa where film holder and a beam indicating device guide the film and the long axis of the root to be parallel to each other , and the x - ray beam passes perpendicular to both root and film , but this does not occur in the case of opg .
however , in the present study opg was found to be more accurate in evaluating the type of impaction ( iopa vs. opg = 88%:94% ) and canal relationship to the apex of the root of the impacted molar ( iopa vs. opg = 74%:86% ) .
though in most cases panoramic was more accurate in determining the exact type of impaction and the canal relationship to the tip of the impacted molar , these values were not statistically significant on analysis .
the clinical examination after extraction of the impacted tooth included direct visualization of the canal in the extraction socket and grooving in the roots of the extracted tooth , if any .
however , in most cases this method of clinically determining the inferior alveolar canal relationship to the root tip may not be accurate and it needs further confirmation with sophisticated modalities such as cbct .
kositbowornchai et al . in their study on comparisons of various radiographic modalities for determining root tip and
canal relationship suggested that opg alone does not meet the need of determining the relationship between the inferior alveolar canal and the roots of lower third molar .
the visibility of number of roots is more obvious in an iopa compared to opg , and hence the panoramic radiographic images are inadequate to detect multiple roots of impacted third molars this result is acceptable with previous studies .
the major disadvantage of both iopa and opg is that they provide two - dimensional information of tooth and its periapical structures .
comparitively dental cbct is highly sensitive and is significantly superior to the so called conventional radiographs for predicting local anatomy around the impacted third molar .
it is an excellent method for localizing the canal , and its relationship to the lower third molar roots especially when there is interruption of white line and when superimposition is seen , because of an added advantage that reformatted images can be generated through the mandibular body in any plane with cbct .
in addition , the alveolar bone loss is better revealed in cone beam ct if the impacted mandibular third molar is periodontally involved cases than compared to iopa . to achieve atraumatic surgical extraction avoiding bizarre complications such as injury to inferior alveolar nerve causing paresthesia cbct will be more helpful than opg .
it is suggested to analyze opg more carefully and use cbct in high risk patients .
the identification of the presence of mandibular retromolar canal is obvious in cbct for extraction of mandibular third molar , whereas the roots appears intimately close to the mandibular canal on opg . in traditional radiographs
, the superimposition of anatomic structures and thickness of the roots may obscure many anatomical and pathological details .
cbct is capable of eliminating this effect and the details are so clear that external root resorption can be identified even in early stages . being expensive , limited availability , and increased radiation exposure makes dental cbct to be chosen very rarely for a preoperative radiographic evaluation of impacted mandibular third molars , making iopa and opg as a better choice for opting .
to conclude , though iopa has a marginal angle over opg in assessing various parameters [ table 1 ] , only the number of roots have a greater accuracy ( p < 0.0013 ) in iopa than opg .
however , the opg is the radiograph of choice when the patient is associated with trismus .
| aims and objectives : this study was conducted to compare the clinical assessment of impacted third molars of mandible with panaromic radiograph ( opg ) and intraoral periapical radiograph ( iopa ) and to assess the efficacy of iopa and .
moreover , we corroborated the opg and iopa findings of impacted mandiblar third molar root apex to inferior alveolar canal.materials and methods : a total of 200 patients with pericoronitis were examined who were indicated for surgical extraction , among which 50 patients were selected for the study .
all the patients underwent a radiographic survey with a digital opg and iopa of impacted mandibular third molars , along with clinical survey for anatomic relationship , type of impaction , space available , position in relation to second molar , number of roots , root curvature , and proximity of nerve canal .
the data was subjected to statistical analysis .
the statistical package for social sciences version 4.0.1 software was used for analyzing the collected data.results:the study revealed that iopa was more accurate in determining a majority of the factors affecting the third molar surgery , including relationship of the external oblique ridge ( iopa vs opg = 96%:90% ) , anteroposterior relation with ramus ( iopa vs opg = 70%:66% ) , vertical depth of impaction ( iopa vs opg = 72%:68% ) , number of roots ( p = 0.013 ) , morphology of roots ( iopa vs opg = 96%:90% ) ; however , opg was found to be accurate in evaluating the type of impaction ( iopa vs opg = 88%:94% ) , canal relation , along with root of impacted molar ( iopa vs opg = 74%:86%).conclusion : to conclude , although iopa has a marginal angle over opg in assessing various parameters , only the number of roots have a greater accuracy ( p < 0.0013 ) in iopa than with opg
. however , the opg is the better choice to be considered when the patient is associated with trismus . |
chronic instability of the major leg joints occurs frequently in athletes who play soccer ,
basketball , rugby , and other sports that involve repetitive joint movements1 , 2 .
the risk of injury appears to depend on the functional differences between the stable and
unstable sides in almost all movement patterns , including sudden stops or direction changes .
the knee and ankle joints are the major areas susceptible to injury in such conditions , and
functional damage to the leg is a frequent cause of pain .
instability of the knee joints leads to restricted movement within the range of motion
( rom ) , which then results in a cycle of repeated instability in the joints3 , 4 .
consequently , improper functional movements change the load applied to the legs5 and can lead to injury to the
musculoskeletal system of the legs .
ankle joint sprains are common among active people , and
acute sprains tend to repeatedly recur6,7,8
.
moreover , > 70% of individuals who experienced an ankle sprain also experience similar
symptoms such as additional and repeated instability from re - injury and functional
abnormalities9 .
even when not
considering structural instability , the development of functional instability indicates the
vulnerability of knee and ankle joints to injury during repetitive activity patterns10 and during active movements involving
sudden participation in exercise and high intensity exercise11 , which may present as chronic problems in elite athletes12,13,14 .
successful drop landing requires muscle strength , stability , and additional capabilities of
the major joints , which are important factors influencing protection against injury to the
joint facets15 , 16 .
moreover , jumping and landing , which occur frequently during
sporting events , can be soft or rigid , depending on the biomechanical energy loss17 , 18 , and can be a cause of injury along with instability19 , 20 .
as the force of impact of ground reaction force ( grf ) is greater
during rigid landing than during soft landing , the presence of any abnormality during
single - leg landing can be easily identified ; moreover , based on these findings , the causes
of such a decrease in major joints and muscles of the lower extremity can also be
determined21 .
accordingly , the instability of the major leg joints may manifest in various forms during
daily activities or participation in exercise12 ,
14 , 18 .
if individuals continue to participate in complex functional
activities without improving the instability , the instability can subsequently progress to
an abnormality and can consequently result in complex injuries .
the purpose of this study
was to assess the stability of functional movements in the major leg joints in order to
elucidate basic biomechanical data of the potential predictors of functional instability in
the knee and ankle joints of soccer players based on the results of single - leg drop
landing .
in this study , the subjects were 24 collegiate soccer players who had experienced injuries
related to knee and ankle joint instability but were currently able to participate in
matches without pain or dysfunction .
the mean age , height , and weight of the participants
were 21.3 1.4 years , 179.3 5.3 cm , and 75.0 6.3 kg , respectively .
all the subjects
understood the purpose of this study and provided written informed consent prior to
participants in the study in accordance with the ethical standards of the declaration of
helsinki .
eight - motion analysis cameras ( 2 raptor and 6 eagle cameras , motion analysis corp .
, snata
rosa , ca , usa ) were employed for recording biomechanical measurements of the knee and ankle
joints during single - leg drop landing . to ensure accurate testing ,
the participant was
positioned at the center of the location where the motion would be performed prior to
measurement , and the cameras were set up as follows ; 3 cameras each on the left and right
sides and 1 camera each on the front and back sides within 5 m of the participant ; the
reference coordinate points were adjusted with the height of the lens in order to completely
cover the entire rom . in addition , calibration was also performed establish spatial
coordinates .
the sampling rate of the shooting speed of the motion analysis cameras was set
at 120 frames / s , and the accuracy was set at within 0.3 mm .
moreover , for biomechanical
measurements , a force plate ( or-5 - 2000 , amti inc . , watertown , ma , usa ) was used , with a
sampling rate of 1,200 hz / s .
the motion analysis and grf data were synchronized via an
analog - digital converter ( ni usb-6218 , instruments hungray , debrecen , hungary ) . to calculate the variables of the human body , 19 reflective markers were attached to the
legs ( mid sacral , right and left asis ( anterior superior iliac spine ) , right and left thigh
and shank , right and left lateral epicondyle of the knee , right and left lateral malleolus
ankle joint , right and left toe and heel , right and left medial epicondyle of the knee , and
right and left medial malleolus ankle joint ) , and an anatomically static posture was
measured for approximately 3 s. thereafter , 4 markers attached to the inner sides of the
left and right knee and ankle joints were removed , and measurements were taken using a total
of 15 markers . for the measurements ,
a drop - landing movement was performed individually from
above a vertical plane that was approximately 30 cm based on previous studies2 , 22,23,24
the stable side was measured first , followed by the unstable side . to assess stability , a
motion involving the maintenance of balance for over 3 s after landing was used for the
analysis .
cortex 4 was used for data processing , whereas a linked rigid body system was used to
convert the center point of the body segments into coordinates .
the center of gravity and
center position of the body were calculated using body segment index parameter data .
planar
( two - dimensional ) data obtained from the 8 motion analysis cameras were converted to
three - dimensional data via the nonlinear transformation ( nlt ) method . to eliminate noise
errors during data processing , a cutoff frequency of 8 hz
was used for butterworth low - pass
digital filtering by cortex 4 . moreover , smoothing was performed on the grf data with a
cut - off frequency of 50 hz . in the present study ,
the global coordinates for the major joints in the leg were defined
as the x ( anteroposterior ) , y ( mediolateral ) , and z ( vertical ) axes . for biomechanical
analysis
, 3 events were used : the point of generating grf or initial foot contact on the
plate ( ic ) , the point of achieving maximum vertical value of the grf or maximum ground
reaction force ( mgrf ) , and the point of achieving maximum knee joint flexion or maximum knee
flexion ( mkf ) .
the knee and ankle joint movements during single - leg drop landing were
analyzed in the sagittal and coronal planes .
accordingly , the movements considered included
flexion ( + ) and extension ( ) of the knee joints and dorsiflexion ( + ) and plantar flexion
( ) of the ankle joints in the sagittal plane , as well as valgus ( + ) and varus ( ) movements
of the knee joints and inversion ( + ) and extraversion ( ) of the ankle joints in the coronal
plane .
all the measured data were calculated as means and standard deviations using ibm spss
statistics 20.0 ( ibm corp . , armonk , ny , usa ) . to compare the differences in the basic
biomechanical levels between the stable and unstable sides for single - leg drop - landing based
on the prior history of injury , the independent sample t - test was used , with p<0.05
indicating statistical significance .
the results of the comparison of angles of the knee and ankle joints in the sagittal and
coronal planes at the 3 events during single - leg drop landing , which could help predict leg
injuries , are as shown in table 1table 1.comparisons of the angles of the knee and ankle joint ( unit : deg)eventjointsagittal planefrontal planestableunstablestableunstableicknee13.65.013.45.55.83.12.33.5ankle31.76.334.67.210.86.316.97.7mgrfknee31.06.532.36.59.04.03.54.1ankle8.26.08.36.23.67.95.37.8mkfknee53.38.948.59.610.45.13.55.3ankle4.45.12.44.91.77.94.37.1values are shown as the meansd , * * * p<0.0001 .
ic : initial foot contact on plate ;
mgrf : maximum vertical ground reaction force ( grf ) ; mkf : maximum knee flexion .
the knee and ankle joint angles in the sagittal plane did not significantly
differ between the stable and unstable sides , whereas those in the coronal plane showed
significant differences at all 3 events . after dividing the grf during the vertical movement
at the 3 events by the body weight of the participant for standardization and comparison ,
there was no significant difference between the bilateral sides at any of the three events
( table 2table 2.comparisons of vertical grfs ( unit : bw)eventvertical grf ( bw)stableunstableic0.20.20.20.2mgrf3.10.73.10.4mkf1.70.51.60.4values are shown as the meansd .
grf : grounf reaction force ; ic : initial foot contact
on plate ; mgrf : maximum vertical grf ; mkf : maximum knee flexion ) .
ic : initial foot contact on plate ;
mgrf : maximum vertical ground reaction force ( grf ) ; mkf : maximum knee flexion values are shown as the meansd .
grf : grounf reaction force ; ic : initial foot contact
on plate ; mgrf : maximum vertical grf ; mkf : maximum knee flexion
the purpose of this study was to elucidate basic biomechanical data of the potential
predictors of instability causing leg injury based on the results of single - leg drop
landing . by comparing the knee and ankle joint angles at 3 events ( ic , mgrf , and mkf ) ,
the
knee joint movement on the stable and unstable sides in the sagittal plane showed gradual
flexion .
in particular , at the mkf , the mean angle of knee flexion on the stable side was
53.3 , which represented a difference of 4.8 from the value ( 48.5 ) on the unstable side .
such an increase in the knee flexion angle was consistent with the results from previous
studies , which indicated that such increases can reduce damage to the anterior cruciate
ligament ( acl ) by promoting a more stable landing motion and can have a positive influence
on ankle joint support25 , 26 .
in contrast , the ankle joints on the stable side showed
plantar flexion from ic to the mgrf , followed by dorsiflexion at mkf . moreover ,
the ankle
joints on the unstable side showed a similar angle and movement as the stable side at all 3
events , although the ankle joint on the unstable side showed approximately 2 of plantar
flexion at mkf relative to the stable side .
the increased rom during dorsiflexion of the
ankle joints holds the tibia at the front , when load is applied , to increase the stability
of the ankles , which is consistent with the findings in previous studies10 , 12 .
these findings support the observation of greater stability on the
stable side than on the unstable side . in the coronal plane ,
the stable side of the knee joint showed gradual varus movement from
ic to mkf , whereas the unstable side of the knee joint indicated a difference from the
stable side in terms of a tendency for valgus movement from ic to mkf . during landing , the
presence of a large valgus angle can induce damage to the acl and medial ligament .
hence ,
the tendency for valgus movement in the present study may be attributed to the restriction
of stable support .
the ankle joints on the stable side showed inversion from ic to the mgrf ,
followed by extraversion at mfk , whereas those on the unstable side showed extraversion to
inversion .
moreover , comparisons of the data between the unstable and stable sides indicated
significant differences .
it is speculated that such different between both sides may be due
to the restriction of the rom of inversion and the improved stability of the ankle
joints27 .
accordingly , the changes in the movements of the knee and ankle joints during drop landing
in the present study support the results of previous studies , wherein multiple lesions in
the longitudinal and lateral directions , along with pain , were reported to occur following
repeated motion that requires weight bearing in athletes and in active youths2 , 11 , 28 .
thus , a representative injury mechanism
of the knee joint reportedly occurs , primarily when the knee is flexed and in the valgus
state29 .
moreover , in order to absorb
the grf that is generated during landing , the ankle joint undergoes movement from plantar
flexion to dorsiflexion30 .
the knee is an
important joint for absorbing shock during drop landing , and is closely associated with the
grf31 . as represented by the change in
angles in the present study , an increase in the flexion and internal rotation of the knees
and ankles during drop landing would absorb the impact delivered to the body , thus
facilitating stable landing . based on the findings from previous studies and the current
analyses , no significant differences were observed in the 3 events , and the mkf on the
stable side appeared to be 0.1 bw lower than that on the unstable side .
these results are
consistent with those of a previous study32 , which reported that an increase in the size of the maximum
vertical grf increases the force delivered to each joint at contact with the ground and
hence easily exposes them to injury . therefore ,
as suggested in the report by kernozek et
al.33 , increasing the flexion angle of
the knee joints can be viewed as a strategy for preventing injury during landing . in studies on unstable knee and ankle joints involving landing after jumping , it has been
observed that some athletes frequently engage in jumping and landing movements in
competitive practices and competition10 , 15 . to achieve a more stable landing ,
an
unnecessary expression of muscle strength occurs , which may also serve as another factor of
instability6 , 14 .
in fact , chronic instability can be caused by various factors and
can progress to abnormalities when other functional activities are performed12 , 14 .
moreover , the entire body , not just the joints , is involved in
absorbing the impact during landing ; hence , future studies that explain the correlations
with even more joint functions are needed .
strategic rehabilitation programs based on
information obtained through such fundamental studies are needed to improve instability . | [ purpose ] the purpose of this study was to determine potential predictors of functional
instability of the knee and ankle joints during single - leg drop landing based on the prior
history of injury .
[ subjects and methods ] the subjects were 24 collegiate soccer players
without pain or dysfunction . to compare the differences between the stable and unstable
sides during single - leg drop landing , 8 motion analysis cameras and a force plate were
used .
the cortex 4 software was used for a biomechanical analysis of 3 events .
an
independent t - test was used for statistical comparison between both sides ; p<0.05
indicated significance .
[ results ] the knee joint movements showed gradual flexion in the
sagittal plane .
the unstable - side ankle joint showed plantar flexion of approximately 2
relative to the stable side . in the coronal plane ,
the unstable - side knee joint differed
from the stable side in its tendency for valgus movement .
the unstable - side ankle joint
showed contrasting movement compared with the stable side , and the difference was
significant . regarding the vertical ground reaction force ,
the stable side showed maximum
knee flexion that was approximately 0.1 bw lower than that of the unstable side .
[ conclusion ] increasing the flexion angle of the knee joint can help prevent injury during
landing . |
chronic pelvic pain ( cpp ) in women is commonly described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or to lead to medical care.1 - 2 cpp may originate from one or more organ systems or pathologies and may have multiple contributing factors.3 the prevalence may vary from country to country . in primary care ,
the prevalence of cpp is found to be comparable to that of asthma and back pain , with values of 3.7% , 3.8% , and 4.1% , respectively.4 in a us study , the prevalence among women of reproductive age reached 24%,4 - 8 whereas in new zealand , grace , and zondervan identified a prevalence of 25.4% when considering a three - month duration of symptoms.9 this elevated prevalence was confirmed by latthe et al . in a recent review of this condition.10 the direct and indirect costs of this condition amount to over two billion dollars per year , and the condition is responsible for 10% of all gynecologic visits , 40 to 50% of all gynecologic laparoscopies and 12% of all hysterectomies.5 cpp has a direct impact on marital status as well as on the social and professional lives of women , and it thus constitutes an important public health problem . in brazil ,
international studies have demonstrated a high prevalence of persistent pain in women from brazil , including approximately 36% in rio de janeiro.11 according to the health ministry of brazil , in 1997 there were 1.8 million gynecologic visits and approximately 300,000 hospital admissions of women aged 15 to 69 with complaints associated with cpp.12 in a survey of diseases and/or health problems among working women in so paulo , gomes & tanaka13 reported that 13% of them mentioned abdominal and pelvic pain among their major complaints .
recently , some studies have concluded that drug or alcohol abuse , abortions , inflammatory pelvic disease , cesarean sections , and psychological diseases are associated with cpp.14 the etiology of cpp in women is not clear , and cpp usually involves a complex interaction between the gastrointestinal , urinary , gynecologic , musculoskeletal , and neurologic and endocrine systems .
it is also influenced by psychological and sociocultural factors.6 several other factors may be associated with the condition , including ( 1 ) neuroplastic changes occurring in the posterior horn of the spinal cord as a consequence of electrophysiological , biochemical , and metabolic changes promoted by the initial noxious stimulus , ( 2 ) cross - sensitivity between viscera that share the same innervation , and ( 3 ) development of a visceromuscular reflex that may culminate not only in dysfunctional repercussions but also in the development of myofascial syndrome and the generation of new pain points.15 - 16 although there is a consensus that cpp has a high prevalence among women , there are a limited number of studies in our country , which can be attributed to the lack of a precise diagnosis , the heterogeneity of the group of diseases that result in cpp , and the variation of primary diseases in different populations.17 considering that knowledge about the magnitude and the demographic and socioeconomic characteristics of cpp contributes to its prevention , control , and treatment , the aim of the present study was to estimate the community prevalence of cpp in women living in ribeiro preto ( a southeastern city in the state of so paulo , brazil with a population of about 700,000 people ) , as well as to identify independent factors associated with it .
the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) .
women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp .
the target population consisted of working - class women treated by the public health system .
a total of 1,306 women over 14 years of age were contacted , but 38 of them refused to participate .
the women who were contacted were selected randomly from the population using their residential address . all subjects ( or those responsible for the subjects ) gave written informed consent to participate in the study .
we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions .
the variables investigated were age ; body mass index ( bmi ) ; number of pregnancies ; parity ; having been subjected to episiotomy , to forceps delivery , or to cesarean - section delivery ; abortions ; abdominal surgery ; umbilical and laparoscopy incisions ; oblique , longitudinal . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ;
per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief . because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification .
interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study .
the data collected by the interviewers were entered during the interview and sent to an electronic database .
were evaluated by experts , and their diagnosis was confirmed before inclusion in the database .
five percent of the subjects were selected at random and re - interviewed to check data consistency .
cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . for this study , we defined cpp as having at least one weekly episode with an intensity higher than 3 cm on a 10-cm visual analog scale .
women who had been pregnant in the previous 12 months were excluded from the analyses .
the intensity of dysmenorrhea was classified according to its effect on the ability to work , the coexistence of systemic symptoms , and the need to use any kind of analgesic medication .
classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . for analysis , we considered only moderate and intense dysmenorrhea to be positive .
dyspareunia was defined as pelvic pain during sexual intercourse or during a period of 24 hours after sexual intercourse .
the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . for analysis
the clinical measurement of pain severity was made using a 10-cm visual analog pain scale ranging from least possible pain ' to worst possible pain ' .
the dagostino and pearson test was used to determine whether data followed a gaussian distribution .
data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) .
data were analyzed using the fisher exact test or chi - square test ( qualitative variable ) and the unpaired t test after confirmation of normal distribution ( quantitative variables ) .
we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case .
logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp .
the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 .
the study was approved by the research ethics committee of the university hospital , faculty of medicine of ribeiro preto , university of so paulo ( hcfmrp - usp ) .
women were recruited from the western district of ribeiro preto between april 2008 and march 2009 for two particular reasons : first , the region presents the same social and demographic patterns as those of the major parts of the city ( socioeconomic level , distribution of age , sex , and ethnicity ) , and second , the university hospital provides medical evaluation and treatment for women with a diagnosis of cpp .
the target population consisted of working - class women treated by the public health system .
a total of 1,306 women over 14 years of age were contacted , but 38 of them refused to participate .
the women who were contacted were selected randomly from the population using their residential address . all subjects ( or those responsible for the subjects ) gave written informed consent to participate in the study .
we chose a questionnaire as the instrument for data collection because it could be applied to all segments of our population , which is very heterogeneous , consisting of both illiterate and literate people , and also because it allowed us to explain the objectives of the research and to answer any questions .
the variables investigated were age ; body mass index ( bmi ) ; number of pregnancies ; parity ; having been subjected to episiotomy , to forceps delivery , or to cesarean - section delivery ; abortions ; abdominal surgery ; umbilical and laparoscopy incisions ; oblique , longitudinal . and transverse incisions ; perineal surgery ; sedentary lifestyle ( women who engaged in activities such as running , walking , pedaling , dancing , or other sport activity for at least three hours a week were considered active ) ; previous sexual activity ; current sexual activity ; contraception ; sexual desire ; lubrication ; orgasm ;
per capita income ; employment status ; dysmenorrhea ; dyspareunia ; having been a victim of violence ( physical and sexual ) ; stable marital relationship ; educational level ( schooling was stratified as low when women had completed the eighth grade in primary school ) ; mood ( depression or anxiety based on previous medical diagnosis or criteria for the detection and diagnosis of psychiatric disorders in primary medical care settings)18 and comorbid health conditions ( e.g. , migraine and low back pain ) ; constipation ( roma iii criteria);19 urinary symptoms ( bladder irritation : increased voiding episodes per day , urgency , pain ) ; nocturia ; alcoholism ( excessive intake during the weekend with frequent drunkenness and/or daily consumption with or without drunkenness ) ; smoking ( current or former daily smoker ) ; excessive coffee intake ( more than 6 cups a day ) ; illicit drug use ; menstrual status ; pain intensity ( visual analogue scale ) ; pain duration ( in months ) and pain frequency ( at least once a week ) ; and self - medication for pain relief .
because our population is racially diverse , it is difficult to determine the ethnicity accurately , and therefore , we did not perform an ethnic analysis because of the high probability of misclassification .
interviewers who were not linked to the city health care programs were trained and selected by the researcher responsible for the study .
the data collected by the interviewers were entered during the interview and sent to an electronic database .
were evaluated by experts , and their diagnosis was confirmed before inclusion in the database .
five percent of the subjects were selected at random and re - interviewed to check data consistency .
cpp in women has been described as continuous or intermittent pain in the anatomic pelvis ( anterior abdominal wall at or below the umbilicus ) that lasts for at least six months , is not exclusively related to menstruation or sexual intercourse , and is sufficiently severe to cause functional disability or lead to medical care . for this study , we defined cpp as having at least one weekly episode with an intensity higher than 3 cm on a 10-cm visual analog scale .
women who had been pregnant in the previous 12 months were excluded from the analyses .
the intensity of dysmenorrhea was classified according to its effect on the ability to work , the coexistence of systemic symptoms , and the need to use any kind of analgesic medication .
classifications included absence ( absence of pain during the periods ) ; mild ( occasional pelvic discomfort that does not impair daily activity , occasional need for medication ) , moderate ( pain lasting almost throughout the menstrual period that impairs daily activity and is responsive to the use of medication ) , and intense ( pain lasting throughout the menstrual period , with significant limitation of daily activity , frequent use of potent analgesics , and without effective improvement ) . for analysis
dyspareunia was defined as pelvic pain during sexual intercourse or during a period of 24 hours after sexual intercourse .
the intensity of dyspareunia was classified according to the disruption of sexual activity during intercourse , as follows : absence ( absence of pain during sexual contact ) , mild ( tolerable pain , does not lead to the interruption of sexual contact ) , moderate ( intense pain sufficient to lead to the interruption of sexual contact ) , and intense ( pain that hinders sexual contact ) . for analysis
the clinical measurement of pain severity was made using a 10-cm visual analog pain scale ranging from least possible pain ' to worst possible pain ' .
the dagostino and pearson test was used to determine whether data followed a gaussian distribution .
data with and without a normal distribution were represented respectively by the mean ( standard deviation ) and median ( range ) .
data were analyzed using the fisher exact test or chi - square test ( qualitative variable ) and the unpaired t test after confirmation of normal distribution ( quantitative variables ) .
we first selected only the significant variables identified ( p<.10 ) and then assigned values of 0 or 1 to the absence or presence of these variables in each case .
logistic regression was used to identify the significant independent variables and to estimate the simultaneous impact of these factors on the assessment of cpp .
the results were expressed as the odds ratio ( or ) and 95% confidence interval ( 95% ci ) , with the level of significance set at p<.05 .
the prevalence of cpp was 11.5% ( 147/1,278 ) . considering only women of reproductive age , the prevalence was 15.1% ( 127/841 ) .
82.4% of healthy women ( 932/1,131 ) and 90.5% of women with cpp ( 133/147 ) stated that they make regular clinic visits at basic health centers ( at least two appointments per year ) .
only 4.1% of women with the disease , however , knew their diagnosis before the interview ( n = 6/147 ) .
the average duration of the pain was 51.861.2 [ 6 - 360 ] months : 6 - 12 months in 34.0% of the women ( 50/147 ) , 13 - 36 months in 28.6% ( 42/147 ) , and over 36 months in 37.4% ( 55/147 ) .
the mean intensity of pain obtained with a visual analogue scale ( vas ) was 58.523.4 mm .
the intensity was 30 - 50 mm in 52.4% of the women ( 77/147 ) , 51 - 70 mm in 17.7% ( 26/147 ) , and 71 - 100 mm in 29.9% ( 44/147 ) . in total
, 44.9% of the women reported a spontaneous onset of pain ( 66/147 ) ; in 3.4% ( 5/147 ) , the pain was related to food intake ; in 8.5% ( 9/106 excluding those who were not sexually active ) , the pain was related to intercourse ; in 21.8% ( 32/147 ) , the pain was related to physical activity ; in 30.6% ( 30/98 excluding those who were menopausal ) , the pain was related to menstruation ; in 2.0% ( 2/98 ) , the pain was related to ovulation ; in 0.7% ( 1/147 ) , the pain was related to stress ; and in 1.4% , it was related to other factors ( 2/147 ) .
the frequency of self - medication was 1.4% ( 16/1131 ) and 24.5% ( 111/147 ) among healthy and cpp women , respectively .
the common occupations were maidservant , hairdresser , and seamstress , with no possibility of grouping the women according to their occupations .
a univariate analysis of the variables investigated is presented in table 2 . in the logistic regression ,
the factors independently associated with cpp were dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level .
in the present study , we detected a one - year prevalence of 11.5% for cpp among women from ribeiro preto and a prevalence of 15.1% in women of reproductive age .
to our knowledge , this is the first time that a high prevalence of this disease has been reported in brazil .
the presence of chronic pain was found to be 48.4% in women from salvador , brazil.20 although that study described pain at various sites , it made no reference to abdominal or pelvic pain .
a recent review published by latthe et al.10 has shown that the worldwide prevalence of this condition ranges from 2% to 24% , which places brazil among the countries with a higher prevalence of cpp .
zondervan et al . , for example , observed a community prevalence of 24.0% in women between 18 and 49 years of age.7 in that study , the questionnaire response rate was 74% .
a higher response rate is natural among women with the disease , which may explain the difference in prevalence compared with our study , which had a response rate of 97.9% ( 1,278/1,306 ) .
we believe that our data are representative of the entire ribeiro preto community because the social - demographic indicators of the covered area are similar to those of the general population of the city ( socioeconomic level , age distribution , sex , and ethnicity ) .
a notable result was that only 4% of the women with cpp stated that they had previously received a specific diagnosis , even though 90% of them attended a basic health center .
three different factors may have contributed to this finding : whether the physician is able to provide the correct diagnosis ; whether the relationship between the patient and the physician enables effective communication and explanation of the diagnosis ( which other studies have commented on21 ) ; and whether the women complain about their clinical conditions to physicians .
studies have shown that gender is an important determining factor of pain complaints in women and might be the reason for diagnosis underestimation at primary care centers.22 the frequency of self - medication was also a notable finding .
approximately one quarter of the women used some kind of medication ( especially analgesics and anti - inflammatory drugs ) for the relief of their symptoms in an indiscriminate way .
this self - medication predisposes them to side effects and requires financial expenditures that do not guarantee clinical improvement , especially over the long term.23 we believe that this percentage may be underestimated because people responding to an interviewer may not want to report the use of their pain medication .
additionally , more than one third of the women had symptoms consistent with a diagnosis of cpp for over three years , and the same proportion had symptoms that are considered severe ( vas>70 mm ) .
although we have no data to support this speculation , it is plausible that the high prevalence of cpp is associated with high rates of absenteeism ( about half of these women have paid employment outside the home ) , which has a direct impact on social and economic life .
we identified the following as factors independently associated with chronic pelvic pain : dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level .
dyspareunia is an important element of sexual dysfunction that ranges in prevalence from 7% to 75% depending on the diagnostic criteria and the associated clinical conditions , such as cpp.24 - 29 in a recent study , we observed that dyspareunia was associated with pelvic muscle tenderness.30 although pelvic muscle tenderness may be a primary cause of cpp,31 we hypothesize that it is more often secondary to cross - talk communication between the viscera and muscles32 through neurogenic inflammation caused by the release of inflammatory mediators at the periphery in response to the antidromic stimulus over time.33 similarly , current sexual activity may be linked in some way to dyspareunia , although this relationship is still unclear .
another hypothesis is that women with dysmenorrhea have a lower pain threshold , which may favor the onset of illness.34 moreover , mechanisms of viscero - visceral hyperalgesia between organs probably involve the sensitization of viscero - viscero - somatic convergent neurons.35 the most notable factor observed in the present study is the association of cpp with abdominal surgery , particularly because we observed that two - thirds of the women studied had undergone a previous abdominal surgery , and more than 40% of the women had previously undergone a cesarean section surgery . considering the high rates of cesarean section surgery in our country36 - 37 ( including this sample of our community ) , and the previous detection of an association with cpp by our group38 and by latthe et al . in a recent systematic review,14
we stress the need for detailed studies regarding this possible causal relationship between cpp and cesarean section .
some studies suggest the possibility of an association between cpp and adhesions.39 however , the correlation between pelvic pain and adhesions is uncertain because adhesiolysis has not been shown to be effective in achieving pain control.40 thus , we emphasize the importance of recognizing abdominal myofascial syndrome as a differential diagnosis.41 anxiety and depression disorders are frequently concomitant with chronic pain , particularly in women , in both developed and developing countries.42 - 43 we have also observed a direct relationship between depression , anxiety , chronic pelvic pain , and quality of life.44 in addition to being a risk factor for cpp , mood disorders may make it more difficult for a women to engage in cognitively or emotionally demanding rehabilitation.45 because the individual experience of pain is personal and subjective , it is probably affected by emotional states and , therefore , by psychosocial factors .
there is some evidence that it is the stress of living with chronic pain , and not personal or family predisposition , that causes depression in these patients.46
the idea that pain , particularly chronic pain , can lead to feelings of frustration , worry , anxiety , and depression seems obvious .
there is also evidence , however , for reverse causality , in which negative moods and emotions can lead to or exacerbate pain.47 it therefore remains uncertain whether depression / anxiety precedes or is a consequence of chronic pain.48 low back pain is usually comorbid with cpp .
this association probably reflects the same pathophysiological mechanisms,49 although each can reduce the thresholds and thus contribute to the development of the other .
painful bladder syndrome and constipation are nongynecologic conditions that may cause or exacerbate cpp ( level of evidence a).2 the prevalence of constipation identified in our sample of healthy women ( 22.7% ) was similar to that observed by oliveira et al.50 because the women reported that constipation was present before cpp , we may infer that this condition may at least contribute to the development of cpp . though this was beyond the scope of our study ,
some studies indicate that constipation may be secondary to the dysfunction of the levator ani muscle,51 - 52 which is a common condition in cpp .
several studies in the literature have shown that a lower educational level is linked to a higher prevalence of chronic pain.20,53 because cpp increases with age and most education takes place in early life , it is likely that a lower educational level increase the risk for the development of cpp , or that both are the consequences of some other undescribed factors .
we have not , however , reached final conclusions about the causal relationship between these factors and cpp . a thorough investigation and other different types of studies are necessary to corroborate our results .
we conclude that the prevalence of cpp in women from ribeiro preto is very high , even though only 4% sufferers are aware of their diagnosis .
this confirmation is crucial for the prevention , early diagnosis , control , and resolution of cpp .
the authors are grateful to cnpq ( conselho nacional de desenvolvimento cientifico e tecnologico ) for financial support . | introduction : chronic pelvic pain is a disease that directly affects the social and professional lives of women.objective:to estimate the prevalence of this clinical condition and to identify independent factors associated with it in women living in ribeiro preto , brazil.methods:a one - year cross - sectional study was conducted in a population sample of 1,278 women over the age of 14 years .
the target population was predominantly composed of women who are treated by the public health system .
the questionnaire was administered by interviewers who were not linked to the city health care programs .
the prevalence of the morbidity was estimated .
first , we identified the significant variables associated with pelvic pain ( p<0.10 ) and then we attributed values of 0 or 1 to the absence or presence of these variables .
logistic regression analysis was used to identify and estimate the simultaneous impact of the independent variables .
the results were expressed by odds ratio and their 95% confidence interval with p<0.05.results : the disease was found in 11.5% ( 147/1,278 ) of the sample .
the independent predictors were dyspareunia , previous abdominal surgery , depression , dysmenorrhea , anxiety , current sexual activity , low back pain , constipation , urinary symptoms , and low educational level.conclusion:the prevalence of chronic pelvic pain in ribeiro preto is high and is associated with conditions that can usually be prevented , controlled , or resolved by improvement of public health policies and public education . |
chronic intraventricular encapsulated hematoma is a rare entity and the exact mechanisms of capsule formation , progressive expansion and resolution have remained uncertain . here
damage to any of these vascular or neural elements from direct compression , ischemia or invasion can result in a multitude of signs and symptoms .
these include cranial nerve deficits , features of elevated intracranial pressure from hydrocephalus , changes in higher cognitive function or even autonomic dysregulation .
computed tomography ( ct ) and magnetic resonance imaging ( mri ) are excellent diagnostic tools and are essential in surgical planning .
since a neoplasm could not be ruled out preoperatively , the lesion was totally removed by right frontal craniotomy with a transcallosal approach in order to eliminate the mass effect .
the general medical condition of the patient is another important consideration in defining surgical strategy . after an extensive review of the literature
a 30-year - old male was admitted to our hospital in october 2008 with a 4-day history of fever , photophobia , diplopia and cervical pain . on admission ,
the brain ct scan showed a round - shaped homogenous mass in the lateral ventricle with a peripheral zone of high density , mild perifocal edema and a ring enhancement with mass effect .
brain mri showed an extra - axial round - shaped lesion in the middle line , arising from the roof of the third ventricle with definite edges and with a significant mass effect .
1 ) . after contrast injection , ring enhancement of the lesion could be observed .
a yellowish mass was found with glistering appearance and a hard wall that was difficult to be operated without micro - knifes and ultrasonic aspirator .
the mass was filled with engine oil - like fluid and removed totally without difficulties because of the plane with the surrounding tissue .
histological examination revealed a hematoma which was consistent with early development of granulation tissue , neovascularization , accumulation of fibroblasts , histiocytes and hemosiderin deposits ( fig .
chronic encapsulated intracerebral hematoma ( ceih ) is a rare lesion which is believed to be caused by angiographically occult vascular malformations ( aovm ) such as arteriovenous malformation ( avm ) , venous angiomas , cavernous malformations , capillary telangiectasia , mixed or unclassified angiomas [ 2 , 3 ] .
mri can be used to demonstrate that a lesion is only a hematoma and is valuable in follow - up and in differentiating these hematomas from neoplasms .
differential diagnosis by location includes astrocytoma , colloid cyst , craniopharyngioma , teratoma , choroid plexus papilloma , cysticercosis , dermoid , choroids plexus carcinoma and avm . in our case , | this case report is a presentation of a chronic intraventricular encapsulated hematoma in an adult who presented with gait disturbance , fever , palsy of n. iii , n. vi and n. vii , and photophobia .
ophthalmoscopy revealed a bilateral papilledema . computed tomography scans and magnetic resonance imaging revealed an intraventricular mass in the lateral ventricle .
preoperative diagnosis could not rule out a neoplasm .
the lesion was totally removed with open surgery and was found to be a chronic intraventricular encapsulated hematoma .
a chronic intraventricular encapsulated hematoma is a rare entity .
after the removal of the mass , the clinical symptoms of the patient gradually resolved . |
vignon and colleagues prospectively assessed the ability of transesophageal echocardiography ( tee ) to predict paop higher than 18 mmhg in mechanically ventilated patients with an inserted pulmonary artery catheter . in a first group , they analyzed simple doppler variables derived from transmitral flow ( tmf ) and pulmonary venous flow ( pvf ) and performed the usual measurements and calculations ( maximal velocity and velocity time integral of e ( the maximal velocity of early diastolic tmf ) and a ( the maximal velocity of late diastolic tmf ) wave , e / a ratio , e wave deceleration time ( edt ) , maximal velocity and velocity time integral of s ( the maximal systolic pvf velocity ) and d ( the maximal diastolic pvf velocity ) wave , s / d ratio , atrial filling fraction and systolic fraction of pulmonary venous flow ( sfpvf ) ) .
the correlations between doppler variables and pulmonary artery occlusion pressure ( paop ) were better in patients with depressed left ventricular ( lv ) systolic function than in those with normal lv systolic function .
paop could be predicted by e / a > 1.4 , edt > 100 ms , atrial filling fraction > 31% and sfpvf > 44% , with similar sensitivity and specificity and acceptable positive and negative predictive values . in a second group these cutoff values
were prospectively evaluated for prediction of paop higher than 18 mmhg . additionally , they measured maximal early diastolic velocity of lateral mitral annulus by tissue doppler ( ea ) and color m - mode doppler flow propagation velocity ( vp ) . an e / ea ratio < 8 and an e / vp ratio < 1.7 were predictive for paop > 18 mmhg , but the use of these additional variables did not improve the correct estimation of paop .
elevated paop reflects an increase of lv end - diastolic pressure due to lv diastolic and/or systolic dysfunction / failure .
paop less than 18 mmhg , if measured , supports criteria for the definition of acute respiratory distress syndrome and acute lung injury .
clinical and radiological estimation of paop is uncertain in cardiac patients and almost impossible in intensive care unit patients [ 2 - 5 ] .
paop measurement by pulmonary artery catheter is , for various reasons , not commonly used in cardiac failure and critically ill patients . on the other hand , tee and transthoracic echocardiography ( tte )
are increasingly used for diagnostic and hemodynamic assessment and in critically ill patients , allowing noninvasive estimation of paop by doppler technique .
the first group includes relatively simple variables ( e , a , e / a , edt , sfpvf ) derived from analysis of diastolic tmf and pvf .
the second group includes ea and vp ; both variables are preload independent and are used to correct the e velocity for relaxation changes ( e / ea and e / vp ratio ) .
use of tte was limited because of inadequate visibility ; many patients had to be excluded because of inadequate doppler signal recordings .
technical improvements and the use of harmonic imaging now allow measurement of tmf and pvf in the majority of patients , but tee is still frequently used , especially in mechanically ventilated critically ill patients .
tmf and pvf variables measured by tte are accurate for the estimation of lv filling pressure and cardiac index in patients with depressed cardiac function and heart failure , but in patients with normal systolic lv function tissue doppler derived variables show better correlation with paop [ 9 - 11 ] . in patients who have undergone cardiac surgery and in critically ill patients ,
tee - derived sfpvf and e / ea correlate well with left atrial pressure and paop [ 12 - 14 ] .
the study by vignon and coworkers shows that in patients with acute lung injury , simple doppler variables derived from tmf and pvf by tee predicted elevated paop better than atrial filling fraction and edt and that the use of additional and more advanced variables ( ea and vp ) did not improve the accuracy of prediction . an important practical limitation of the study is the fact that 20% of patients could not be studied because of cardiac problems , and that in a further 10% of patients , some variables could not be recorded . concerning the study , the following questions should be considered .
despite the fact that paop is not transmural pressure and does not accurately reflect preload and volume responsiveness , it is still used as a supportive criterion for the diagnosis of acute respiratory distress syndrome and heart failure .
noninvasive estimation of paop is feasible by using tte / tee - derived simple doppler variables , but not in every patient . despite technological improvements in past years
, adequate doppler tracing can not be obtained by tte in many critically ill patients .
. it would be interesting to compare tte and tee simultaneously for paop estimation in a large group of critically ill patients . besides this , in a certain subset of patients , noninvasive estimation of paop
is not possible and invasive measurement of paop , if needed , is still necessary .
taking into account that tte or tee should be performed in the majority of intensive care unit patients for initial hemo - dynamic assessment , the systematic estimation of paop by simple analysis of tmf and pvf would undoubtedly increase the overall quality of this .
the use of additional variables ( ea , vp ) , which are routinely not measured in the intensive care unit setting , is not necessary for paop estimation in patients with impaired global systolic lv function , but can improve its estimation in patients with normal systolic function and diastolic dysfunction / failure .
a = maximal velocity of late diastolic tmf ; d = maximal diastolic pvf velocity ; e = maximal velocity of early diastolic tmf ; ea = tissue doppler diastolic velocity of mitral annulus ; edt = e wave deceleration time ; lv = left ventricular ; paop = pulmonary artery occlusion pressure ; pvf = pulmonary venous flow ; s = maximal systolic pvf velocity ; sfpvf = systolic fraction of pvf ; tee = transesophageal echocardiography ; tmf = transmitral flow ; tte = transthoracic echocardoigraphy ; vp = color m - mode doppler flow propagation velocity .
| the measurement of pulmonary artery occlusion pressure ( paop ) is important for estimation of left ventricular filling pressure and for distinction between cardiac and non - cardiac etiology of pulmonary edema .
clinical assessment of paop , which relies on physical signs of pulmonary congestion , is uncertain .
reliable paop measurement can be performed by pulmonary artery catheter , but it is possible also by the use of echocardiography .
several doppler variables show acceptable correlation with paop and can be used for its estimation in cardiac and critically ill patients .
noninvasive paop estimation should probably become an integral part of transthoracic and transesophageal echocardiographic evaluation in critically ill patients .
however , the limitations of both methods should be taken into consideration , and in specific patients invasive paop measurement is still unavoidable , if the exact value of paop is needed . |
the proportions of seniors are steadily increasing . on july 1 , 2015 , approximately one in six canadians ( 16.1% or 5,780,900 persons ) was at least 65 years old , and according to population projections , the number of persons aged 65 years and older is expected to continually increase and account for 20.1% of the population by july 1 , 2024 .
however , seniors over the age of 65 are likely to have one or more chronic illness , which can compromise their prospect of independence and indicate the need for health - care professionals ( hcps ) capable of addressing these needs in a variety of care settings . the largest proportion of hcps providing care and services to seniors are unregulated staff ( a.k.a .
nursing aides or assistants [ nas ] , resident care attendants or personal support workers ) .
these hcps are faced with the rising challenge of caring for an increasingly complex group of seniors with diverse biomedical and psychosocial needs . yet , gerontological education is not a priority in most health - care programs .
the integration of gerontological courses into health - care programs is scarce and , although seniors have more contacts with hcps , there is a lack of hcps with geriatric expertise .
furthermore , competencies offered in health - care programs often lack clearly expressed standards for gerontology practice .
present gaps in hcp competencies hinder the necessary efforts to build and attract a well - equipped workforce in seniors care , resulting in recruitment and retention challenges .
thus , there is a need for improved and innovative programs to accommodate the demand for gerontological expertise among health - care students .
this manuscript presents the living classroom ( lc ) , an innovative pedagogical approach , collaboratively created by conestoga college and schlegel villages , a continuum of seniors care in ontario , canada .
the lc presents an inter - professional ( ip ) learning approach whereby a post - secondary educator ( pse ) program is delivered within the context of a nursing home ( nh ) , with team members consisting of faculty , students , nh teams , residents , and families who engage with each other within a culture of interactive learning . in one ontario college , the na program provides students with a thorough understanding of person - centred are delivery in a variety of settings .
students progress from novice engagement to a professional level so that , upon graduation , they are competent and confident to participate in care as directed .
na students receive theoretical learning ( lectures ) , as well as applied and experiential learning ( skills lab , simulated environments , and clinical placements ) .
the transfer of knowledge from a theoretical concept to an applied skill or behaviour is often difficult for students , but essential if one wants to successfully graduate and practise .
this knowledge transfer is particularly challenging in gerontology , as seniors often present with complex care needs requiring comprehensive care .
baumbusch and andrusyszyn state that students must first acquire gerontological knowledge in the classroom , so that they may then develop skills specific to the care for seniors
. however , of equal importance is the clinical aspect of learning , as it has been found that students are more likely to apply their theoretical learning to care of seniors if they work with clinical experts in the field .
it is clear that both didactic and clinical components of learning have their place in gerontological education . when these learnings occur independently , disconnects happen between education and practice .
for example , na students are taught to transfer a senior from a bed to a chair in a theoretical manner , followed by practice sessions with fellow students . once students can perform this skill adequately ,
learning is moved to a practice setting , in which students are expected to integrate theoretical learning into a personal care encounter .
this situation is very different from the theoretical or lab learning setting , as the student is now expected to help a senior with a transfer , yet the senior presents with real mobility impairments and potential other frailties .
transfer , but also maintain a person - centred conversation , observe mobility and other capabilities , and determine and adjust the care plan based on these observations .
it is not possible to fully develop these essential skills in a classroom or even a controlled lab setting with healthy participants ; this independent learning leads to a theory - practice divide .
the council of ontario universities has termed this disconnect of didactic and clinical education as a crisis in education of hcps . in order to maximize the knowledge transfer ,
both didactic and clinical components need to be integrated into an immersive form of learning as opposed to keeping these as separate forms of knowledge acquisition . in turn , this integrated learning enhances the development of a strong workforce and drives quality care .
successful gerontological learning in actual care settings has been described by several authors . in the 1980s , a model for the teaching nursing home ( tnh ) was developed and found to be effective in improving care for nh residents by supporting nursing students clinical education in that setting .
( the researchers described a partnership model developed between a nursing college and four nhs .
positive outcomes included increased professionalism for the nh team , identification of areas for growth within the nhs , increased awareness of how the nh team s practice influenced new nurses , and increased interest among students in choosing a nh environment as a career .
wieland and colleagues described an academic nh program designed to improve care of residents , provide ip training for a medical team and allied health students , and stimulate research .
students were paired with an expert within their respective field to integrate what they learned in the classroom .
benefits of the program included significant decreases in resident transfers to acute care hospitals , as well as noteworthy improvements in residents functional status , satisfaction , and morale .
lastly , mezey and lynaugh created a five - year tnh program to improve quality of nh care , create a supportive environment for undergraduate and graduate nursing students and care teams , and promote clinical research .
funding ended in 1987 ; by then the program included 12 nhs and 11 university schools of nursing .
the program resulted in 1.5 to 2 new full - time positions in each nh primarily nurse practitioners and clinicians specializing in geriatrics and these positions focused on education , quality assurance and infection control .
the researchers analysis of the tnh program revealed many benefits : resident hospitalization dropped 7% in the tnh while rising 5% in homes not linked to schools , and overall quality of care increased .
furthermore , the program cost was neutral ; the cost of hiring of practitioners / clinicians was offset by reduced resident hospitalization .
although shown to be effective , the idea of the tnh faded in the 1990s because of a lack of funding .
the concept has recently re - emerged in canada as a viable approach , with a stronger emphasis on creating work - integrated learning opportunities for students to improve recruitment and retention of graduates in geriatric care settings .
in one ontario college , the na program provides students with a thorough understanding of person - centred are delivery in a variety of settings .
students progress from novice engagement to a professional level so that , upon graduation , they are competent and confident to participate in care as directed . na students receive theoretical learning ( lectures ) , as well as applied and experiential learning ( skills lab , simulated environments , and clinical placements ) .
the transfer of knowledge from a theoretical concept to an applied skill or behaviour is often difficult for students , but essential if one wants to successfully graduate and practise .
this knowledge transfer is particularly challenging in gerontology , as seniors often present with complex care needs requiring comprehensive care .
baumbusch and andrusyszyn state that students must first acquire gerontological knowledge in the classroom , so that they may then develop skills specific to the care for seniors . however , of equal importance is the clinical aspect of learning , as it has been found that students are more likely to apply their theoretical learning to care of seniors if they work with clinical experts in the field .
it is clear that both didactic and clinical components of learning have their place in gerontological education . when these learnings occur independently , disconnects happen between education and practice .
for example , na students are taught to transfer a senior from a bed to a chair in a theoretical manner , followed by practice sessions with fellow students .
once students can perform this skill adequately , learning is moved to a practice setting , in which students are expected to integrate theoretical learning into a personal care encounter .
this situation is very different from the theoretical or lab learning setting , as the student is now expected to help a senior with a transfer , yet the senior presents with real mobility impairments and potential other frailties .
transfer , but also maintain a person - centred conversation , observe mobility and other capabilities , and determine and adjust the care plan based on these observations .
it is not possible to fully develop these essential skills in a classroom or even a controlled lab setting with healthy participants ; this independent learning leads to a theory - practice divide .
the council of ontario universities has termed this disconnect of didactic and clinical education as a crisis in education of hcps . in order to maximize the knowledge transfer ,
both didactic and clinical components need to be integrated into an immersive form of learning as opposed to keeping these as separate forms of knowledge acquisition . in turn , this integrated learning enhances the development of a strong workforce and drives quality care .
successful gerontological learning in actual care settings has been described by several authors . in the 1980s , a model for the teaching nursing home ( tnh ) was developed and found to be effective in improving care for nh residents by supporting nursing students clinical education in that setting .
( the researchers described a partnership model developed between a nursing college and four nhs .
positive outcomes included increased professionalism for the nh team , identification of areas for growth within the nhs , increased awareness of how the nh team s practice influenced new nurses , and increased interest among students in choosing a nh environment as a career .
wieland and colleagues described an academic nh program designed to improve care of residents , provide ip training for a medical team and allied health students , and stimulate research .
students were paired with an expert within their respective field to integrate what they learned in the classroom .
benefits of the program included significant decreases in resident transfers to acute care hospitals , as well as noteworthy improvements in residents functional status , satisfaction , and morale .
lastly , mezey and lynaugh created a five - year tnh program to improve quality of nh care , create a supportive environment for undergraduate and graduate nursing students and care teams , and promote clinical research .
funding ended in 1987 ; by then the program included 12 nhs and 11 university schools of nursing .
the program resulted in 1.5 to 2 new full - time positions in each nh primarily nurse practitioners and clinicians specializing in geriatrics and these positions focused on education , quality assurance and infection control .
the researchers analysis of the tnh program revealed many benefits : resident hospitalization dropped 7% in the tnh while rising 5% in homes not linked to schools , and overall quality of care increased .
furthermore , the program cost was neutral ; the cost of hiring of practitioners / clinicians was offset by reduced resident hospitalization .
although shown to be effective , the idea of the tnh faded in the 1990s because of a lack of funding .
the concept has recently re - emerged in canada as a viable approach , with a stronger emphasis on creating work - integrated learning opportunities for students to improve recruitment and retention of graduates in geriatric care settings .
in 2009 , conestoga college and schlegel villages embarked on a partnership to develop a pedagogical approach for contextual based learning for nursing and na students in the context of a nh .
conestoga college is a community college in ontario , canada that offers polytechnic education through a full range of career - focused education and applied research programs to prepare students for success .
schlegel villages is an owner and operator of nhs and retirement villages across ontario , canada , with the mission , to provide holistic health care in a home environment , with emphasis on optimal health and life purpose for each resident .
we developed a lc conceptual framework , where na students , college faculty , and nh staff , residents , and families engage in the implementation of a culture of learning and mentoring within the physical context of a nh .
this conceptual framework is unique , as it situates the learner within an environment where new knowledge , culture , relationships , behaviours , and practices are modelled , as opposed to exposing the learner to these concepts later on in the curricular program .
students are immersed for the entirety of their program in the actual milieu in which they will practice .
first , the lc aims to demonstrate strong partnerships between a post - secondary educator , an nh , and the community , to develop meaningful and positive change in the education of hcps who will provide care to seniors and their families .
second , the lc aims to create welcoming environments embracing interaction and mentoring among students , college faculty , nh staff , residents , and families to build and strengthen a culture of learning .
the final aim of the lc is to integrate students into an environment where they learn and reflect on the life of seniors residing in nhs . over the last six years
a second lc opened in september 2015 , welcoming another 96 students . over the years ,
the collaborative has refined its conceptual framework and is now confident that others can benefit from this knowledge .
the framework consists of four layers : exploring the potential for the lc , developing the foundations for the lc , implementing the lc , and promoting and sustaining the lc .
the layers are represented in 10 building blocks , discussed in further detail in figure 1 .
exploring the potential for the lc forms the basis for all the layers that follow .
it encompasses three core building blocks : agreeing on the lc definition , determining operational viability , and identifying win - win opportunities and developing shared values .
agreeing on a lc definition describes both partners engaging in the development of a clear conceptual definition to promote a shared understanding of the lc .
this building block aims to promote realization that a lc is more than a classroom located within a nh ; it is an approach to integrated learning through experiences , a culture of learning , and supportive leadership .
the second building block determines the operational viability and includes an assessment of the legal underpinnings for the presence of a post - secondary educator in the nh , market viability ( enrolment capacity , workforce needs ) , operational viability ( scope of education programs and human resource implications ) , and physical resources and financial viability ( clarity on fiduciary relationships , space agreements outlining insurance and risk management considerations , and documented financial expectations ) .
the third building block relates to identifying win - win opportunities and developing shared values , and describes the process of identifying successful collaborators and exploring shared objectives for the lc ( learning from role models , nh staff acts as mentors ) , the shared reality of a lc ( local access to education , recruitment pool for nh ) , and direct benefits of the lc ( increased support for nh learning , opportunities to expand access to clinical sites ) .
the second level of the lc framework encompasses the foundational requirements in three building blocks : formalizing a collaboration , committing to the physical space of the lc , and creating standard operating protocols and formal agreements .
the collaboration requires a high level of mutual trust and strong communication channels between the collaborators and internal and external stakeholders .
this discussion refers to the locations and spaces available ( labs , classrooms , washrooms ) , as well as the esthetics of the environment , the responsibility for care equipment and teaching technology , utilities , housekeeping and maintenance , faculty and student space and supports , safety , parking , and signage .
operating procedures were developed prior to the lc opening , and required refinement once the lc was operational .
examples include procedures related to mandatory vaccinations for hcp students at the beginning of the lc ( as opposed to the start of the first clinical placement for other students ) or the distinction between students attending the nh as a volunteer or as a student .
as well , it was determined that some existing operating procedures , both for the nh ( introducing the lc to new residents ) and for the educator ( hand hygiene practices ) , required revisions to include both perspectives . as for the formal agreements ,
two agreements are recommended to protect the interests of the collaborative : a use of space agreement and a clinical affiliation agreement .
building on the previous layers , this third layer focuses on implementing an lc , and consists of three building blocks : identifying and connecting college faculty and nh teams , integrating curriculum and nh processes , and defining communication and coordination mechanisms .
the first building block describes the importance of identifying and connecting college faculty and nh teams .
successful teaching and learning teams require both the faculty and the nh team to be dedicated to the lc .
all members on the team must understand their individual roles , while maintaining an understanding of the values and objectives of the lc .
both faculty and members of the nh team develop strong relationships with all stakeholders and exemplify the values of student - centred learning for person - centred care . providing consistent staffing by the college and
a curriculum is the heart of any given educational program and consists of a series of planned instruction that is coordinated in a manner that is designed to result in students achievement of specific knowledge and skills and the application of knowledge . in the lc
, additional integrated experiences are added to promote the experiential learning within a nh setting .
the third and last building block in this layer describes the communication and coordination mechanisms to implement the lc .
this building block refers to the process of informing all stakeholders on a regular basis , to ensure that information is exchanged to help organize , implement , and evaluate all processes , resources , and services to sustain the lc .
of special importance are the resident and family communication mechanisms to highlight the significance of their engagement in the lc .
residents and their families are crucial partners in the creation of positive learning environments , and have extensive contributions to the students understanding of person - centred care and a positive culture in aging .
the final level of the framework discusses the evaluation and strategies for promotion and sustainability of the lc .
this level consists of promoting the lc , developing strong connections with community partners , and monitoring of success . promoting
once the lc is operational , the focus changes to developing strategies to sustain momentum , improve quality , and promote continuous innovation .
this requires both organizations to work together to intentionally develop new possibilities for advancing the lc initiative in ways that are mutually beneficial .
part of this work lies in the forming of community connections in a variety of ways . to be successful
, the lc must be recognized as a community resource , and specific attention must be given to the breadth of student learning and career preparedness , nh team development , and resident and family engagement .
several strategies are used to represent the impact of the lc on people and systems , how this has been measured , and how the findings have been incorporated for further enhancement .
this framework described the four layers of exploring the potential for the lc , developing the foundations for the lc , implementing a lc , and promoting and sustaining the lc , represented by 10 building blocks .
the partners context , students , college faculty , nh teams , residents , and families all play vital roles in making the lc a success .
many teachable moments are created beyond knowledge and skill acquisition , including interpersonal communication and relationship building , team culture , workload management , ip relations and more .
in 2009 , conestoga college and schlegel villages embarked on a partnership to develop a pedagogical approach for contextual based learning for nursing and na students in the context of a nh .
conestoga college is a community college in ontario , canada that offers polytechnic education through a full range of career - focused education and applied research programs to prepare students for success .
schlegel villages is an owner and operator of nhs and retirement villages across ontario , canada , with the mission , to provide holistic health care in a home environment , with emphasis on optimal health and life purpose for each resident .
we developed a lc conceptual framework , where na students , college faculty , and nh staff , residents , and families engage in the implementation of a culture of learning and mentoring within the physical context of a nh .
this conceptual framework is unique , as it situates the learner within an environment where new knowledge , culture , relationships , behaviours , and practices are modelled , as opposed to exposing the learner to these concepts later on in the curricular program .
students are immersed for the entirety of their program in the actual milieu in which they will practice .
first , the lc aims to demonstrate strong partnerships between a post - secondary educator , an nh , and the community , to develop meaningful and positive change in the education of hcps who will provide care to seniors and their families .
second , the lc aims to create welcoming environments embracing interaction and mentoring among students , college faculty , nh staff , residents , and families to build and strengthen a culture of learning .
the final aim of the lc is to integrate students into an environment where they learn and reflect on the life of seniors residing in nhs .
over the last six years , the lc has been successful in meeting its objectives . to date
a second lc opened in september 2015 , welcoming another 96 students . over the years ,
the collaborative has refined its conceptual framework and is now confident that others can benefit from this knowledge .
the framework consists of four layers : exploring the potential for the lc , developing the foundations for the lc , implementing the lc , and promoting and sustaining the lc .
the layers are represented in 10 building blocks , discussed in further detail in figure 1 .
exploring the potential for the lc forms the basis for all the layers that follow .
it encompasses three core building blocks : agreeing on the lc definition , determining operational viability , and identifying win - win opportunities and developing shared values .
agreeing on a lc definition describes both partners engaging in the development of a clear conceptual definition to promote a shared understanding of the lc .
this building block aims to promote realization that a lc is more than a classroom located within a nh ; it is an approach to integrated learning through experiences , a culture of learning , and supportive leadership .
the second building block determines the operational viability and includes an assessment of the legal underpinnings for the presence of a post - secondary educator in the nh , market viability ( enrolment capacity , workforce needs ) , operational viability ( scope of education programs and human resource implications ) , and physical resources and financial viability ( clarity on fiduciary relationships , space agreements outlining insurance and risk management considerations , and documented financial expectations ) .
the third building block relates to identifying win - win opportunities and developing shared values , and describes the process of identifying successful collaborators and exploring shared objectives for the lc ( learning from role models , nh staff acts as mentors ) , the shared reality of a lc ( local access to education , recruitment pool for nh ) , and direct benefits of the lc ( increased support for nh learning , opportunities to expand access to clinical sites ) .
the second level of the lc framework encompasses the foundational requirements in three building blocks : formalizing a collaboration , committing to the physical space of the lc , and creating standard operating protocols and formal agreements .
forming a collaboration is situated in a business relationship model . the business structure for a lc encompasses a shared vision and team work .
the collaboration requires a high level of mutual trust and strong communication channels between the collaborators and internal and external stakeholders .
this discussion refers to the locations and spaces available ( labs , classrooms , washrooms ) , as well as the esthetics of the environment , the responsibility for care equipment and teaching technology , utilities , housekeeping and maintenance , faculty and student space and supports , safety , parking , and signage .
operating procedures were developed prior to the lc opening , and required refinement once the lc was operational .
examples include procedures related to mandatory vaccinations for hcp students at the beginning of the lc ( as opposed to the start of the first clinical placement for other students ) or the distinction between students attending the nh as a volunteer or as a student .
as well , it was determined that some existing operating procedures , both for the nh ( introducing the lc to new residents ) and for the educator ( hand hygiene practices ) , required revisions to include both perspectives . as for the formal agreements ,
two agreements are recommended to protect the interests of the collaborative : a use of space agreement and a clinical affiliation agreement .
building on the previous layers , this third layer focuses on implementing an lc , and consists of three building blocks : identifying and connecting college faculty and nh teams , integrating curriculum and nh processes , and defining communication and coordination mechanisms .
the first building block describes the importance of identifying and connecting college faculty and nh teams .
successful teaching and learning teams require both the faculty and the nh team to be dedicated to the lc .
all members on the team must understand their individual roles , while maintaining an understanding of the values and objectives of the lc .
both faculty and members of the nh team develop strong relationships with all stakeholders and exemplify the values of student - centred learning for person - centred care . providing consistent staffing by the college and
a curriculum is the heart of any given educational program and consists of a series of planned instruction that is coordinated in a manner that is designed to result in students achievement of specific knowledge and skills and the application of knowledge . in the lc
, additional integrated experiences are added to promote the experiential learning within a nh setting .
the third and last building block in this layer describes the communication and coordination mechanisms to implement the lc .
this building block refers to the process of informing all stakeholders on a regular basis , to ensure that information is exchanged to help organize , implement , and evaluate all processes , resources , and services to sustain the lc .
of special importance are the resident and family communication mechanisms to highlight the significance of their engagement in the lc .
residents and their families are crucial partners in the creation of positive learning environments , and have extensive contributions to the students understanding of person - centred care and a positive culture in aging .
the final level of the framework discusses the evaluation and strategies for promotion and sustainability of the lc .
this level consists of promoting the lc , developing strong connections with community partners , and monitoring of success . promoting
once the lc is operational , the focus changes to developing strategies to sustain momentum , improve quality , and promote continuous innovation .
this requires both organizations to work together to intentionally develop new possibilities for advancing the lc initiative in ways that are mutually beneficial .
part of this work lies in the forming of community connections in a variety of ways . to be successful
, the lc must be recognized as a community resource , and specific attention must be given to the breadth of student learning and career preparedness , nh team development , and resident and family engagement .
several strategies are used to represent the impact of the lc on people and systems , how this has been measured , and how the findings have been incorporated for further enhancement .
this framework described the four layers of exploring the potential for the lc , developing the foundations for the lc , implementing a lc , and promoting and sustaining the lc , represented by 10 building blocks .
the partners context , students , college faculty , nh teams , residents , and families all play vital roles in making the lc a success .
many teachable moments are created beyond knowledge and skill acquisition , including interpersonal communication and relationship building , team culture , workload management , ip relations and more .
the lc is an ip approach whereby a pse program is delivered within the context of an nh , with the team members consisting of faculty , students , nh teams , residents , and families , who engage with each other within a culture of interactive learning .
students are intentionally involved to develop their knowledge , behaviours , and skills in care for seniors and their families .
this manuscript presents the conceptual framework and the operationalization for the lc designated for na programs .
the outcomes of the integration between learning and caregiving have been mutually beneficial , both for the college and the nh , in that integration of knowledge transfer is established , promoted , and rewarded . the paradigm behind the development of the first lc was to promote knowledge transfer within rich environments for work - integrated learning so students would be encouraged and well prepared for providing care to seniors . as noted by others , the idea of a nh is extremely valuable to promote knowledge transfer .
within an lc , students gain more and different knowledge , and demonstrate an increased comfort and confidence in working with residents , families , and teams .
as well , students are exposed to a variety of learning opportunities and feel that their contributions are valued because they function as part of a care team while learning .
this integrated approach of combining didactic and clinical components of learning has proven to be successful for a student s learning . college faculty
are involved in the development of an appropriate lc curriculum , while respecting all standard program requirements for each of the programs .
faculty integrate the context and expertise of the nh teams , residents , and families into the learning with the aim to continually increase gerontological knowledge , confidence , and competence .
because of the nh context , faculty have the ability to integrate all aspects of learning ; especially those situations that are difficult to simulate in a theoretical environment ( i.e. , a resident fall , a death of a resident , a quality audit ) , as well as having access to the highest expertise and the newest practices , regulations , and resources in the field .
this observation has been made by previous researchers in the domain of teaching nursing homes .
the nh team is presented with many opportunities to participate in mentoring of students and provides support and constructive feedback .
benefits for the nh team extend beyond active participation in developing the next workforce generation ; because of the students presence , the nh team is expected to present themselves as role models in relationships , ip collaborations , and evidence - informed care delivery .
the phenomenon of increased quality of care because of mentorship roles has been discussed before , but is confirmed by the lc approach .
the incorporation of meaningful activities for residents allows them to be part of something bigger , and encourages seniors to share wisdom .
this engagement provides a sense of belonging which is greatly beneficial to nh residents , as discussed by gilhooly and colleagues .
some researchers propose that meaningful activity leads to decreases in depression and anxiety for people with dementia and increases nutritional intake , mobility , and cognition .
relationship - building among lc students and residents is significant as it provides students with the opportunity to see the person with abilities behind the resident with ailments , allowing for a different perception of seniors . ensuring that these relationships are valued contributes to the career longevity of an na .
most importantly , the lc provides several benefits to the group of nh residents and families .
previous research emphasized the important value of personal connections between students and seniors as this influences students career decisions .
long - term advantages of the lc approach include a larger and better prepared workforce to care for seniors .
detailed findings of a mixed methods evaluation study will be published soon , but overall findings have been promising .
students involved in the lc demonstrated higher knowledge , behaviours , and skills in gerontology upon graduation in comparison to their counterparts at other campuses , and students had higher job satisfaction and work adaption skills once employed , as compared to other recent graduates .
the intentional involvement of nh residents and staff in teaching was successful , and hiring statistics indicated that the majority of lc graduates found employment in seniors care .
as the lc is an on - going development , some limitations should be discussed .
it is important to point out that an evaluative study yet to be published was conducted with participants in one lc and therefore , generalization of the findings may be limited .
further research studies need to include more rigorous evaluations of the lc including resident outcomes , quality of care measurements , and sustainability of the lc .
a second limitation is related to the small scope of the current lcs . to the best of our knowledge
, there is only one college that is engaged with two nhs in this endeavour .
this small sample limits the opportunities for broader discussions to optimize the current lc concepts . to address this concerns and encourage others to engage in this type of innovative learning
innovative learning approaches , such as the lc , have great potential to attract students to gerontology , and influence career choices by offering learning environments where students gain real - life experience . student learning embedded in health - care settings offers students a scope of education beyond that of any typical classroom setting and the ability to engage in practice while learning . with such contributions , nh environments can become innovative hubs for learning , and the input from nh teams , residents , and lc students all contribute to integrated learning , a better prepared workforce , and ultimately , better care for seniors .
lc graduates have the capability and motivation to provide quality care and services for seniors in care settings , and are well - positioned to become future leaders in their field . | backgroundevidence - informed care to support seniors is based on strong knowledge and skills of nursing assistants ( nas ) . currently , there are insufficient nas in the workforce , and new graduates are not always attracted to nursing home ( nh ) sectors because of limited exposure and lack of confidence .
innovative collaborative approaches are required to prepare nas to care for seniors.methodsa 2009 collaboration between a nh group and a community college resulted in the living classroom ( lc ) , a collaborative approach to integrated learning where na students , college faculty , nh teams , residents , and families engage in a culture of learning .
this approach situates the learner within the nh where knowledge , team dynamics , relationships , behaviours , and inter - professional ( ip ) practice are modelled.resultsas of today , over 300 na students have successfully completed this program . na students indicate high satisfaction with the lc and have an increased intention to seek employment in nhs .
faculty , nh teams , residents , and families have increased positive beliefs towards educating students in a nh.conclusionthe lc is an effective learning approach with a positive and high impact learning experience for all .
the lc is instrumental in contributing to a capable workforce caring for seniors . |
i novoroeneta predstavlja stres , a metoda kontakt koa na kou ( kkk ) i m a brojne pozitivne efekte .
cilj studije je da se odreivanjem nivoa salivarnog kortizola pre i nakon primene kkk proceni efekat na stres majke i novoroeneta i da se proceni uspenost uzimanja malih uzoraka salive kod novoroeneta .
kkk , iz malog uzorka salive , 50 l , kvantitativnim elisa testom visoke osetljivosti ( 0,0828 nmol / l ) , za merenje niskih koncentracija kortizola .
kortizol kod majki opada nakon kkk : najvie vrednosti su izmerene pre , a najnie posle primene kkk metode , i razlike u vrednostima su znaajne i tokom 1 .
kkk detektovan pad kortizola kod 14 ( 40% ) , a porast kod 21 novoroeneta ( 60% ) , a tokom 5 .
kkk pad kod 16 ( 45,7% ) , a porast kod 19 novoroenadi ( 54,3% ) , bez potvrene znaajnosti razlike .
pad vrednosti kortizola dokazuje redukciju stresa kod majki pri primeni kkk , dok varijabilne vrednosti kortizola kod novoroenadi ne upuuju na redukciju stresa i ukazuju na potrebu za daljim istraivanjima .
primenjena metoda uzorkovanja salive se pokazala jednom od najoptimalnijih u odnosu na veliinu uzorka , trajanje uzimanja uzorka i uspenost .
preterm birth is the most common cause of mother - infant separation due to the necessary medical treatment of the newborn in the neonatal intensive care unit .
mothers of preterm infants are exposed to higher amounts of stress and experimental research has proven that prolonged separation causes an increase in the cortisol level of the young and hyperactivity of the hypothalamic - pituitary - adrenal axis with long - term consequences ( 13 ) . due to repeated exposure to stress and painful procedures during their treatment , extremely premature infants can have altered patterns of cortisol response even at 8 months corrected age ( 46 ) . considering the longterm side effects of high cortisol levels
high quality of mother care , high sensitivity and responsiveness to infant signals can reduce stress and therefore also the cortisol reactivity in neonates ( 7 , 8) .
ssc method recommended for low birth weight infants ( lbw ) , among many other benefits , can also minimize the negative effects of stress and mother - infant separation ( 9 , 10 ) .
correlation of salivary cortisol with plasma free cortisol fraction allowed the non - invasive research of cortisol level in neonates , with saliva sampling and measuring low cortisol levels in small saliva samples representing a special challenge ( 1114 ) .
the aim of this study was to assess the influence of the late type of ssc on stress level in mothers and their infants by determining the level of salivary cortisol before and after the first and before and after the fifth ssc session . as a secondary outcome , the efficacy of collecting small volume saliva samples in lbw neonates was also estimated .
the prospective study was conducted at the institute of neonatology in belgrade during the one year period from 2013 to 2014 .
inclusion criteria were birth weight under 2500 g , mothers presence and their agreement to participate in the study .
exclusion criteria were treatment with corticosteroids , anticonvulsants or sedatives , congenital anomalies , severe forms of intracranial hemorrhage , consciousness disorders .
the study was approved by the ethics committee of the institute of neonatology and it was conducted with prior written notice and parental informed consent .
ssc was initiated once the infant was in stable clinical condition and it was performed daily for five consecutive days , during two hours , each time from 10 to 12 a.m. , due to the diurnal rhythm of maternal cortisol .
salivary cortisol level in mothers and newborns was determined as basal and reactive on 4 occasions : before and after the first , and before and after the fifth ssc .
the cortisol level was determined using the quantitative elisa kit ( salimetrics , lcc , state college , pa , usa ) , which is intended for research purposes only , with high sensitivity ( 0.0828 nmol / l ) that allows measurement of low concentrations of cortisol .
the measurement was done in duplicate using very small saliva samples of 25 l ( 50 l in total ) .
the samples from the same person before and after the ssc were determined in the same series .
saliva was collected using eye sponge by alcon , a hydrocellulose micro sponge with plastic grip , which has great absorption power and ability to re - release saliva ( 9095% ) .
saliva samples were collected without the usage of any salivation stimulating agents , always by the same trained nurse .
one hour before the sampling newborns were not fed and mothers did not consume any food , caffeine or nicotine .
eye sponge was held under the tongue during 3 to 5 minutes , until the swelling of the sponge became noticeable .
it was then placed in a 5 ml polypropylene cuvette with the lid on and centrifuged at 6000 rpm for 15 minutes .
the acquired saliva sample was collected using a pipette and frozen at 20 c until analyzed .
statistical analysis was performed using ibm spss statistical software ( spss for windows , release 21.0 , spss , chicago , il ) .
data is expressed as mean values with standard deviations or medians with minimum and maximum values for continuous variables .
changes in salivary cortisol values in mothers and infants before and after ssc were determined by the wilcoxon two related samples test .
salivary cortisol level was determined in 35 mother - infant pairs whose basic demographic characteristics were median ( range ) : gestational age 31 ( 2537 ) weeks , birth weight 1545 ( 7452450 ) g , apgar score at 1 min 7 ( 19 ) , apgar score at 5 min 8 ( 69 ) , age at the beginning of ssc 27 ( 1362 ) days , 15 male ( 42.9% ) , 20 female ( 57.1% ) newborns .
the highest levels of maternal cortisol were measured before the first and the lowest after the first ssc session and the difference was highly significant ( p<0.001 ) .
cortisol level in mothers also decreased during the fifth ssc session and the difference in these values was also significant ( p<0.001 ) ( figure 1 ) .
the percentage change in mothers salivary cortisol level was also analyzed : during the first ssc the mean value of percentage change in cortisol was 57.0 ( 18.5 ) , while during the fifth ssc the mean percentage change value is lower at 52.7 ( 22.7 ) . by analyzing the basal cortisol level in mothers before the first and before the fifth ssc , we established that basal cortisol level during the fifth ssc was lower but the difference did not reach statistical significance ( p=0.437 ) . during ssc the salivary cortisol level in newborns either increases or decreases . during the first ssc
a decrease in cortisol level was detected in 14 ( 40% ) and an increase in 21 ( 60% ) newborns , and during the fifth ssc a decrease was detected in 16 ( 45.7% ) and an increase in 19 ( 54.3% ) newborns ( figure 2 ) . by analyzing the difference in cortisol level change in newborns , the significance of this difference was not confirmed , either during the first ( p=0.831 ) or during the fifth ( p=0.961 ) ssc session . unlike the basal cortisol levels in mothers ,
the measured basal cortisol levels in newborns were higher during the fifth than during the first ssc ; however , this difference was not significant ( p=0.554 ) .
ssc 1 first ssc , ssc 5 fifth ssc , o outliers , * extreme values salivary cortisol changes in infants during ssc .
ssc 1 first ssc , ssc 5 fifth ssc , o outliers , * extreme values during saliva sampling in newborns , it was necessary to collect 4 samples from each of the 35 neonates , a total of 140 samples .
a total of 187 samples was collected , out of which 140 had a volume of 70 ml , with sampling time from 3 to 5 minutes .
despite numerous evidence of the positive effects of ssc , a number of studies estimating the salivary cortisol level in lbw infants during ssc of late intermittent type is moderate ( 10 , 15 ) . while estimating the stress level by measuring salivary cortisol in term infants , takahashi et al .
have proved that groups with early ssc , immediately after delivery , with duration from 60 to 120 minutes , had significantly lower cortisol levels .
sampling was conducted using the original sorbettes , sample volume was not listed and cortisol level was determined using a commercial high sensitivity eia - kit ( salimetrics , llc , pennsylvania , usa ) ( 16 ) . in our study , after ssc the salivary cortisol level in infants either increased or decreased : what made the difference was the population examined , with cortisol being measured in lbw infants with gestational age up to 37 weeks and with ssc being introduced at the average age of 27 days . when comparing the results of both studies , the immaturity of hypothalamic - pituitary - adrenal axis and insufficiently developed circadian rhythm of cortisol in preterm infants should be considered ( 5 , 17 , 18 ) .
measured salivary cortisol in 11 preterm infants during ssc and massage and they proved a significant and consistent decrease in cortisol level after only one 20 minute ssc session , with the effects of massage being variable ( 19 ) . in our study , however , neither the first nor the following ssc sessions , each lasting significantly longer , caused a significant and consistent cortisol level decrease . the question whether the study group being three times bigger ( 35 compared to 11 infants ) has allowed more precise insight in salivary cortisol change , along with the longer session time , remains open .
( 20 ) have compared the influence of family centered care ( constant presence of parents ) in relation to standard care in 289 preterm infants by measuring the salivary cortisol before and after nappy change as a stressful intervention .
significant differences regarding cortisol reactivity were not proven ; however , the correlation between the basal and reactive cortisol levels in mothers and newborns in the family centered group compared to the standard care group was confirmed ( 20 ) .
these results emphasize the importance of parental presence in achieving the mother - infant synchronization and stress reduction and this study certainly is important considering the size of the test group . the same group of authors has compared the effects of continuous ssc to standard care in 37 mother - infant pairs by determining salivary cortisol during nappy change as a stressful intervention . in infants from the ssc group , at the age of one month , cortisol reactivity was lower , whereas at the age of four months the cortisol level in mothers and infants were correlated , which indicates the stress reductive effect of ssc ( 21 ) .
22 ) have compared the salivary cortisol level in mothers and infants before and after heel prick , during heel lancing procedure and during ssc and they found no increase in the cortisol level of mothers and infants , which indicates the stress regulatory effect of ssc .
results of the clinical study in which the cortisol level was examined in 17 mothers and their premature born babies during ssc document that the reactive cortisol level in mothers was 32% lower after the first and 22% lower after the fourth ssc session , while the cortisol level in infants was either increasing or decreasing ( 23 ) .
results of our study were similar to those stated above , with the decrease of salivary cortisol level in mothers being more prominent , which can be connected to the longer use of ssc and longer duration of sessions , while the cortisol level in infants has shown inconsistent changes also in our study . in these studies ,
have used their own original modification of the radioimmunoassay ( ria ) method for determining salivary cortisol level , in order to achieve a lower detection limit and smaller saliva sample volume up to 10 l .
saliva sample was collected using two joined cotton swabs with a plastic grip , sampling time was not listed and the sampling efficacy has varied during different phases of the study in the range from 70.6% to 88% ( 21 , 23 , 24 ) .
the same group of authors also documented the difference in sampling using cotton swabs with plastic and with wooden grip , with the recommendation to avoid the ones with a wooden grip , unless the samples are being centrifuged immediately , since the obtained cortisol levels were otherwise lower ( 25 ) .
the study that compared saliva sampling using cotton swabs and eye sponge in adults and newborns indicated the advantage of using eye sponge in the sense of collecting an adequate sample volume , comfort and simplicity ( 26 ) .
saliva sampling in preterm newborns represents a challenge because of deficient salivation in comparison to the necessary sample volume and sampling is considered to be successful if a sample volume , sufficient enough to determine the cortisol level , is collected in the shortest time possible .
the highest sampling efficacy of 99% was published when using visipear sorbettes ( salimetrics lcc ) during 20 minutes and the lowest efficacy of 46% when using cotton applicators during 5 to 8 minutes . even in term newborns and with the prior use of salivation stimulating agents ,
therefore , according to the results of sampling analysis in six studies , the use of sorbettes during 20 minutes is recommended while sampling ( 27 ) . in the present study we used the eye sponge ,
sampling time was short and the optimal sample volume of 70 l was achieved , along with relatively high sampling efficacy compared to the data published in the literature . only morelius et al .
( 25 ) have cited a smaller sample volume , however , the sampling time was unknown , sampling efficacy was lower and the method used was a modified ria method .
cortisol level decrease during ssc proves the stress reduction in mothers , and with repeated ssc sessions the effect is even more prominent .
variable cortisol levels in lbw infants do not indicate stress reduction during ssc , which implies the need for further research . the saliva sampling method used in this study
was proven to be one of the most optimal considering the sample volume , sampling time and efficacy and it can be considered a method with significant advantages . | summarybackgroundsalivary cortisol measurement is a non - invasive method suitable for use in neonatal research .
mother - infant separation after birth represents stress and skin - to - skin contact ( ssc ) has numerous benefits .
the aim of the study was to measure salivary cortisol in mothers and newborns before and after ssc in order to assess the effect of ssc on mothers and infants stress and to estimate the efficacy of collecting small saliva samples in newborns.methodssalivary cortisol was measured in 35 mother - infant pairs before and after the first and the fifth ssc in small saliva samples ( 50 l ) using the high sensitivity quantitative elisa - kit ( 0.0828
nmol / l ) for low cortisol levels detection .
samples were collected with eye sponge during 3 to 5 minutes.resultscortisol level in mothers decreased after ssc : the highest levels were measured before and the lowest after ssc and the differences in values were significant during both the first ( p<0.001 ) and the fifth ssc ( p<0.001 ) . during the first ssc the cortisol level decrease was detected in 14 ( 40% ) and an increase in 21 ( 60% ) newborns , and during the fifth ssc a decrease was detected in 16 ( 45.7% ) and an increase in 19 ( 54.3% ) newborns , without confirmed significance of the difference .
saliva sampling efficacy using eye sponge was 75%.conclusionscortisol level decrease in mothers proves the stress reduction during ssc , while variable cortisol levels in infants do not indicate stress reduction and imply the need for further research .
the used sampling method appeared to be one of the most optimal considering the sample volume , sampling time and efficacy . |
the advent of various new techniques like ureteroscopy ( urs ) , shock wave lithotripsy ( swl ) , and percutaneous renal surgery has revolutionized the treatment of ureteral calculi and rendered a need for open surgical lithotomy .
however , these minimally invasive techniques have not been able to completely substitute for open surgery in selected cases of large , hard , long standing , and impacted ureteral calculi . since the initial report by clayman and colleague ( 1 ) in 1991 , laparoscopic surgery has been used for many types of urologic surgery involving ureterolithotomy . in particular , the retroperitoneal approach has become established in laparoscopy since gaur ( 2 ) developed a balloon dissection technique for the retroperitoneum .
many groups have described cases requiring retroperitoneal laparoscopic ureterolithotomy ( rplu ) and several series have examined the transperitoneal and/or rplu as replacements for open ureterolithotomy .
we have performed many retroperitoneal laparoscopic operations since 1997 , and have treated 12 cases for large impacted upper ureteral stones using rplu , and first reported on the safety and efficacy of the technique in a series of laparoscopic nephrectomies for nonfunctioning kidney due to renal tuberculosis ( 3 ) .
however , we experienced an unexpectedly high open conversion rate in our rplu series , which was unrelated with the learning curve .
between february 1998 and july 2004 , 12 patients ( 10 men and 2 women , mean age 45.2 yr [ range 25 - 62 ] ) underwent rplu for upper ureter stones ( 10 left , 2 right ) with moderate to severe hydronephrosis .
five patients had undergone previous treatment , including 3 patients treated by swl only and 2 patients that had been treated by urs and swl .
one of two patients treated with both eswl and urs had undergone eswl 20 times during one year .
one of the seven patients had undergone open ureterolithotomy at the site of this ureteral stone 10 yr previously .
patients ' details are summarized in table 1 patients were positioned in a full lateral position .
the transversalis fascia was incised , and the possible pararenal space was developed bluntly as far as possible with a finger with the aim of pushing the peritoneum forward and creating sufficient space for the introduction of a balloon dilator .
the dilator was placed into the retroperitoneal space under digital control and inflated with normal saline .
after balloon deflation and removal , a 10 mm hasson trocar was inserted and a pneumoretroperitoneum was created .
twelve and 5 mm trocars were inserted under laparoscopic vision at the midclavicular and posterior axillary lines , and three ports were located on the transverse line in preparation for open conversion . after identifying the ureteral stone , the ureter was incised directly over the stone longitudinally , and the stone was removed using a grasping forceps .
the ureterotomy was closed with 4 - 0 vicryl using an intracorporeal suture and a double - j ureteral stent was inserted endoscopically in the lithotomy position after laparoscopic ureterolithotomy . in two patients ,
the transversalis fascia was incised , and the possible pararenal space was developed bluntly as far as possible with a finger with the aim of pushing the peritoneum forward and creating sufficient space for the introduction of a balloon dilator .
the dilator was placed into the retroperitoneal space under digital control and inflated with normal saline .
after balloon deflation and removal , a 10 mm hasson trocar was inserted and a pneumoretroperitoneum was created .
twelve and 5 mm trocars were inserted under laparoscopic vision at the midclavicular and posterior axillary lines , and three ports were located on the transverse line in preparation for open conversion . after identifying the ureteral stone , the ureter was incised directly over the stone longitudinally , and the stone was removed using a grasping forceps .
the ureterotomy was closed with 4 - 0 vicryl using an intracorporeal suture and a double - j ureteral stent was inserted endoscopically in the lithotomy position after laparoscopic ureterolithotomy . in two patients ,
the reason for open conversion was a failure to identify the ureter due to severe adhesion in 5 cases and a vascular injury in one case .
in detail , the first open conversion case was our first retroperitoneal laparoscopic operation . in this case
after four consecutive successful laparoscopic surgeries the second open conversion occurred because it was difficult to continue the laparoscopic operation due to peritoneal tearing caused by severe adhesion around the ureter .
subsequently , in the two cases in which urs and/or swl had been performed before laparoscopy , severe adhesion around ureter forced conversion to open ureterolithotomy .
the fifth open conversion was due to a vascular injury that occurred while identifying the ureter in severely adhesive fat tissue in a case with a long standing large stone ( more than 6 months , 21 mm ) .
the last open conversion case was the case that had received open ureterolithotomy 10 yr ago previously at the same site . in this case
we could not identify the ureter due to severe adhesion with the retroperitoneum ( table 1 ) .
table 2 compares the laparoscopically successful cases and the open conversion cases with respect to patient demographics .
the mean age and the mean stone size were higher and larger in the open conversion group than in the successful laparoscopically group .
the number of cases that had received previous treatment were two in the successful laparoscopically group and three in the open conversion group , respectively .
intraoperative blood loss was negligible ( mean 50 ml ) and no transfusion was required .
nasogastric tubes were removed immediately after operation and foley urethral catheters were removed during the morning of the day following the procedure .
postoperative pain was well controlled with a mean dosage of 30 mg of ketoprofen. the period of hospital stay ranged from 2 to 7 days ( mean 4.6 days ) .
the average period of ureteral stenting was 19.5 days ( 10 - 36 days ) .
one patient with ureteral stenting developed a urine leakage , which resolved after 5 weeks of ureteral stent indwelling .
the surgical management of urinary stone disease has dramatically changed since the introduction and development of percutaneous renal surgery , and due to significant achievements in shockwave lithotripsy , refinements in ureteroscopy , and technical advancements in the available modalities of intracorporeal lithotripsy ( 4 ) .
several authors have reported that the rates of open stone surgery since the establishment of shockwave lithotripsy and subsequent endoscopic advancements are only 0.3 to 5.4% ( 5,6 ) .
while most patients with renal and ureteral stones can be treated using less invasive techniques , open stone surgery continues to represent a reasonable alternative for a small segment of the urinary stone population ( 4 ) .
the indications for open stone surgery have been reported as being complex stone disease , less invasive ( endourologic or swl ) treatment failures , anatomic abnormalities , obesity , comorbid disease , and patient preference ( 7 ) .
after gaur described retroperitoneal laparoscopic surgery facilitated by a hydraulic balloon dilatation system in 1992 ( 2 ) , straightforward access became available through the retroperitoneum .
thereafter , several authors have tried to replace open ureterolithotomy as a transperitoneal or retroperitoneal laparoscopic procedure for ureteral stone disease , because of the minimal invasiveness of the laparoscopic technique .
rplu has advantages over transperitoneal access by obviating compromise of the peritoneum and mobilizing the viscera , and by preventing urine spillage into the peritoneal cavity .
however , the retroperitoneal approach offers a limited working place and it is often difficult to find the ureter due place and it is often difficult to find the ureter due to lack of anatomical landmarks .
in fact , harewood et al . ( 9 ) reported in their series that two of three retroperitoneal approach cases were converted to transperitoneal laparoscopy because of a limited working place .
( 10 ) reported the lowest success rate ( 75% ) in their rplu series , and explained that open conversion happened early in their series and that it was related to the learning curve .
in addition , rassweiler et al . ( 11 ) reported five cases of rplu among 200 retroperitoneal cases , but felt that the procedure needed to be evaluated further .
our results also show a high open conversion rate . taken together these results suggest rplu is not easily performed .
however , compared to several series that reported open conversion rates of < 10%(9,12 - 14 ) our series resulted in an unusually high open conversion rate .
thus , we feel obliged to explain why we believe that open conversion happened so frequently in the present study . first the learning curve should be considered , but the time course of open conversions during our series shows that open conversions were not related to the learning curve .
second , technical shortcomings could have been contributed , but our team reported the first series of laparoscopic nephrectomies for a nonfunctioning kidney by renal tuberculosis , which was recognized as a contraindication for laparoscopy , and proved the safety and efficacy of the technique ( 3 ) .
thus , we do not believe that the six open conversion cases in the present study were due laparoscopic technique shortcomings .
open conversion group patients were older and had larger stones than those in the laparoscopically successful group , although this difference was not significant because of the small sizes of the groups involved .
thus , we can not be sure that age and stone size are related to the difficulty of performing rplu , and further study is needed to identify the roles of age and stone size in rplu .
pretreatments like urs and swl may make rplu more difficult to perform , but little difference was observed in this respect between the two groups , which suggests that previous swl and/or urs treatments were not the cause of open conversion . from the above reasoning , we concluded that rplu is inherently difficult to perform , like laparoscopic nephrectomy for xanthogranulomatous pyelonephritis , which was reported not to be feasible versus open surgery ( 15 ) .
this hypothesis is supported by the fact that long - standing and large ureter stones are usually accompanied by severe adhesion around a stone ; we experienced severe adhesions in all twelve cases regardless of pretreatment history . however conversion to open surgery does not necessarily indicate a complication . unlike laparoscopic nephrectomy for xanthogranulomatous pyelonephritis
, our rplu series did not cause severe postoperative complications and were not related to protracted operation times .
thus , we do not agree that rplu is inherently too complicated to be feasible . in conclusion ,
half of our cases were converted to open surgery indicating that rplu is not easily performed , because , in the present study , it was indicated mainly for ureter stones with severe adhesion or after failed swl and/or urs .
nevertheless , it should be considered a primary procedure before open ureterolithotomy for large impacted ureter stones and for ureteral stones after failed swl and/or urs because rplu has the definite advantage of reduced invasiveness . | we evaluated the role of retroperitoneal laparoscopic ureterolithotomy ( rplu ) for upper ureter stones . between february 1998 and july 2004
, 12 patients ( 10 men and 2 women ) underwent rplu for upper ureter stones ( mean size 18.1 mm , range 10 - 25 ) .
rplu was carried out in 5 patients as a salvage treatment after failed shock wave lithotripsy ( swl ) ( 3 ) and both of failed swl and ureteroscopy ( urs ) ( 2 ) , and in 7 patients as primary treatment for large impacted stones .
total 6 of 12 cases were converted to open surgery .
the reason of open conversion was failure of locating ureter due to severe adhesion in 5 cases and vascular injury in 1 case .
in 6 successful cases , mean operation time , mean estimated blood loss , and mean postoperative hospital stay were respectively 109 min ( 90 - 120 min ) , 50 ml ( 10 - 100 ml ) , 4.6 days ( 2 - 7 days ) .
there was no serious postoperative complication except for one patient who showed delayed urinary leakage but was cured with conservative management .
our experience suggested that rplu was not easy to perform simply because it was indicated mainly in ureter stones with severe adhesion or after failed swl and/or urs .
nevertheless , it can be considered as a primary procedure before open ureterolithotomy . |
every patient with acute respiratory distress syndrome ( ards ) is hypoxemic by definition . in these patients , mechanical ventilation ( mv )
is often life - saving . repetitive cyclic stretch however , results in regional overdistension or / and derecruitment which is associated with a number of severe complications termed ventilator - induced / associated lung injury ( vi / ali ) .
the attributable mortality of vi / ali has been estimated to be at least 9% , and despite evidence that high concentrations of oxygen ( fractional inhaled concentrations of oxygen [ fio2 ] greater than 50% ) , can lead to hyperoxic acute lung injury ( hali ) , oxygen therapy remains a cornerstone of management .
little is known about " permissive hypoxemia " and for the most part , clinicians will optimize positive end expiratory pressure ( peep ) to enable reductions in fio2 , accepting oxygen saturations in the mid to high 80 's .
in the first three icu days , most ards patients are ventilated with average fio2 > 59% ( mean fio2 delivered on day 1 = 70% ) , but it is not uncommon for the most severely ill to require much higher fio2 concentrations ( 100% ) for prolonged periods or frequent intervals . in a previous issue of critical care , li and colleagues elucidate the potential mechanisms regulating interactions between injury cascades resulting from hyperoxia and high tidal volume ventilation . using gene - deficient models and specific inhibitors of intracellular signaling pathways , this author group demonstrate that the combination of hyperoxia and high tidal volume ventilation results in augmented lung injury , evidenced by indices of increased lung inflammation , microvascular permeability , and lung epithelial apoptotic cell death .
the combined detrimental effect of oxygen and repetitive cyclic stretch was shown to result in the activation of specific intracellular signaling pathways .
the paper by li and colleagues is part of a growing body of literature suggesting that the response of the mechanically ventilated lung to biochemical or biomolecular stimuli is profoundly altered by the coexistence of injurious stimuli that synergize at the cellular level as well as at the tissue level .
more importantly , the findings suggest that interference or cooperation of signals may have critical physiological consequences such as activation of death pathways
. studies on various model systems have shown that a relatively small number of transcription factors can set up strikingly complex spatial and temporal patterns of gene expression .
this pattern creation is achieved mainly by means of combinatorial or differential gene regulation ; that is , regulation of a gene by two or more transcription factors simultaneously or under different conditions .
li and colleagues offer insight into the specific molecular details of the mechanisms of combinatorial regulation of hyperoxia and high tidal volume . in their model , mitogen - activated protein kinase erk1/2 ,
c - jun nh2-terminal kinases , and downstream binding of the transcription factor ap-1 were responsible for orchestrating the molecular response and cellular physiological consequence of hali plus vi / ali whilst lung stretch alone is dependent on activation of the jnk pathway , high volume plus hyperoxia mediated its detrimental effect via jnk and erk 1/2 activation . despite a historical emphasis on nf-b - dependent inflammation - related genes as mediators of injury , li and colleagues ' paper suggests that the augmented response seen when high volume and hyperoxia coexist appears to be nf-b independent .
the molecular implication from their paper is that individual stimuli exert intracellular effects via independent signaling pathways that may converge or diverge at specific molecular ' nodes ' or ' hubs ' critical control points and potential targets for therapy .
moreover , molecules that were previously perceived as reflecting redundancy in the response represent a sophisticated system that probably depends on the ' message ' carried rather than the messenger .
the clinical implications of deciphering injury specific intra - cellular signaling is that it provides novel insight into the potential for future molecular treatment of injury - specific stimuli .
exposure to hyperoxia is a well - established model of lung injury characterized by the development of pulmonary edema and inflammation .
the development of hyperoxic lung injury was until recently thought to require the generation of reactive oxygen species , which leads to alveolar epithelial and endothelial cell death by both apoptosis and necrosis .
disturbance of cell - death pathways , either local ( pulmonary ) or distal ( kidney and intestines ) , has also been implicated in the pathogenesis of vi / ali and ensuing mof , and this disturbance is a key feature of the alveolar remodeling process during recovery from injury . in contrast to the literature on vi / ali , where increased expression of cytokines and chemokines has been thought to be related to increase epithelial cell apoptosis , in studies of hali , overexpression of cytokines or chemokines ( for example , tnf- , il-1 , il-6 , cxc - chemokine receptor 2 , and il-11 ) , growth factors ( for example , insulin growth factor and keratinocyte growth factor [ kgf ] ) , or the subunit of the na , k - atpase have been shown to protect animals from hyperoxia by attenuating death signals . moreover , this effect appears to be independent of potential antioxidant effects , since antioxidants by themselves do not reverse or prevent all of the manifestations of hali and since the ability of certain cytokines to inhibit hyperoxia - induced cell death is independent of major alterations in lung antioxidants .
this evidence raises crucial questions about how different injury signal components are integrated ; what are the morbidity and mortality defining manifestations of vi / ali and/or hali and which , where and when should " outcome - defining " pathways be blocked .
fio2 = fractional inhaled concentration of oxygen ; hali = hyperoxic acute lung injury ; il = interleukin ; nf = nuclear factor ; tnf = tumor necrosis factor ; vi / ali = ventilator - induced / associated lung injury .
| in patients with acute respiratory distress syndrome ( ards ) , supportive therapy with mechanical ventilation and oxygen is often life saving .
further acute lung injury however , is an unfortunate consequence of oxygen therapy as well as mechanical injury secondary to ventilator induced / associated lung injury ( vi / ali ) . in this issue of critical care , li et al .
expand on the intra - cellular signaling pathways regulating interactions between injury cascades resulting from hyperoxia and high tidal volume ventilation .
the findings , suggest that interference or cooperation of different signals may have critical consequences as evidenced by indices of increased lung inflammation , microvascular permeability , and lung epithelial apoptotic cell death . |
a 42-year - old overweight immunocompetent woman with a history of hypertension and mild psoriasis and no travel history was seen twice in the emergency department-11 days prior to admission and again 8 days prior to admission - with tachycardia , tachypnea , and left infrascapular back pain that radiated to her chest .
her white blood count was 8.52 k/l ( normal range , 3.7 to 10.4 k/l ) , and chest radiograph and computed tomographic ( ct ) scan were negative for any bony abnormalities or cardiopulmonary pathology .
the patient was given muscle relaxants and narcotic medication on both occasions and discharged home .
five days prior to admission , the patient reported using her arms to help stand up and immediately feeling a " pop " in her shoulder with associated pain .
her upper back pain had diminished ; however , her shoulder was noted to be erythematous , warm , and diffusely tender to palpation .
the patient was prescribed cefadroxil 1 g twice daily for 10 days of treatment for presumed cellulitis of the shoulder and anterior chest .
the patient again returned to her primary care physician with a fever , increased pain , and decreased range of motion in her shoulder .
she had an ultrasound - guided aspiration of the left ac joint , blood cultures were taken , and she was admitted to the hospital .
her admission testing revealed a white blood cell count of 18 k/l , a platelet count of 660 k/l ( reference range , 140 to 385 k/l ) , a c - reactive protein concentration of 9.9 mg / dl ( normal range , 11.8 to 15.1 mg / dl ) , erythrocyte sedimentation rate of 60 mm / hr ( normal range , < 25 mm / hr ) , and blood cultures negative for growth . a ct scan of the patient 's shoulder revealed no signs of osteomyelitis . a magnetic resonance imaging ( mri )
scan without contrast of the left shoulder revealed increased fluid collection on short ti inversion recovery ( stir ) images in the anterior deltoid and supraspinatus , as well as in the ac joint ( fig .
cultures from the ac joint revealed a staphylococcus aureus susceptible to methicillin , but due to a penicillin allergy , she was initially treated with intravenous ( iv ) vancomycin therapy and later changed , based on susceptibility testing , to 900 mg clindamycin iv 3 times a day while in the hospital .
a large amount of necrotic tissue and pus was drained from the anterior deltoid and supraspinatus muscles , and a small amount from the ac joint .
the clavicle was soft with cystic changes compatible with osteomyelitis , and a small fragment of the distal clavicle was excised .
immediately after surgery , the patient felt better , had improved shoulder range of motion and decreased erythema , and was discharged on hospital day 5 on 300 mg oral clindamycin 3 times a day for 25 days to complete 5 weeks of antibiotic treatment . on her follow - up appointment 2 weeks after discharge , she was feeling better and had no further signs of infection , and at 5 months had a full range of her shoulder function without signs of recurrent infection . over 4 or 5 days prior to her admission to hospital , a 50-year - old woman developed increasing pain erythema , and swelling over the left sc joint .
the patient 's past medical history was noteworthy for a remote history of ivda , hypothyroidism , and hypertension .
pertinent findings were pain and swelling over the left sc joint with pain in the left contiguous scm muscle , but no definite fluctuance .
joint aspiration revealed frank pus , and gram - positive cocci were seen on a gram stain . on admission
the patient had a blood pressure of 132/78 mmhg , pulse of 105 beats per minute , respiration rate of 18 breaths per minute , and temperature of 37.4. initial laboratory results revealed no evidence of drug abuse on urine testing , a white blood cell count of 16.3 k/l , 91% polymorphic nuclear cells , and an sc joint aspirate with 140 k / ul white blood cells .
the patient 's initial antibiotics , iv vancomycin and iv piperacillin tazobactam , were therefore changed to oxacillin 2 g iv every 6 hours . on day 2 of hospitalization
the tenseness and swelling over the sc joint worsened , and was incised and drained of purulent material .
the next day , with increasing pain and swelling of the scm muscle , an mr scan revealed fluid collection on stir images in the sc joint , edema of the proximal bone , and liquefaction of the entire scm muscle from the sc joint to the base of the skull ( fig .
2 ) . this muscle was , therefore , incised inferiorly , drained of a large amount of purulent material , and was packed with iodoform gauze .
subsequently , the patient has responded to iv antibiotics directed against the s. aureus , first with 2 weeks of oxacillin iv followed by 4 weeks of outpatient ceftriaxone iv 2 g per day . at follow - up ,
her joint and left neck pain had improved , but atrophy of the scm muscle was noted . in more than 12 month
a 42-year - old overweight immunocompetent woman with a history of hypertension and mild psoriasis and no travel history was seen twice in the emergency department-11 days prior to admission and again 8 days prior to admission - with tachycardia , tachypnea , and left infrascapular back pain that radiated to her chest .
her white blood count was 8.52 k/l ( normal range , 3.7 to 10.4 k/l ) , and chest radiograph and computed tomographic ( ct ) scan were negative for any bony abnormalities or cardiopulmonary pathology .
the patient was given muscle relaxants and narcotic medication on both occasions and discharged home .
five days prior to admission , the patient reported using her arms to help stand up and immediately feeling a " pop " in her shoulder with associated pain .
her upper back pain had diminished ; however , her shoulder was noted to be erythematous , warm , and diffusely tender to palpation .
the patient was prescribed cefadroxil 1 g twice daily for 10 days of treatment for presumed cellulitis of the shoulder and anterior chest .
the patient again returned to her primary care physician with a fever , increased pain , and decreased range of motion in her shoulder .
she had an ultrasound - guided aspiration of the left ac joint , blood cultures were taken , and she was admitted to the hospital .
her admission testing revealed a white blood cell count of 18 k/l , a platelet count of 660 k/l ( reference range , 140 to 385 k/l ) , a c - reactive protein concentration of 9.9 mg / dl ( normal range , 11.8 to 15.1 mg / dl ) , erythrocyte sedimentation rate of 60 mm / hr ( normal range , < 25 mm / hr ) , and blood cultures negative for growth . a ct scan of the patient 's shoulder revealed no signs of osteomyelitis . a magnetic resonance imaging ( mri )
scan without contrast of the left shoulder revealed increased fluid collection on short ti inversion recovery ( stir ) images in the anterior deltoid and supraspinatus , as well as in the ac joint ( fig .
cultures from the ac joint revealed a staphylococcus aureus susceptible to methicillin , but due to a penicillin allergy , she was initially treated with intravenous ( iv ) vancomycin therapy and later changed , based on susceptibility testing , to 900 mg clindamycin iv 3 times a day while in the hospital .
a large amount of necrotic tissue and pus was drained from the anterior deltoid and supraspinatus muscles , and a small amount from the ac joint .
the clavicle was soft with cystic changes compatible with osteomyelitis , and a small fragment of the distal clavicle was excised .
immediately after surgery , the patient felt better , had improved shoulder range of motion and decreased erythema , and was discharged on hospital day 5 on 300 mg oral clindamycin 3 times a day for 25 days to complete 5 weeks of antibiotic treatment . on her follow - up appointment 2 weeks after discharge , she was feeling better and had no further signs of infection , and at 5 months had a full range of her shoulder function without signs of recurrent infection .
over 4 or 5 days prior to her admission to hospital , a 50-year - old woman developed increasing pain erythema , and swelling over the left sc joint .
the patient 's past medical history was noteworthy for a remote history of ivda , hypothyroidism , and hypertension .
pertinent findings were pain and swelling over the left sc joint with pain in the left contiguous scm muscle , but no definite fluctuance .
joint aspiration revealed frank pus , and gram - positive cocci were seen on a gram stain . on admission
the patient had a blood pressure of 132/78 mmhg , pulse of 105 beats per minute , respiration rate of 18 breaths per minute , and temperature of 37.4. initial laboratory results revealed no evidence of drug abuse on urine testing , a white blood cell count of 16.3 k/l , 91% polymorphic nuclear cells , and an sc joint aspirate with 140 k / ul white blood cells .
the patient 's initial antibiotics , iv vancomycin and iv piperacillin tazobactam , were therefore changed to oxacillin 2 g iv every 6 hours . on day 2 of hospitalization
the tenseness and swelling over the sc joint worsened , and was incised and drained of purulent material .
the next day , with increasing pain and swelling of the scm muscle , an mr scan revealed fluid collection on stir images in the sc joint , edema of the proximal bone , and liquefaction of the entire scm muscle from the sc joint to the base of the skull ( fig .
2 ) . this muscle was , therefore , incised inferiorly , drained of a large amount of purulent material , and was packed with iodoform gauze .
subsequently , the patient has responded to iv antibiotics directed against the s. aureus , first with 2 weeks of oxacillin iv followed by 4 weeks of outpatient ceftriaxone iv 2 g per day . at follow - up ,
her joint and left neck pain had improved , but atrophy of the scm muscle was noted . in more than 12 month
the ac and sc joints , sliding and saddle joints , respectively are non - weight - bearing and have minimal surface area .
pyomyositis and septic arthritis of either end are rare entities probably due to their unique anatomy .
although treatment modalities are similar for septic arthritis and pyomyositis , variations do exist , and rapid , accurate diagnosis can save both life and limb .
differentiating pyomyositis from septic arthritis is difficult because , as in these patient 's presentations , one can mimic the other,4 ) so 2 distinct pathologic possibilities exist .
one possible explanation for these patients ' symptoms is initial primary pyomyositis with secondary septic arthritis of the clavicular joints .
although primary pyomyositis is described as a rare disease in temperate climates , the epidemiology , clinical symptoms , course , hypothetical pathophysiology , and treatment have been well documented.1,2,3,4,5,6 ) primary pyomyositis is defined as an acute deep bacterial infection of large skeletal muscles not secondary to a contiguous skin , bone , or soft - tissue infection .
originally described in tropical climates in immunocompetent hosts , pyomyositis in immunocompromised patients living in temperate climates is becoming more prevalent .
in fact , up to 75% of pyomyositis in temperate climates has been associated with underlying disease with immunodeficiency , such as diabetes mellitus , aids , ivda , or malignancy.6 ) infective agents vary on immune state as well as other predisposing factors .
immunocompetent patients in temperate climates are affected by s. aureus 75% of the time , often is associated with younger age , a prior traumatic event such as vigorous exercise , and underlying skin diseases.1 ) in contrast , immunocompromised patients are also susceptible to more unusual organisms , such as haemophilus influenzae , bartonella species , pseudomonas species , enterobacteriaceae , and anaerobes .
although the pathophysiology of pyomyositis has not yet been defined , it is believed to be due to a transient bacteremia that localizes in the muscle tissue .
muscle tissue is usually resistant to infectious agents , but in the face of an injury , the alteration of muscle structure makes it more susceptible , explained by the latin term " locus minoris resistentiae .
" increased blood supply to the damaged tissue may provide hematogenous access for the bacteria and their required nutrients .
presentation of pyomyositis has been described in 3 stages : invasion , suppuration , and sepsis .
the invasive stage begins in the first few days of onset as a dull and crampy pain.2 ) a patient may have a low - grade fever and mild leukocytosis . if the infection is promptly diagnosed in the invasive stage , antibiotic therapy alone may suffice , and the selection of which should be based on culture and susceptibilities and/or the epidemiology .
anti - gram - positive antibiotic therapy directed against s. aureus and beta streptococci is usually adequate for immunocompetent patients , whereas immunocompromised patients should also receive coverage for gram - negative organisms .
unfortunately , 90% of patients are not treated in the invasive stage and progress in 1 to 3 weeks to the suppurative or purulent stage , as in our cases .
pus is palpable as an abscess in the muscle ; however , it is typically firm , not fluctuant as was found in these cases .
laboratory test results demonstrate an elevated erythrocyte sedimentation rate and leukocytosis.2 ) treatment at this stage includes drainage of the abscess in addition to antibiotic therapy .
the picture of primary pyomyositis fits our first patient 's presentation , which began with a vague crampy pain and progressed to abscess formation in the supraspinatus and anterior deltoid fitting the time - frame of the documented stages of primary pyomyositis .
she was affected by the most common pathogen that causes pyomyositis , methicillin - susceptible s. aureus ( mssa),5 ) and had an underlying skin disorder , which has been associated with mssa pyomyositis.1 ) however , to our knowledge , only 1 case of pyomyositis leading to septic arthritis of the sc joint has been reported , and none of the ac joint .
this seems more likely in the second case who had a remote history of ivda .
secondary pyomyositis has been reported less often than primary pyomyositis and has been associated with non - s .
although primary and secondary pyomyositis present similarly , less than 20% of patients with secondary pyomyositis recover with antibiotic therapy alone.6 ) to our knowledge , only 25 cases of septic arthritis of the ac joint have been recorded.7,8,9 ) while not as rare , sc joint septic arthritis has been reported , to be mistaken for pyomyositis.4 ) the epidemiology of septic arthritis is similar to that of pyomyositis .
risk factors include ivda , local glucocorticoid injection , diabetes , aids , and undergoing chemotherapy for myeloma .
in review of ac joint septic arthritis , patients range in age from 17 to 79 years , with an average age of 54 years . unlike our cases
diagnosis of sepsis involving either clavicular joints is made by aspiration , with or without ultrasound guidance .
mri is also useful in assessing septic arthritis and helps greatly , as in our cases , with assessment of surrounding muscle for suppuration and contiguous osteomyelitis .
overall , s. aureus is responsible for the arthritis in more than 50% of cases .
other pathogens include various streptococcal species , enterobacteriaceae , atypical mycobacterium , haemophilus parainfluenzae , candida parapsilosis , cryptococcus , and ochribactrum anthropi .
septic arthritis of the ac joint has also been associated with abscess formation between the surrounding muscle .
treatment options include iv antibiotics and surgical debridement with or without distal clavicle excision.7,8,9,10 ) the exact pathology of our patients ' septic arthritis of the ac and sc joints along with contiguous pyomyositis is unclear , and their clinical manifestations were , on review of the literature , unusual .
the concomitant presence of 2 rare entities made determining their primary causation difficult ; however , prolonged antibiotic therapy for septic arthritis is similar to that for pyomyositis , and treatment with surgical drainage and debridement , eradicated their infections . | acromioclavicular ( ac ) and sternoclavicular ( sc ) septic arthritis with contiguous pyomyositis are rare , especially in immunocompetent individuals .
we report a case of septic ac joint with pyomyositis of the deltoid and supraspinatus muscles and a separate case with septic sc joint with pyomysitis of the sternocleidomastoid muscle . both patients had similar presentations of infections with staphylococcus aureus and
were successfully treated with surgical incision and drainage followed by prolonged antibiotic therapy . |
endothelial dysfunction ( ed ) , which is characterized by an imbalance between relaxing and contracting factors , procoagulant and anticoagulant substances , and between pro - inflammatory mediators , may play a particularly significant role in the pathogenesis of atherosclerosis . under physiological conditions ,
the vascular endothelium produces many substances that are closely involved in hemostasis , fibrinolysis , growth factor synthesis , and the regulation of vessel tone and permeability .
one of these substances is von willebrand factor ( vwf ) , which is synthesized by and stored in endothelial cells .
vwf is a multimeric glycoprotein , and it is essential for platelet aggregation and adhesion .
numerous clinical and experimental reports suggest that a high vwf level reflects endothelial damage or ed .
mean platelet volume ( mpv ) , which is routinely determined by complete blood count analyzers , is a parameter reflecting the platelet size .
large platelets are more thrombogenic and active than normal sized platelets since they have a higher content of granules .
mpv , a determinant of platelet activation , is a newly emerging risk factor for atherothrombosis .
elevated mpv levels have been identified as an independent risk factor for myocardial infarction in patients with coronary heart disease and for death or recurrent vascular events after myocardial infarction .
moreover , increased platelet size has been reported in patients with vascular risk factors such as diabetes mellitus , and in patients with acute ischemic stroke , essential hypertension , obesity .
there have been few studies of the relationship between vwf and mpv . to the best of our knowledge , there has been no study on the relationship between levels of vwf and mpv in subjects with isolated ifg .
therefore , we investigated the relationship between von vwf and mpv in subjects with isolated ifg .
this study was performed in the population of our previous study ( 48 subjects with isolated ifg and 48 normoglycemic healthy controls matched for age , sex , and body mass index , who attended our outpatient clinic ) .
we performed a retrospective analysis of the data of our previous study to determine if there is a relationship between levels of vwf and mpv levels .
plasma vwf levels were measured quantitatively by sta - liatest [ ( diagnostica stago ( france ) ] .
we measured mpv in blood samples collected in tubes containing citrate ( 1:4 v / v ) in order to avoid the platelet swelling induced by edta ; samples were analyzed within 1 hour .
( between each group ) and power = 80% , a sample size of > 36 subjects per group was needed to detect an actual difference .
two - group comparisons ( ifg vs. control ) were performed with independent t - tests .
this study was performed in the population of our previous study ( 48 subjects with isolated ifg and 48 normoglycemic healthy controls matched for age , sex , and body mass index , who attended our outpatient clinic ) .
we performed a retrospective analysis of the data of our previous study to determine if there is a relationship between levels of vwf and mpv levels .
plasma vwf levels were measured quantitatively by sta - liatest [ ( diagnostica stago ( france ) ] .
we measured mpv in blood samples collected in tubes containing citrate ( 1:4 v / v ) in order to avoid the platelet swelling induced by edta ; samples were analyzed within 1 hour .
( between each group ) and power = 80% , a sample size of > 36 subjects per group was needed to detect an actual difference .
two - group comparisons ( ifg vs. control ) were performed with independent t - tests .
the main characteristics and laboratory results of the study population are reported in table 1 .
the level of vwf was significantly higher in the ifg group than in the normoglycemic control group ( 111.0852.78% vs. 74.0848.47% , p=0.001 ) .
mpv was significantly higher in the isolated ifg group than in the control group ( 8.490.83 vs. 7.990.57 , p=0.002 ) .
vwf level was positively correlated with mpv level in the ifg group ( r=0.452 , p=0.001 ) ( figure 1 ) .
the present study demonstrated that there was significant correlation between vwf and mpv in subjects with isolated ifg .
these results demonstrate that there was a relationship between endothelial dysfunction and platelet activation in subjects with isolated ifg .
endothelial dysfunction may be triggerring the same mechanisms , and stimulate the production of large platelets in the bone marrow . on the other hand
ihara et al reported positive correlation between vwf and mpv in patients with ischemic heart disease and in patients with aortic aneurysm .
however , with the current data it is not possible to say whether high vwf levels are the cause of large platelet volume .
multiple mechanisms are involved in platelet activation of patients with diabetes mellitus , which can be categorized as follows : 1 ) hyperglycemia , 2 ) insulin deficiency and resistance , 3 ) associated metabolic conditions , and 4 ) other cellular abnormalities , and there may be similar mechanisms for ifg .
several mechanisms have been proposed to contribute to the increased platelet activity caused by hyperglycemia , including the following : 1 ) non - enzymatic glycation of platelet membrane proteins that decrease membrane fluidity , which may increase platelet adhesion ; 2 ) osmotic effect of glucose that activates platelet gp iib / iiia and p - selectin expression ; 3 ) activation of protein kinase c , a mediator of platelet activation , and 4 ) glycation of circulating low - density lipoproteins ( ldl ) , which increases intracellular calcium concentration and nitric oxide ( no ) production .
first , the study population was rather small , thus large - scale studies would be helpful in order to make further comments on the relation between mpv and vwf .
second , unfortunately , we could not perform the measurement of other ed and platelet activation markers .
the third limitation is that our analyses were based on a single baseline determination that may not reflect the patients status over long periods .
in conclusion , our results suggest that vwf seems to be related to platelet volume in subjects with isolated ifg . | summarybackgroundto understand the heterogeneity of platelets , we investigated the correlation between von willebrand factor ( vwf ) and mean platelet volume ( mpv ) in subjects with isolated impaired fasting glucose ( ifg).material / methodswe selected 48 subjects with isolated ifg and 48 normoglycemic control subjects matched for age , sex , and body mass index .
we measured levels of vwf and mpv in all subjects.resultsthe levels of vwf and mpv were significantly higher in the isolated ifg group than the control group ( p<0.05 ) also , vwf level was positively correlated with mpv level in subjects in the isolated ifg group ( r=0.452 , p=0.001).conclusionsour results suggest that vwf seems to be profoundly related to platelet volume in subjects with isolated ifg . |
the divide and conquer technique first described by gimbel1,2 in the late 1980s , has been gradually refined and tested over the years to be safe and reliable.3 it involves formation of deep grooves within the lens to allow for fracturing into smaller segments within the capsular bag using the phacoemulsification ( phaco ) tip and a secondary instrument .
this technique is commonly taught first to surgical trainees , due to its advantages of requiring less bimanual manipulation , greater anterior chamber stability , and high reliance on visual cues,4 compared to more difficult techniques such as the chop technique .
one of the challenging steps of this technique is determining adequate groove depth during sculpting to produce a thin enough posterior plate for effective splitting of the nucleus .
too shallow a groove and the nucleus fails to split , disrupting groove wall integrity , and making further attempts more difficult .
current methods of groove depth assessment are several , and include utilizing phaco tip diameters .
the literature differs in the estimates of how many phaco tips should fit vertically inside a groove , ranging from 24.46 other visual cues include observing the red reflex , with brightening red reflex suggesting thinning posterior plate;46 as well as observing for groove floor linear striations parallel to trench long axis , meeting at posterior y - suture.7 visual techniques include utilizing good stereopsis , with better depth perception at lower magnifications.5 the parallax technique has also been described , which involves observation of the posterior opacities relative to base of groove when small side to side nuclear displacements are made .
less movement would indicate a deeper groove.5,8 this is useful in posterior cortical or subcapsular cataracts where red reflex may be of limited use .
use of phaco tip diameters is a recognized technique to physically estimate depth however , variables such as phaco probe type and exact lens thickness dimensions can help determine more accurately how many phaco tips depths is the optimum for a particular case .
patients consisted of those undergoing routine cataract surgery and were selected based on appropriateness for trainee operators .
factors considered included ability to remain immobile , minimal preexisting ocular disease , clear corneas , deep anterior chambers , lens stability , and medium density nuclear cataracts .
written informed consent was obtained from all patients undergoing cataract operations , including material used for teaching and publication purposes , in our hospital ( hawkes bay hospital , hastings , nz ) . given the intraoperative phaco probe depth technique described is commonly already in routine practice , our paper focuses on adding theoretical calculations to allow for the method to be utilized more effectively and safely . as such ,
this methodology was deemed low risk and out of scope with the nz health and disabilities ethics committee , with no application for ethics approval made .
in the preoperative stage , appropriate patients are determined for trainee operators . as part of routine preoperative cataract assessment ,
biometry is done and measurements of lens thickness are often available . with the modern intraocular lens formulas and biometry devices ,
lens thickness measurements are becoming more commonplace in ophthalmic practice , and are likely to be incorporated into future biometric preoperative measurements routinely .
knowledge of phaco probe dimensions is crucial for determining probe tip vertical height and official manufacturer product dimensions is ideal .
we routinely use a 0.9 mm kelman 45 mini - flared abs tip ( alcon laboratories , inc . , fort worth , tx , usa ) .
the described 0.9 mm is the internal diameter of the tip aperture whilst the outside diameter is actually approximately 1.2 mm , which will also be the vertical height of the phaco tip aperture .
for those mathematically inclined , one could calculate the aperture diameter for any angled tip , ( assuming the probe body diameter was known and the tip was not flared ) via the trigonometric equation to calculate the hypotenuse of an angle as shown in figure 1 . during preoperative planning ,
an estimate of the required number of phase tip depths for each case can be made , aiming to produce a groove of sufficient depth to enable cracking but maintaining a safe distance from the posterior capsule .
for example , if a patient s biometry with a lens thickness of 4.47 mm , using a phaco tip of height of 1.2 mm , an estimated three and half phaco tips would provide adequate depth and still be a safe distance away from the posterior capsule . keeping this figure in mind
, the initial sculpting phase also requires careful recognition of how much of the anterior cortex is removed or disrupted , as measurement will be referenced to the original anterior cortex height .
as the grooves are deepened , measurement of depth is done by placing the phaco tip at the deepest central part of the groove in a fashion so that the aperture is vertically oriented as shown in figure 2 , thus achieving expected vertical height of the probe .
the top of the probe tip is then measured against the groove wall , and the probe is lifted to place the bottom of the tip at the previously noted wall height position .
this continues until the probe reaches the height of the original anterior cortex and from here further grooving can be decided if the required number of probe tip depths have not been reached .
once the required depth has been reached , splitting of the nucleus proceeds as normal .
the main results of this described technique allow the operator to calculate the exact number of appropriate probe depths during grooving for any individual patient , as long as preoperative biometry and phaco tip dimensions are known . at the time of writing
, this technique has been utilized successfully in a small handful of patients during trainee operated surgery .
with the current available methods of estimating phaco groove depth , many are utilized more effectively once a surgeon gains experience and familiarity with intraoperative findings . to a trainee surgeon
however , the knowledge of just how bright a red reflex needs to be before adequate depth is reached is perhaps more difficult to obtain .
this can be further complicated by cases where posterior cortical or subcapsular cataract can limit use of red reflexes . our technique is independent of preexisting lens characteristics or intraoperative findings which may or may not occur in different cases , so can be applied in all cases .
this technique can also be helpful when teaching cataract surgery using a microscope that has a monocular view through the side arm viewing eyepiece .
the use of phaco tips as a measurement device provides a physical way to gauge groove depths and can be utilized by both established and trainee cataract surgeons .
previous texts have recommended grooves of anywhere between 2 and 4 phaco tip depths,46 giving potential groove depths of approximately 2 mm to 4 mm but knowing that cataract lens thickness will vary between individuals ( one study showing thickness variations of 4.260.55 mm),9 these current recommendations may not be ideal . by knowing preoperatively the physical dimensions of the phaco probe tip , together with lens thickness readings obtained from biometry , our technique can
more accurately predetermine an individualized safe groove depth for any patient undergoing cataract surgery . the ideal thickness for successful
lens cracking relies not only on groove depth , but also on other factors such as lens physical characteristics like nuclear density .
this technique should allow those that employ it in phacoemulsification to sculpt to a safe groove depth in order to optimize nucleus splitting and minimize potential for posterior capsule breach .
for the trainee cataract surgeon , it has been demonstrated that early in their training , the rates of pc rupture are higher compared to cases performed when more experience has been gained.10 this technique should prove especially valuable for early trainees , where knowledge of real time grooving depth can be compared to the desired safe groove depth , allowing for increased confidence , efficiency , and procedural safety awareness .
by combining the biometric measurements of an individual patient s lens thickness together with known phaco tip dimensions , individualized safe groove depths can be theoretically determined and applied during divide and conquer cataract surgery . | aimto describe a useful technique utilizing lens thickness from biometric data as well as phacoemulsification ( phaco ) probe tip dimensions , in order to more accurately determine safe groove depth during divide and conquer techniques for cataract surgery.methodssingle center , selection of patients for cataract surgery deemed low risk and suitable for surgical teaching cases .
individual lens thickness measurements from biometry were calculated with known phaco tip dimensions to give an individualized safe number of phaco tip depths for grooving during divide and conquer .
this technique was then applied during cataract surgery.resultsutilization of this technique allows calculation and determination of an appropriate number of phaco tip depths of grooving for each individual patient .
this technique was applied as a teaching tool for surgical trainees , with subsequent successful safe cataract surgeries completed .
no posterior capsule ruptures were noted for these cases.conclusionby combining the biometric measurements of an individual patient s lens thickness together with known phaco tip dimensions , individualized safe groove depths can be theoretically determined and applied during divide and conquer cataract surgery . |
an autogenous arteriovenous fistula ( avf ) is the optimal form of vascular access for hemodialysis ( hd ) due to the longer duration of patency , fewer infections , and lower mortality [ 1 , 2 ] .
the presence of occlusive neointimal hyperplasia at the anastomosis and/or the outflow veins , which may be accelerated by chronic kidney disease , has been considered to be the leading cause of avf failure , while the major problem associated with the creation of an avf is the fact that fistulas do not necessarily mature into a usable vascular access , requiring subsequent revision and construction of another access . the caliber of the vessels used for fistula creation is a pivotal factor predicting the presumable maturation of the constructed fistula , and veins less than 2.5 mm in diameter have poor outcomes .
this report describes the balloon - assisted creation and maturation of an autogenous radial - cephalic arteriovenous fistula ( rcf ) in a patient with small - caliber veins and a radial artery of approximately 2 and 1.5 mm in diameter , respectively .
a 78-year - old female with end - stage renal failure caused by diabetic nephropathy had been undergoing hd treatment for 9 years .
her medical history included arteriosclerosis obliterans , an old myocardial infarction , and complete atrioventricular block , which was managed by the implantation of a definitive pacemaker via the right subclavian vein .
she first began undergoing hd through a left upper arm brachial - cephalic avf , since a forearm rcf created before initiating hd did not mature despite repeated surgical revisions .
the fistula failed 2 years later because of a thrombotic occlusion presumably induced by frequent postdialytic hypotension resulting from excessive interdialytic weight gain .
the patient underwent numerous vascular access interventions , including percutaneous transluminal angioplasty and stenting for thrombotic or stenotic lesions , not only in accessory veins , but also in the central venous system .
the peripheral venous access in the left upper extremity seemed to be exhausted 5 years after the initial intervention , and the right brachial artery was superficially repositioned .
the peripheral venous system in the right upper extremity also did not seem to be feasible to use as the return route , so the patient was dialyzed with a silicone tunneled cuffed catheter inserted through the right femoral vein .
however , thrombotic catheter occlusion developed 5 months after the insertion , and the catheter could only be used for the return route .
finally , a balloon - assisted procedure was used to try to create and expedite the maturation of the autogenous right forearm rcf
. small - caliber veins of approximately 2 mm and a radial artery of 1.5 mm in diameter were confirmed by preoperative digital subtraction angiography ( dsa ) .
the cephalic vein and radial artery were exposed through a transverse incision at the wrist using magnification glasses ( 3.0 ) .
the vein was then freed by wide dissection , and the collateral branch was ligated , followed by a longitudinal venotomy of the posterior wall .
the anterior wall of the radial artery was exposed without any dissection of its trunk , and a 5-mm longitudinal arteriotomy was made . a side - to - end , artery - to - vein anastomosis
palpation just after the creation of the anastomosis failed to confirm the patency of the fistula , and subsequent dsa revealed the stenosis of the juxta - anastomotic vein .
next , the stenotic lesion of the juxta - anastomotic radiocephalic vein was crossed with a 0.018-inch hydrophilic guide wire , and a 3-mm ( 40 mm long ) balloon catheter ( sterling balloon dilatation catheter , boston scientific , natick , mass . ,
usa ) was inflated to 6 atm for at least 1 min , with one overlapping angioplasty under direct visualization ( fig .
the patient underwent subsequent balloon angioplasty on days 20 and 60 to aid in the maturation of the radial artery and the usable portion of the rcf .
balloon angioplasty was performed under radiological guidance on day 20 , with a retrograde radial arterial approach using a 0.018-inch platform combined with a 4-mm balloon catheter for the radial artery ( inflated to 4 atm for 10 s ) , anastomotic region ( inflated to 6 atm for 20 s ) , and the juxta - anastomotic region ( inflated to 8 atm for 20 s ; fig .
angioplasty was performed on day 60 , using a 5-mm balloon catheter from the anastomotic to the juxta - anastomotic lesion .
follow - up fistulography was performed 3 months later , when the rcf became functional and the tunneled cuffed catheter was removed , revealing the matured radial artery and cephalic vein ( fig .
the right rcf has been functional for more than 6 months since the final fistulography .
a wrist avf including an rcf has been the first choice of vascular access for chronic hd , since it is simple to create and preserves more proximal vessels for future access placement with fewer complications than other fistula types .
however , a high level of surgical experience appears to affect the primary success and patency rate of an rcf , thus suggesting that the construction of such access is technically demanding . on the other hand
, the availability of a distal cephalic vein and radial artery will become even more limited for arteriovenous fistula construction with the increasing average age of the end - stage renal failure population .
the survival rate of a successfully created forearm autogenous rcf is excellent , and the 2-year complication - free survival of such a fistula is approximately 75% , while another study by miller et al . demonstrated that the overall success rate of forearm rcf is rather poor .
therefore , it is not unusual that the left forearm rcf created in the current patient before initiating hd did not mature despite repeated surgical revisions .
several studies have evaluated factors that might predict fistula maturation , and preoperative vascular mapping has been shown to improve the rate of fistula placement and overall surgical success rate .
in addition , the criteria for the placement of an autogenous fistula have included a minimum artery diameter of 2 mm , a minimum vein diameter of 2.5 mm , and a lack of stenosis or thrombosis in the draining vein or central veins [ 3 , 8 ] .
the experience in the current patient with a suboptimal artery measuring approximately 1.5 mm in size and veins measuring 2.0 mm in diameter suggests the clinical benefit of balloon - assisted creation and maturation of an autogenous right forearm rcf .
although one may argue that perioperative radiological intervention could be dangerous due to the risk of disruption of the anastomosis , intraoperative interventions are not exceptional and are occasionally indicated in clinical settings .
the most common reason is limited percutaneous vascular access , such as with lesions proximal or distal to a stenosed or occluded portion that is simultaneously treated by vasculotomy . the directly visualized intraoperative procedure may avoid the development of several adverse events , such as vascular perforation or rupture
variability in the techniques used for creating avfs has made it difficult to objectively analyze the results and develop best practice recommendations , despite a large amount of literature on the subject and continued technological advances .
in addition , there are no clear protocols to address accessible vessel maturation with regard to an avf .
the application of intraoperative balloon angioplasty and the subsequent interventional approach has allowed the maturation of an autogenous avf with suboptimal vessels that otherwise could not have been used [ 13 , 14 ] .
the mechanisms of successful balloon angioplasty with vascular access stenoses involves vessel stretching and dissection , which are similar to the mechanisms that have been validated with morphological evaluation of arterial balloon angioplasty [ 15 , 16 ] . on the other hand ,
the vascular access portions dilated by repeated balloon angioplasty are thought to heal mostly by fibrosis , and eventually to become large - diameter fibrous segments [ 15 , 16 ] .
further studies and more extensive clinical experience are required to better determine the optimal interval and appropriate duration of balloon angioplasty not only for post - dilation healing , but also for expediting the maturation of an autogenous avf with small - caliber vasculature .
| the major problem associated with the creation of an arteriovenous fistula ( avf ) , which is the optimal form of vascular access for hemodialysis , is the fact that fistulas do not necessarily mature into a usable vascular access , requiring subsequent revision and construction of another access .
the caliber of the vessels used for fistula creation is a pivotal factor predicting the presumable maturation of the constructed fistula , and veins less than 2.5 mm in diameter have poor outcomes .
this report describes the balloon - assisted creation and maturation of an autogenous radial - cephalic avf in a patient with a small - caliber vein and a radial artery measuring approximately 2 and 1.5 mm in diameter , respectively .
the clinical impact of percutaneous radiological intervention for expediting the maturation of an autogenous avf with small - caliber vessels is also discussed . |
percutaneous liver biopsy is known to have risk of major complications , in particular , hemorrhage , due to the highly vascular liver tissue and limited access for hemostasis at biopsy site .
transarterial embolization and laparotomy are the mainstay of management in the current practice in case of massive post biopsy hemorrhage .
however , they require special angiographic suite or operation theater which may not be easily accessible or readily available .
we report a case of massive post biopsy hemorrhage , which could not be controlled by trans arterial embolization and patient could not undergo laprotomy .
we performed this innovative application of ultrasound ( us)-guided percutaneous microwave ablation to the post biopsy bleeding site to achieve hemostasis .
a 61-year - old man , smoker and social drinker with beta - thalassemia trait , who presented with painless jaundice for 1 month . on admission , his liver function was severely deranged ( bilirubin : 532 , alkaline phosphatase : 876 , alanine transaminase : 98 ) .
hepatitis serology , alpha - fetoprotein , and carcinoembryonic antigen tests were all negative . international normalized ratio ( inr ) and platelet count were normal , but prothrombin time ( pt ) and activated partial thromboplastin ( aptt ) were moderately raised .
an urgent us - guided percutaneous liver biopsy was arranged . a 20-gauge temno needle via coaxial system was used for liver biopsy .
three samples of tissue were obtained from the same site at segment 5 of the liver , followed by gelfoam injection along the track biopsy tract to reduce post biopsy hemorrhage .
the patient went into shock after eight hours during the observation period after liver biopsy .
urgent contrast enhanced computed tomography ( ct ) revealed large intraperitoneal hemorrhage and contrast extravasation from biopsy site [ figure 1 ] .
urgent trans arterial embolization via femoral artery approach attempted but failed due to marked stenosis at origin of the celiac trunk [ figure 2 ] .
laparotomy was contraindicated due to high risk of mortality as the patient developed disseminated intravascular coagulopathy ( dic ) with severely deranged clotting profile .
after discussion between the intervention radiology team and surgical team , urgent us was arranged to localize the site of bleeding from biopsy tract on color doppler study .
urgent us - guided percutaneous microwave ablation was performed after localization of active bleeding site by color doppler us [ figure 3 ] . under us
guidance , microwave needle was inserted into the bleeding zone percutaneously through subcostal approach [ figure 4 ] .
coagulation was done with the acculis mta system , using 140 kw for 6 min , aiming at ~ 4.5 cm 5.5 cm .
successful hemostasis was observed on both color doppler us and contrast enhanced ct performed immediately after microwave ablation . a 61-year - old man with shock after liver biopsy .
contrast enhanced computed tomography of the abdomen demonstrates massive hemoperitoneum and active contrast extravasation from the anterior surface of segment 5 of the liver ( arrow ) .
celiac angiogram of the patient showed marked stenosis and tortuous course of the celiac trunk , common and main hepatic arteries , and the intrahepatic arteries ( arrows ) .
gray scale sagittal ultrasound image of liver with color flow doppler demonstrates active bleeding from the segment 5 of liver corresponding to the biopsy site ( arrow ) .
ultrasound - guided percutaneous microwave ablation performed with microwave needle targeted at the bleeding zone at segment 5 of the liver ( arrow ) demonstrates post ablation changes with cessation of bleeding .
the patient showed improved hemodynamics on the following day with stable hemoglobin level and improved clotting profile .
subsequently , liver biopsy report revealed hepatic amyloidosis , and the patient was wait listed for liver transplant .
unfortunately , patient 's liver function showed continued rapid deterioration and had developed multiorgan failure after admission and unfortunately , succumbed due to cardiac arrest on day eleven after admission .
liver biopsy is an important diagnostic procedure for investigation of liver disease and liver tumors .
however , it is associated with known risks including major bleeding , bile leak , bowel and gallbladder perforation , and pneumothorax .
it is estimated that the complication rate for major bleeding requiring blood transfusion or other procedures is about 1.01.7% and overall mortality rate is about 0.130.33% .
the risk of bleeding is higher in patients with impaired clotting profile and increases serum bilirubin .
currently , angiographic embolization and surgery remain the major treatments of choice for control of post biopsy liver bleeding .
microwave ablation has been initially developed to achieve hemostasis in laparotomy patients during hepatic resection and is a simple , rapid , and definite hemorrhage control method .
percutaneous microwave coagulation therapy ( pmct ) is now mainly used in tumor ablation therapy including liver , lung , renal , and bone malignancies .
no report regarding application of percutaneous microwave ablation for treatment of active bleeding in solid organs has been reported in the literature .
an experimental study in 2008 , using rabbit liver models with active bleeding produced by biopsy needle , showed that percutaneous microwave ablation could significantly decrease blood loss in animal models , and it is a simple and fast method to control blood loss in liver injuries with active bleeding . comparing microwave ablation and existing thermo ablative technologies including radiofrequency ablation , microwave has the advantage of achieving a consistently higher tissue temperature and covering larger tissue volume , thus theoretically allowing faster ablation time and larger area of coagulation . in this case ,
active contrast extravasation was demonstrated on ct and angiogram . however , angiographic embolization failed due to difficult vascular access and anatomy , with atherosclerosis and calcifications along the aortic wall , and irregular stenosis of the hepatic arteries . with dic and unstable hemodynamic ,
thus , we attempted to do an us - guided percutaneous microwave ablation , which not been previously reported .
the prerequisite for this procedure is that the site of bleeding has to be identifiable on us , which in this case could be demonstrated by color doppler study showing a focus of active bleeding from the hepatic surface [ figure 2 ] .
the highest power ( 140 w ) and longest duration ( 6 min ) recommended for liver ablation by the acculis mta system were applied to reach the largest tissue volume of ablation in the shortest period .
after the first 6 minutes of ablation , no further active bleeding from the previous site of biopsy could be demonstrated on color doppler us .
contrast enhanced ct done after percutaneous microwave ablation confirmed that homeostasis was achieved successfully . regarding the risk of bleeding
, we suspect that the cause of massive post biopsy hemorrhage in our case could be related to underlying coagulopathy associated with amyloidosis .
the patient had normal inr and platelet count , but the pt and aptt were abnormal .
coagulopathy is frequent in patients with amyloidosis due to factor x deficiency , decrease vitamin k - dependent clotting factors , and increase antithrombin activity fibrinolysis and dic .
amyloid can also deposit in vascular wall , resulting in weak and stenosed vessels ; thus , angiographic embolization was difficult .
we believe that it has wide potential applications in solid organ hemostasis , especially in cases with contraindications for transarterial embolization , such as severe liver failure , portal vein thrombosis , and difficult vascular access or laparotomy ( e.g. , coagulopathy , unstable hemodynamic , risk of general anesthesia ) .
moreover , percutaneous microwave ablation may be performed at bedside without the need for going to the operation theater or patients not suitable to undergo angiographic suite ; thus , it can be readily used when the previously mentioned setups are not available and in nonmajor hospitals or even in the emergency department .
it may also play a role in other causes of solid organ hemorrhage , such as in trauma or ruptured vascular tumors including hepatocellular carcinoma and renal angiomyolipoma .
moreover , the long - term efficacy and whether there will be delayed bleeding remain unknown .
so far , this is the first reported case using pmct for liver hemostasis ; thus , our experience is very limited .
this is the first reported case of massive post biopsy liver hemorrhage successfully controlled by us - guided percutaneous microwave ablation .
| percutaneous microwave coagulation therapy has been one of the major new developments in tumor ablation .
microwave ablation has also been used intraoperatively to achieve hemostasis at surgical margins in laparotomy .
however , the use of microwave ablation for coagulation and hemostasis through percutaneous approach has not been described in the literature . here
, we report a case of hepatic amyloidosis with massive post biopsy liver hemorrhage , which could not be by transarterial embolization , and subsequently controlled by ultrasound - guided percutaneous microwave ablation . to the best of our knowledge ,
this is the first reported case of this technology application in human . |
family members are classically grouped into three subclasses including one that inhibits apoptosis , a second that induces apoptosis and a third contains that family members , such as bim , that only have a bh3 domain that binds antiapoptotic family members to promote apoptosis . bcl-2
exerts opposing effects with regards to apoptosis compared with bim , consistent with their opposing effects on cell adhesion and migration .
the removal of a single allele of bim is sufficient to prevent the degenerative disorders caused by bcl-2 deficiency [ 3 , 4 ] .
bim expression is also essential for apoptosis of a wide range of growth factor deprived cells , including endothelial cells and pericytes .
therefore , improper modulation of apoptosis could impact the development and/or the pathogenesis of many diseases including diabetic retinopathy .
murine retinal vascular development proceeds after birth and is complete by postnatal day 21 ( p21 ) .
remodeling and pruning of the retinal vasculature continue until p42 [ 7 , 8 ] .
pro- and anti - apoptotic factors regulate apoptosis during retinal vascular development , remodeling , and regression [ 810 ] .
bim influences not only apoptosis but also cell adhesion , migration , and extracellular matrix ( ecm ) protein expression . the ability of bim to impact both apoptosis and the ecm milieu is integral to its role during retinal vessel remodeling , and regression .
however , whether bim expression impacts retinal endothelial cell and pericyte function in a similar manner remains to be determined .
our previous studies demonstrated that bim modulates retinal vascular development , remodeling , and regression .
bim deficient ( bim ) mice demonstrated precocious formation of the retinal vascular plexus , increased vascular density , and attenuated hyaloid vessel regression .
loss of bim expression also protected the retinal vasculature from hyperoxia - mediated vessel obliteration and neovascularization during oxygen induced ischemic retinopathy ( oir ) .
these studies indicated that bim expression is essential for retinal vascular remodeling but did not implicate a specific vascular cell type .
thus , understanding the role bim plays in modulating endothelial cell and pericyte function will give us further insight into how aberrant regulation of retinal vessel development and remodeling can be avoided to preserve vision .
endothelial cells and pericytes play specialized roles in the retina during the development and maintenance of the vasculature .
pericyte coverage of vascular sprouts stabilizes the vessels while endothelial cells provide the inner lining of the blood vessel [ 12 , 13 ] .
retinal endothelial cell and pericyte dysfunction can lead to retinal vascular rarefaction and subsequent vision loss as the one that occurs in diabetic retinopathy . here
, we assessed whether the loss of the proapoptotic protein bim differentially impacts the retinal endothelial cell and pericyte function .
we observed an increased vegf expression , ecm protein production , cell migration , proliferation , and adhesion in bim retinal endothelial cells compared with wild - type cells . although the decreased endothelial nitric oxide synthase ( enos ) expression corresponded with decreased nitric oxide ( no ) production in bim retinal endothelial cells , capillary morphogenesis was similar to that observed in wild - type retinal endothelial cells .
in contrast to bim retinal endothelial cells , bim pericytes displayed decreased proliferation compared with their wild - type counterpart .
coculturing wild - type or bim retinal endothelial cells with bim pericytes diminished capillary morphogenesis .
the data presented here demonstrate that the deletion of bim differentially impacts retinal endothelial cell and pericyte function , perhaps through the modulation of response to their microenvironment .
to further investigate the role bim plays in retinal vascular function , we isolated retinal endothelial cells and pericytes from wild - type and bim mice as previously described [ 2 , 1417 ] .
we first examined retinal endothelial cell and pericyte morphology and their expression of cell specific markers to confirm that these cells maintain their endothelial cell and pericyte characteristics .
bim retinal endothelial cells displayed a slightly elongated morphology compared to wild - type cells when plated on gelatin - coated plates ( figure 1(a ) ) .
the morphology of wild - type and bim retinal pericytes was similar ( figure 1(b ) ) .
wild - type and bim retinal endothelial cells expressed the endothelial cell markers ve - cadherin and pecam-1 ( figure 1(c ) ) .
retinal pericytes expressed the pericyte markers platelet derived growth factor - receptor ( pdgfr ) and neuroglia proteoglycan 2 ( ng2 ) ( figure 1(d ) ) .
wild - type and bim retinal endothelial cells and pericytes were incubated with staurosporine to induce apoptosis .
although the basal levels of apoptosis were similar , upon incubation with staurosporine for 24 h , bim endothelial cells and pericytes demonstrated less apoptosis compared to wild - type cells ( figures 2(a ) and 2(b ) ) .
next cell proliferation was examined by counting the number of cells every other day for 2 weeks .
figure 2(c ) shows that bim retinal endothelial cells proliferated at a faster rate than their wild - type counterpart .
in contrast , bim pericytes demonstrated a reduced rate of proliferation compared to their wild - type counterpart ( figure 2(d ) ) .
a confluent monolayer of wild - type and bim endothelial cells ( figure 3(a ) ) and pericytes ( figure 3(b ) ) was wounded , and 5-fluorouracil ( 5-fu ) was added to prevent cell proliferation . both bim deficient retinal endothelial cells and pericytes migrated faster than their wild - type counterparts ( figures 3(a ) and 3(b ) ) . a quantitative assessment of the scratch wound data for endothelial cells and pericytes is shown in figures 3(c ) and 3(d ) , respectively .
enhanced migration of bim endothelial cells and pericytes was also observed using a transwell migration assay as shown in figures 3(e ) and 3(f ) , respectively .
changes in migration of bim retinal vascular cells could be due to altered cell adhesion .
we next examined the ability of wild - type and bim endothelial cells ( figure 4(a ) ) and pericytes ( figure 4(b ) ) to adhere to fibronectin , collagen i , collagen iv , and vitronectin .
bim deficient endothelial cells and pericytes displayed increased adhesion to all of these matrices compared to their wild - type counterpart .
next we analyzed the integrin expression on the surface of retinal endothelial cells and pericytes by facscan analysis ( figures 5(a ) and 5(b ) ) .
wild - type and bim endothelial cells expressed similar levels of 5 , v3 , and 1 integrins on their surface while wild - type and bim retinal pericytes expressed similar levels of 2 , v3 , 1 , and 3 integrins .
thus , the increased adhesion noted in bim retinal vascular cells may be independent of significant changes in the integrin expression and may be due to alterations in the affinity and/or avidity of these integrins .
modulating the ecm milieu can influence vascular cell functions including cell adhesion and migration . to examine whether the lack of bim expression differentially impacts retinal endothelial cell or pericyte ecm expression , serum - free conditioned medium from these retinal vascular cells
retinal endothelial cells from bim mice displayed an increased expression of tenascin c , osteopontin , fibronectin , and tsp1 compared to their wild - type counterpart ( figure 6(a ) ) .
in contrast , bim retinal pericytes displayed similar levels of tenascin c , fibronectin , but an increased tsp1 and a decreased osteopontin expression ( figure 6(b ) ) . the activation of enos and akt1 by vegf promotes angiogenesis [ 1820 ] . in the absence of bim , vegf expression was dramatically increased in retinal endothelial cells and pericytes ( figures 7(a ) and 7(b ) ) . however , in bim retinal endothelial cells , the increased vegf expression did not affect akt , phospho - akt , or hsp90 expression ( figure 7(c ) ) .
bim retinal endothelial cells also demonstrated nearly undetectable enos expression ( figure 7(c ) ) which corresponded with a 25-fold decrease in no production ( figure 7(d ) ) .
thus , vegf activation of enos through the activation of akt is uncoupled in the absence of bim .
capillary morphogenesis is fundamental in vascular development and remodeling in which pericytes play a major role .
the ability of pericytes to migrate and produce survival factors is central to their recruitment along the developing endothelium .
however , pericytes typically do not undergo any substantial morphogenesis in matrigel . in the retina , endothelial cells and pericytes
, we evaluated the ability of pericytes from wild - type and bim mice to impact retinal endothelial cell capillary morphogenesis .
wild - type and bim endothelial cells demonstrated a similar number of branch points in matrigel ( figures 8(a)8(c ) ) .
however , bim endothelial cells formed rather stunted capillary - like tubes compared with wild - type cells ( figures 8(a)8(c ) ) . next , we cocultured wild - type retinal endothelial cells with either wild - type or bim pericytes .
wild - type retinal endothelial cells cocultured with wild - type pericytes demonstrated an improved tubular network compared to wild - type endothelial cells alone ( figures 8(a)8(c ) ) .
in contrast , wild - type retinal endothelial cells cocultured with bim pericytes demonstrated diminished capillary morphogenesis .
bim endothelial cells cocultured with wild - type pericytes demonstrated enhanced capillary morphogenesis while cocultures of bim endothelial cells and pericytes demonstrated diminished capillary morphogenesis ( figures 8(a ) and 8(c ) ) .
thus , coculturing of retinal endothelial cells with bim pericytes resulted in diminished endothelial cell capillary morphogenesis .
next , we addressed whether an increased vegf expression contributed to the inability of wild - type endothelial cells to undergo capillary morphogenesis when cocultured with bim pericytes .
vegfr-1 ( flt-1 ) has a higher affinity for vegf compared to vegfr-2 and can compete for vegf binding to vegfr-2 and its activation .
thus , the soluble form of flt-1 can act as a trap for vegf and prevent its signaling . here , we used sflt-1 fc chimera to demonstrate that the negative impact of bim pericytes on capillary morphogenesis of retinal endothelial cells is driven by the production of excess vegf .
the sflt1 was added to wild - type endothelial cell and bim pericyte coculture experiments , and capillary morphogenesis was monitored ( figure 8(d ) ) .
as shown above , coculture of wild - type endothelial cells with bim pericytes demonstrated decreased numbers of branch points compared to wild - type endothelial cells alone .
the addition of sflt1 to wild - type endothelial and bim pericyte coculture experiments significantly restored branching morphogenesis in matrigel .
the addition of control igg to wild - type endothelial cell and bim pericyte coculture experiments did not impact the number of branch points ( figures 8(d ) and 8(e ) ) .
therefore , enhanced vegf expression contributes to the decreased capillary morphogenesis of endothelial cells cocultured with bim pericytes .
aberrant modulation of apoptosis has a reoccurring theme in many diseases that result in blindness .
the regression of the hyaloid vasculature is an apoptosis - driven process in which bim expression plays a central role .
failure of the hyaloid vasculature to regress is a common congenital ocular malformation that can lead to blindness .
improper modulation of apoptosis can also contribute to retinal vascular rarefaction and/or neovascularization , observed during retinopathy of prematurity and diabetic retinopathy .
thus , understanding how apoptosis is modulated in retinal vascular cells , including endothelial cells and pericytes , will further our understanding of normal and abnormal retinal vascularization and vascular cell autonomous functions .
the retina has the highest numbers of pericytes covering its blood vessels compared to other tissues .
the appropriate interaction of pericytes with each other and endothelial cells is important for vessel maturation and stabilization .
altered pericyte migration or adhesion may disrupt or enhance these interactions leading to vasculopathies or increased vascular stabilization . here , we show that pericyte migration and adhesion are enhanced in the absence of bim perhaps as a result of their increased vegf expression . increased
pericyte migration may lead to greater pericyte coverage of the retinal vasculature , which may contribute to the precocious formation of the deep vascular plexus and protection from hyperoxia - mediated vessel obliteration that we previously observed .
maturation of the retinal vasculature involves remodeling and pruning of vessels into an organized branched network of vessels to meet the demand of the tissue .
modulation of apoptosis by bcl-2 family members facilitates retinal vascular remodeling and regression [ 11 , 24 ] .
bim expression is required for retinal capillary pruning , hyaloid vessel regression , and hyperoxia - induced retinal vessel obliteration . here
, we have extended these findings to delineate the role bim plays in retinal endothelial cell and pericyte function .
we show that in addition to enhanced migration , retinal pericytes from bim mice were resistant to an apoptotic challenge and demonstrated an increased adhesion to extracellular matrices .
extracellular matrix protein expression by pericytes was only modestly impacted by the loss of bim expression .
perhaps an increased pericyte adhesion in the absence of bim enhances pericyte survival and coverage of retinal vessels .
this may explain the resistance of the retinal vasculature of bim mice to developmental pruning or excessive pruning in response to hyperoxia .
therefore , modulating pericyte adhesion and migratory properties may be essential for retinal vascular pruning .
since vegf withdrawal can lead to endothelial cell apoptosis , determining how vegf levels can be modulated by expression of bcl-2 family members may aid in our understanding of normal and aberrant retinal vascularization and pruning .
here , we show that bim deficiency increases vegf levels in both endothelial cells and pericytes .
pericytes act as an angiogenic soup , producing 7-fold higher vegf levels than their endothelial cell counterpart ( figure 7 ) . in the absence of bim ,
vegf expression in retinal endothelial cells increased to nearly twice that observed in wild - type pericytes .
unfortunately , vegf can also inhibit blood vessel maturation and ablate pericyte coverage of nascent sprouts causing vessel destabilization .
the increased vegf produced by bim pericytes may result in the diminished capillary morphogenesis observed when cocultured with either wild - type or bim endothelial cells .
have also defined vegf as an inhibitor of neovascularization which is consistent with the inability of bim mice to undergo neovascularization following hyperoxia - induced ischemia .
however , retinal development in these mice proceeds quite well with precocious formation of the retinal vascular plexus and increased vascular density in contrast to what has been previously shown in fibrosarcomas [ 11 , 25 ] .
thus , the ability of bim to impact cell survival may be , in part , through the modulation of vegf expression .
phosphorylation of enos can mediate the proangiogenic activity of vegf . here , we show an increased vegf expression but a decreased enos expression in bim retinal endothelial cells , consistent with our previous studies in bim kidney and lung endothelial cells .
moreover , our recent studies further support a reciprocal relationship between vegf and enos expressions in endothelial cells .
our previous studies demonstrated that knocking down enos expression in kidney endothelial cells from diabetic mice not only decreased vegf expression but also restored migration and capillary morphogenesis .
thus , local regulation of bim expression may play a central role in vascular pruning through modulating enos and vegf expressions .
our previous studies demonstrated an inability of the retinas from bim mice to undergo retinal vascular pruning leading to increased retinal cellularity and capillary loops .
these mice were also resistant to hyperoxia - mediated vessel obliteration and neovascularization during oir , as well as a hyaloid vessel regression .
thus , bim expression may modulate retinal vascular pruning by localized changes in retinal vascular cell adhesion , migration , and vegf expression ( figure 9 ) thereby , either protecting or inducing pruning by localized apoptosis .
the mice used for these studies were maintained and treated in accordance with our protocol approved by the university of wisconsin animal care and use committee .
immortomice expressing a temperature - sensitive sv40 large t antigen were obtained from charles river laboratories ( wilmington , ma , usa ) .
bim mice ( jackson laboratory , bar harbor , me , usa ) were crossed with immortomice and screened as previously described [ 2 , 17 ] . to isolate retinal endothelial cells , retinas from approximately four- to five - week - old wild type and bim immortomice
were dissected out aseptically and placed in serum - free dulbecco 's modified eagle medium ( dmem ) containing penicillin / streptomycin ( sigma , st .
the retinas were pooled together , rinsed with dmem , minced into small pieces in a 60 mm tissue culture dish using sterilized razor blades , and digested in 5 ml of collagenase type i ( 1 mg / ml in serum - free dmem ; worthington , lakewood , nj , usa ) for 3045 minutes at 37c . following digestion , dmem containing 10% fetal bovine serum ( fbs ) was added and cells were pelleted .
the cellular digests were then filtered through a double layer of sterile 40 m nylon mesh ( sefar america inc .
, hanover park , il , usa ) , centrifuged at 400 g for 10 min to pellet cells , and then washed twice with dmem containing 10% fbs .
the cells were resuspended in 1.5 ml medium ( dmem with 10% fbs ) and incubated with sheep anti - rat magnetic beads precoated with anti - pecam-1 antibody ( mec13.3 , bd biosciences , bedford , ma , usa ) as described previously . after affinity binding ,
bound cells in endothelial cell growth medium were plated into a single well of a 24-well plate precoated with 2 g / ml of human fibronectin ( bd biosciences ) .
endothelial cells were grown in dmem containing 10% fbs , 2 mm l - glutamine , 2 mm sodium pyruvate , 20 mm hepes , 1% nonessential amino acids , 100 g / ml streptomycin , 100 u / ml penicillin , heparin at 55 u / ml ( sigma , st .
louis , mo , usa ) , endothelial growth supplement 100 g / ml ( sigma , st .
louis , mo , usa ) , and murine recombinant interferon- ( r&d , minneapolis , mn , usa ) at 44 units / ml .
cells were progressively passed to larger plates , maintained , and propagated in 1% gelatin - coated 60 mm dishes .
retinal endothelial cells were positive for b4-lectin ( a mouse endothelial cell specific lectin ) and expressed pecam-1 and ve - cadherin as previously described [ 2 , 14 ] .
pericytes were isolated from mouse retina by collecting retinas from one litter ( 6 - 7 pups , 4 weeks old ) using a dissecting microscope .
retinas ( 12 to 14 ) were rinsed with serum - free dulbecco 's modified eagle 's medium ( sigma ) , pooled in a 60 mm dish , minced , and digested for 45 min with collagenase type ii ( 1 mg / ml ; worthington , lakewood , nj , usa ) with 0.1% bovine serum albumin ( bsa ) in serum - free dmem at 37c .
cells were rinsed in dmem containing 10% fetal bovine serum ( fbs ) and centrifuged for 5 min at 400 g .
digested tissue was resuspended in 4 ml of dmem containing 10% fbs , 2 mm l - glutamine , 100 g / ml streptomycin , 100 u / ml penicillin , and the murine recombinant interferon- ( r&d , minneapolis , mn , usa ) at 44 u / ml .
cells , tissue , and medium were evenly divided into 4 wells of a 24-well tissue culture plate and maintained at 33c with 5% co2 .
cells were progressively passed to larger plates , maintained , and propagated in 60 mm dishes .
the experiments described here were performed with two separate isolations of cells with similar results .
apoptosis was determined by measuring caspase activation using a caspase - glo 3/7-assay kit as recommended by the supplier ( promega , madison , wi , usa ) . the assay provides caspase-3/7 devd - aminoluciferin substrate and the caspase 3/7 activity is detected by luminescent signal .
for the assay , cells were plated at 8 10 per well of a 96-well plate . as an oxidative or apoptotic stimulus
, cells were incubated with 10 nm staurosporine ( invitrogen ) , in growth medium for 24 h at 33c .
caspase activity was detected using a luminescent microplate reader ( victa2 1420 multilabel counter , perkinelmer , waltham , ma , usa ) .
cells ( 4 10 ) were plated in 60 mm tissue culture dishes and allowed to reach confluence ( 2 - 3 days ) .
after aspirating the medium , cell layers were wounded using a 1 ml micropipette tip .
plates were then rinsed with pbs , fed with growth medium containing 100 ng / ml of 5-fluorouracil ( 5-fu ) , to rule out potential contribution of differences in cell proliferation .
the distance migrated was determined as a percent of total distance for quantitative assessments as described previously .
matrigel ( 10 mg / ml ; bd biosciences ) was applied at 0.5 ml / well of a 6-well tissue culture dish and incubated at 37c for at least 30 minutes to harden .
cells were removed using trypsin - edta , washed with growth medium once , and resuspended at 1 10 cells per ml in serum - free growth medium .
for coculture experiments , retinal endothelial cells and pericytes were used at a 1 : 1 ratio as we previously described .
cells ( 2 ml ) were gently added to the matrigel coated plates , incubated at 37c , monitored at ~18 h , and photographed using a nikon microscope equipped with a digital camera . in some cases ,
10 g / ml of control igg or sflt1-fc chimera ( r&d systems ) was added to the coculture experiments . for a quantitative assessment of the data
varying concentrations of fibronectin , human type i and iv collagen , and vitronectin ( bd biosciences ) prepared in tbs with camg ( 2 mm each ; tbs with camg ) were coated on 96-well plates ( 50 l per well ; nunc maxisorp plates , fisher scientific ) overnight at 4c .
plates were then rinsed four times with tbs with camg and blocked with 200 l of 1% bsa prepared in tbs with camg for at least 1 h at room temperature .
cells were removed using 1.5 ml dissociation solution , washed once with tbs , and resuspended at 5 10 cells / ml in hepes - buffered saline ( 25 mm hepes , ph 7.60 , 150 mm nacl , and 4 mg / ml bsa ) . after blocking , plates were rinsed with tbs camg once , 50 l of cell suspension was added to each well containing 50 l of tbs with camg , and the cells were allowed to adhere for 90 min at 37c in a humidified incubator .
nonadherent cells were removed by gently washing the plate four times with 200 l of tbs with camg until no cells were left in wells coated with bsa . the number of adherent cells in each well was quantified by measuring the levels of intracellular acid phosphatase .
cells were lysed in 100 l of lysis buffer ( 50 mm sodium acetate ph 5.0 , 1% triton x-100 , and 4 mg / ml p - nitrophenyl phosphate ) and incubated at 4c overnight .
the reaction was neutralized by adding 50 l of 1 m naoh , and the absorbance was determined at 405 nm using a microplate reader ( thermomax , molecular devices ) .
all samples were prepared in triplicate , and the experiments were repeated at least three times with similar results .
cells were plated at 4 10 in 60 mm dishes coated with 1% gelatin ( endothelial cells ) or uncoated ( pericytes ) and allowed to reach nearly 90% confluence in 2 days .
the cells were then rinsed once with serum - free medium and incubated with serum - free dmem for 48 hours .
then , conditioned medium ( 2 ml ) was collected and clarified by centrifugation .
the 45 l of the sample was mixed with appropriate volume of 6x sds buffer and analyzed by sds - page ( 420% tris glycine gel ; invitrogen , carlsbad , ca ) . in some cases ,
total protein lysates were prepared from these cells in a modified ripa buffer ( 142.5 mm kcl , 5 mm mgcl2 , 10 mm hepes , ph 7.4 , 2 mm orthovanadate and 2 mm sodium difluoride , 1% nonidet p-40 , and a complete protease inhibitor cocktail ( roche , mannheim , germany ) ) .
the proteins were transferred to a nitrocellulose membrane , and the membrane was incubated with an antifibronectin ( sigma ) , a rabbit anti - chicken tenascin - c polyclonal antibody ( thermo scientific ; pierce , rockford , il , usa ) , anti - tsp1 monoclonal antibody ( clone a6.1 , neo marker , fremont , ca , usa ) , antiosteopontin ( r&d , minneapolis , mn ) , anti--actin ( thermo scientific ; pierce , usa ) , anti - hsp90 ( cell signaling technology ) , anti - akt ( cell signaling ) antiphospho - akt ( cell signaling ) , antiphospho - enos ( cell signaling ) , and anti - enos ( sc-654 ; santa cruz technology , santa cruz , ca ) .
membranes were washed , incubated with horseradish - peroxidase - conjugated secondary antibody ( 1 : 5000 , jackson immunoresearch laboratories , west grove , pa ) for 1 hour at room temperature , and the protein was visualized according to the chemiluminescent procedure ( chemiluminescence reagent ; ge biosciences ) [ 2 , 14 ] .
the cells were washed once with pbs containing 0.04% edta and incubated with 2 ml of dissociation solution ( sigma ) to remove the cells from the plate .
the cells ( 10 ) were washed with tbs , blocked in tbs containing 1% goat serum on ice for 20 minutes , and incubated with the appropriate dilution of primary antibody , anti - pecam-1 ( bd pharmingen ) , antivascular endothelial ( ve)-cadherin ( alexis biochemical , san diego , ca ) , anti-1 ( millipore ) , anti-5 ( mab1949 ; millipore ) , anti-2 ( chemicon ) , anti-3 ( mab1957 ; millipore ) , anti-v3 ( mab1976z ; millipore ) , control igg ( jackson ) , rabbit anti - ng2 ( ab5320 ; millipore , temecula , ca ) , and rat anti - mouse pdgfr ( ebiosciences , san diego , ca ) . for antibodies that required cell permeabilization ,
cells were removed , washed with pbs , fixed with 2% paraformaldehyde on ice for 30 min , washed with pbs , and resuspended in pbs containing 0.1% triton - x-100 and 0.1% bsa containing appropriate dilution of primary antibody .
the cells were washed with tbs containing 1% bsa and then incubated with the appropriate fitc - conjugated secondary antibody ( jackson immunoresearch , west grove , pa ) for 30 min on ice . after the incubation , the cells were washed twice with tbs containing 1% bsa and resuspended in 0.5 ml of tbs containing 1% bsa .
analysis was performed on a facscan caliber flow cytometer ( becton - dickinson , franklin lakes , nj ) .
transwell filters ( corning , acton , ma ) were coated with 2 g / ml fibronectin in pbs and incubated overnight at 4c .
the bottom of the transwell was rinsed with pbs and blocked with 2% bsa in pbs for 1 h at room temperature .
the transwell was rinsed with pbs , and 500 l serum - free dmem was added to the bottom of each well , and 1 10 cells in 100 l of serum - free medium were added to the top of the transwell membrane . following 4 hours in a 33c tissue culture incubator ,
the cells and medium were aspirated and the upper side of the membrane was wiped with a cotton swab .
the cells that migrated through the membrane were fixed with 4% paraformaldehyde , stained with hematoxylin - eosin , and mounted on a slide .
ten - high power fields ( 200 ) of cells were counted for each condition and the average and standard error of the means were determined .
all samples were prepared in duplicate , and the experiment was repeated at least three times with similar results .
vegf protein levels produced by vascular cells were determined using a mouse vegf immunoassay kit ( r&d systems , minneapolis , mn ) .
cells were plated at 6 10 cells on 60 mm tissue culture dishes and allowed to reach approximately 90% confluence .
the cells were then rinsed once with serum - free dmem and were grown in serum - free medium for 2 days .
conditioned medium was centrifuged at 400 g for 5 min to remove cell debris , and 50 l was used in the vegf immunoassay .
the assay was performed in triplicate as recommended by the manufacturer and was normalized to the number of cells .
the amount of vegf was determined using a standard curve generated with known amounts of vegf in the same experiment .
the assay was repeated twice using two different isolations of endothelial cells with similar results .
cells were plated in black wall clear bottom microtest 96-well plates ( bd # 35 3948 ; 1 10 cells in 100 l ) . the next morning
the medium was changed to endothelial cell or pericyte medium containing 30 m daf - fm diacetate ( invitrogen ; d-23842 ) and 5 g / ml of celltracker red ( invitrogen ; c34552 ) . following a 40 min incubation at 33c
, fresh medium was placed on the cells and the incubation continued for 20 min .
the wells were washed with tbs and the cells in each well were resuspended in 100 l of tbs .
statistical differences between groups were evaluated with an unpaired t - test ( two - tailed ) . | apoptosis of vascular cells , including pericytes and endothelial cells , contributes to disease pathogenesis in which vascular rarefaction plays a central role .
bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix ( ecm ) protein expression .
endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation . here
we set out to determine the cell autonomous impact of bim expression on retinal endothelial cell and pericyte function using cells prepared from bim deficient ( bim/ ) mice .
bim/ endothelial cells displayed an increased production of ecm proteins , proliferation , migration , adhesion , and vegf expression but , a decreased enos expression and nitric oxide production .
in contrast , pericyte proliferation decreased in the absence of bim while migration , adhesion , and vegf expression were increased .
in addition , we demonstrated that the coculturing of either wild - type or bim/ endothelial cells with bim/ pericytes diminished their capillary morphogenesis .
thus , our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function . |
although sanitation
has improved dramatically in the past decade ,
globally 2.4 billion people lack access to improved sanitation .
an
estimated 946 million lack access to any sanitation facility and practice
open defecation , although the actual number
is probably much higher . fecal contamination
of the environment from poor sanitation together with poor handwashing
are responsible for an estimated 577 000 deaths annually .
additionally , there is growing evidence that ,
through environmental enteropathy , open defecation contributes to
more malnutrition than previously thought and
could be responsible for approximately half of child stunting .
many households
construct latrines for improved social status and dignity ; there are also potential gender - equity benefits , the benefit of increased school attendance for girls , and economic benefits including time savings
and increased productivity .
community - led
total sanitation ( clts ) emerged in the year 2000
as a participatory approach to address open defecation .
clts aims
to generate emotional reactions to open defecation during a community
meeting such as shame and disgust and to elicit collective action
on sanitation .
clts is now a well - established
approach that has been implemented in over 50 countries .
the water institute at the university of north carolina
at chapel hill ( unc ) and plan international ( plan ) conducted a systematic
review of journal - published and gray literature .
the review found
few published studies on clts or related approaches , which tended
to focus on impacts .
the gray literature
was more extensive and focused on project settings and processes rather
than impacts .
gray literature included a preponderance of low - quality
study designs and data - collection methods . a randomized controlled
trial of clts in mali reported a 23 percentage
point reduction in open defecation among adults , and a 30 percentage
point increase in latrine ownership .
it
also reported beneficial health impacts in the form of increased height - for - age
and decreased stunting among children despite there being no impact
on diarrheal incidence . a case study in zimbabwe reported that latrine
ownership and use was higher where clts had been implemented than
in other villages .
a total of five evaluations
of india s total sanitation campaign ( tsc ) , which includes
some clts features with the addition of hardware subsidies , have shown
positive impacts on latrine access , although only two demonstrated health impacts .
water- , sanitation- , and hygiene- ( wash ) related health outcomes
are difficult to measure and to attribute to wash interventions , and
a failure to demonstrate health impact does not necessarily indicate
the absence of health benefits .
our study is designed as operational research and has the aim of
generating findings with immediate implications for policy and practice .
to address a gap in the evidence , we investigated how context and
process ( or implementation arrangements ) influence clts effectiveness .
we applied collaborative design by an implementation organization
( plan ) and a research institute ( unc ) , focused on an existing public health program , compared different institutional arrangements for public
service delivery , and here report on
context and the intervention process in addition to outcomes .
health extension workers ( hews ) are tasked
with facilitating clts
in ethiopia , where there have been dramatic reductions in open defecation
since clts was introduced .
a kebele is the lowest
administrative unit in ethiopia and is composed of 2030 villages
and approximately 5000 people .
every kebele in ethiopia has one health
post staffed by one to three hews who typically are from that geographic
area , speak the local language , and share cultural background with
residents .
plan has explored training teachers
as clts facilitators to alleviate the burden on hews and enable increased
follow - up activities with some signs of success .
teachers have demonstrated aptitude for promoting
healthy wash
behaviors previously for household water treatment and handwashing
in kenya , for student handwashing in
china , and for schistosomiasis prevention
in tanzania .
however , to date , there are no
studies published on teachers leading sanitation promotion at the
community level .
we compared two interventions in different regions
of ethiopia : conventional clts , in which health workers and local
leaders provided facilitation , and an alternative intervention , in
which teachers provided facilitation .
clts implementation in ethiopia
consists of the standard three stages from the clts handbook : pretriggering , triggering , and follow - up .
triggering consists
of a community meeting in which outside facilitators use tools ( such
as sanitation mapping ) designed to trigger an emotional
response and a collective desire to improve the situation .
all
of the villages are under the authority of the kebele leaders , who
must approve any development activities within villages and often
coordinate them . in the follow - up , facilitators visit villages to
monitor progress and guide them in eliminating open defecation . in
ethiopia ,
the follow - up includes emphasis on handwashing with soap
or ash at key times .
when ready , a kebele
can request certification of open - defecation - free ( odf ) status by
the government .
the first
group of actors ( conventional clts ) is composed of
hews who lead facilitation and kebele leaders who support them . the
second ( teacher - facilitated clts ) is composed of teachers
facilitating clts . in october 2012 , plan initiated the interventions
by training the two groups of facilitators , who then facilitated clts
for the following year .
the same clts tools and activities were used
by both groups of facilitators ; the only difference was in who facilitated .
plan provided monthly guidance to both groups of facilitators and
occasionally observed their facilitation in communities . in
the two kebeles that received conventional clts , plan trained
one to three hews and eight leaders per kebele ( table 1 ) . in the four teacher - facilitated clts kebeles ,
plan trained 10 to 28 teachers and two leaders per kebele .
a timeline of implementation activities for both interventions is
in the supporting information , and the
teacher training manual and report on detailed implementation activities
written by plan are available online .
the hew trained from kebele 1 in
oromia left their job in may 2013 ( 7 months into implementation ) ;
a replacement hew had not been hired by the follow - up survey .
the
eight kebele leaders from kebele 1 continued facilitating clts for
the duration of the intervention .
teachers and hews were surveyed
at follow - up on their activity level during the clts interventions .
kebele leaders were not surveyed ; their activity
level outside of triggerings is not known .
plan had prior experience implementing clts , training facilitators ,
and collaborating with the government on sanitation and hygiene .
the
government of ethiopia had included clts in national policy and had
institutional support mechanisms in place , such as a memorandum of
understanding between three ministries for coordination on water ,
sanitation , and hygiene .
the catchment
areas for schools and health posts are the same in ethiopia , the kebele ,
which enables teachers to facilitate clts .
teachers are known by community
members ( the intended beneficiaries of clts ) and speak the local language .
development activities are typically coordinated at the kebele level ,
and teachers are familiar with all of the villages within their kebele .
this is a mixed - methods study that uses
quantitative data from a quasi - experimental design and qualitative
data from interviews .
we selected six kebeles from two regions , prematched
them within each region on latrine access and kebele size ( number
of households ) using 2011 census data collected from district government ,
and then manually assigned them to receive conventional or teacher - facilitated
clts ( figure 1 ) .
we
chose prematching because with six study sites , it was more likely
than random assignment to result in similar comparison groups .
prematching
is established as a valuable tool for evaluating community - demand - driven
sanitation policies .
we used a difference - in - difference
estimator to test for a difference in the effectiveness of the two
interventions in changing sanitation practices because it removes
baseline differences in the outcome and accounts for time - invariant
differences between comparison groups .
we used a
multivariable regression , incorporating robust covariate indicators ,
to further account for baseline differences between comparison groups .
because this study was designed to compare two interventions , no do - nothing
control group was included .
timeline and sequence of the quasi - experimental
study design and
execution . while we used prematching , a difference - in - difference
estimator ,
and a multivariable regression to address any baseline differences
between comparison groups ; outcomes can not be attributed solely to
the different interventions because this study involves nonrandom
assignment .
interviews with ngo employees , facilitators , and government
and supplemental data collected from household surveys were used to
understand the implementation process and to explore possible explanations
for the differences in outcomes between interventions .
a pair of regions where plan had prior clts
experience and government collaboration were selected : oromia and
the southern nations , nationalities , and peoples ( snnp ) regions .
one district with no prior clts was selected from each region ( deksis
and dara districts , respectively ) .
a total of three road - accessible
kebeles with no major towns and low reported latrine access in the
2011 census were selected from each district .
the sample size
( number of households ) was set to detect a difference between a 30
and 40 percentage point ( pp ) reduction in open defecation ( i.e. , a
10 pp difference - in - difference , = 0.8 , = 0.05 ) .
on the basis of the average village size , we randomly sampled 75
villages .
all 2444 households within sampled villages were approached
for surveying . at the baseline , 2182 households were surveyed . at
the follow - up ,
2263 households were surveyed ( figure 1 ; sampling details in the supporting information ) .
household surveys , latrine and handwashing
station observations , and health extension worker and teacher surveys
were completed at the baseline ( september 2012 ) and the follow - up
( october 2013 ) .
household surveys covered demographics , sanitation ,
hygiene , interactions , and recall of clts events .
indicators were
selected from review of prior wash research and input
from unc and plan .
sanitation outcomes were assessed by asking heads - of - households
where they primarily defecated and their handwashing practices .
those
reporting using a latrine were asked a series of questions to determine
if it was private , shared , or communal .
hew and teacher
surveys covered clts - related knowledge , attitudes , and practices .
all surveying was completed by an independent contractor with extensive
experience in ethiopia , one team lead per region , and experienced
local surveyors .
household surveys were translated into the local
languages ( oromo and sidama ) ; hew and teacher surveys were translated
into amharic by the contractor and translations checked by an independent
wash specialist and rechecked by plan .
printed survey tools were pretested
during training , piloted in nonproject kebeles , and revised in consultation
with unc .
survey team leaders reviewed
surveys each evening , and plan staff were available to help locate
missed households .
surveyors were audited by plan resurveying one
randomly selected village per kebele ( 2340 households per
audited village ) with 11 questions from the full survey . the
clts process was monitored with checklists filled out by the plan
coauthors and interviews with plan staff ( including coauthors ) and
district government .
interviews with plan staff used open - ended questions
on the relative successes and challenges of the two interventions
and two regions included in the study and lasted 12 h. interviews
occurred 2 , 6 , and 12 months into implementation and 3 months after
implementation ended .
interviews with district government used semistructured
questions on the sanitation - related roles , activities , and experiences
of different district government offices and lasted 0.51 h.
plan staff and government officers spoke english and were interviewed
by the primary author .
odf certification by district government is part
of implementation and was not validated by the researchers .
descriptive statistics
from household surveys
and observations were used to assess differences between the comparison
groups at baseline .
the primary outcome was change in levels of open
defecation , with the household as the unit of analysis .
secondary
outcomes assessed were use of a communal , shared , or private latrine ,
latrine quality , and access to handwashing materials .
latrines that surveyors
observed to be full , collapsed , or had unstable flooring were categorized
as open defecation .
a difference - in - difference ( or treatment - time )
estimator was used to account for baseline differences in outcome
variables .
sanitation practice was modeled using logistic regression
as a function of treatment , survey time point ( time ) , and treatment
time , and four covariates were included to address baseline differences
between comparison groups .
the four covariates ( roofing material ,
household size , water collection time , and discussing sanitation or
hygiene with a neighbor in the past two months ) showed statistically
significant differences at baseline and no multicollinearity ( the
highest variance inflation factor was 1.01 ) and could logically associate
with household sanitation practices ( table 2 ) . to avoid issues with potential endogeneity
the sampling design including village clustering
of outcomes , unequal selection probabilities , and nonresponse rates
was accounted for using the svyset command .
respondents were heads - of - households ,
and females were preferred if available .
all values account
for unequal selection probability , nonresponse rates , and village
clustering .
interviews with government
and plan staff were either transcribed
in real - time or audio - recorded and then transcribed .
transcripts were
analyzed by the primary author for statements regarding kebele leaders ,
hews , teachers , their interactions , and challenges they faced in facilitating
clts .
this study was reviewed and approved by the unc office
of human
research ethics ( study no .
for most variables , the baseline
differences between the comparison groups were insignificant ( table 2 ) . in conventional
clts kebeles ,
average household size was larger , water collection
time longer , and metal roofing more common , as was participation in
village meetings and discussions regarding sanitation and hygiene
with neighbors .
household ownership of a latrine and handwashing station
was also higher in conventional clts kebeles , and fewer practiced
open defecation . prematching did not fully eliminate baseline sanitation
differences , likely because it was based on government census data
from 2011 , which occurred one year before our baseline household surveys
and likely used the improved latrine definition , which does appear
to be well - balanced in our baseline . from baseline to follow - up , the
proportion of households practicing open defecation decreased in both
the teacher - facilitated and conventional clts groups ( figure 2 ) .
conventional clts was associated
with an 8.2 percentage point greater decrease in open defecation than
was teacher - facilitated clts in the full sample ( p = 0.048 ) . the difference - in - difference in open defecation was associated
with minimal change in use of communal or shared latrines , and a 9.0
percentage point greater increase in use of private latrines was associated
with conventional clts ( details and figure in the supporting information ) .
outcomes varied dramatically between
regions ( figure 2 and table 4 ) . in oromia , conventional
clts was associated with a 22.9 percentage point greater decrease
in open defecation than was teacher - facilitated clts ( p = 0.002 ) . in the snnp region
open defecation
before and after conventional and teacher - facilitated
clts interventions in ethiopia .
the confidence
interval is largest for teacher - facilitated clts in oromia because
standard error for binary variables is largest at 50% .
open defecation
is modeled from logistic regression parameters with covariates set
to their means ( full regressions in the supporting information ) .
all analysis accounts for unequal selection
probabilities , nonresponse rates , and village clustering .
did = difference - in - difference . across both interventions , open
defecation decreased by 15.3 percentage
point through an increase in use of communal latrines ( + 1.9 pp ) ,
shared latrines ( + 4.7 pp ) , and private latrines ( + 8.7 pp ) ( figure 3 ) . at baseline ,
33.2%
of households with a metal roof ( an indicator of economic status )
were practicing open defecation , and 48% of households without a metal
roof were practicing open defection . during the clts interventions ,
10.6% of households with a metal roof and 8.9% of households without
a metal roof adopted use of a private latrine ( data in the supporting information ) .
a t test was used to check
for significant differences between baseline and follow - up .
households
reporting the use of latrines not shown to surveyors or latrines with
unstable flooring were classified as open - defecation .
all analysis
accounts for unequal selection probability , nonresponse rates , and
village clustering . across both interventions ,
household ownership of any observed
latrine did not change significantly during the clts interventions ,
nor did ownership of an improved latrine ( a pit latrine with stable
flooring made of a durable material ) ( table 3 )
. however , ownership
of latrines with stable , safe flooring and an intact superstructure
increased .
ownership of latrines with indicators of cleaning and with
available handwashing materials also increased .
changes in latrine
characteristics came about through upgrades of existing latrines and
through some old latrines collapsing and new latrines being built
( data not presented ) .
this analysis covers the 1684 of
1692 privately owned latrines that were observed at baseline and 1779
of 1803 at follow - up .
variables are based on surveyor observations ,
who had descriptions so that latrine categorization was consistent .
a t test was
used to check for significant differences . percentages and p values account for unequal selection probability , nonresponse
rates , and village clustering .
icc = 0.317 for household ownership
of any observed latrine at the village level . based on surveyors observations
and judgment .
latrine
is a separate variable based on the joint monitoring program definition ,
although measurement of improved latrines varies globally .
a total of four kebeles
were certified as odf by district government
during the evaluation ( table 4 ) .
the two remaining kebeles
in the teacher - facilitated clts group were certified odf in 2014 after
the follow - up survey .
the research team did not observe or validate
odf status . in - depth
interviews and monitoring of the clts process
were used
to explore possible explanations for the differences in outcomes between
interventions .
kebele
leader attendance at teacher - facilitated village triggerings was lower
than in conventional clts kebeles ( table 1 ) , possibly because kebele leaders do not
typically work with teachers , and fewer kebele leaders were trained
in these kebeles .
hews are seen
using the kebele structure more effectively because they spend most
of their time in the kebele and have got an already established relationship .
unlike that , teachers seem less effective in using kebele structure ,
though they are using it ... individual teachers were less
active than hews in clts ( an average of 2.4 versus 4.7 days per month ) .
however , the more numerous teachers collectively spent more time on
clts than hews . according to a plan employee ,
... teachers
actually take a shorter time for triggering ... however , ... due
to vacations and exams in schools , teachers have some less time to
conduct follow ups than hews .
government and plan interviewees
further explained that teachers typically report directly to the district
education office and do not have an official relationship with kebele
leaders .
additionally , kebele leaders were noted as the most influential :
political leaders can have a higher role ( in sanitation ) even
though it s not their responsibility .
households
had differing levels of engagement in clts depending on who facilitated .
at the follow - up , households in the conventional clts kebeles reported
higher attendance at triggering meetings and could recall more activities
from the triggering , such as the mapping exercise ( table 5 )
. this could be partly due
to baseline differences ; at baseline , households in the conventional
clts kebeles reported being more active in village meetings .
however ,
this does not affect the effect estimates presented above , as baseline
activity was included as a covariate in outcome regressions .
a plan employee observed that ... teachers
have their own assignments , and they are also expected to do clts .
the same is true for kebele administration and hews . in january
2013 , facilitation stalled for one month in all kebeles while development
activities were restricted to a natural resource conservation campaign .
according to district officials ,
households faced challenges in latrine
construction : the closest place to buy latrine slabs , cement , or pvc
was approximately 2 h of travel from project kebeles .
we found that teachers were willing and active facilitators of
sanitation promotion at a community level in ethiopia .
there have
been no prior assessments of teachers leading sanitation promotion
at the community level .
our findings are consistent with previous
studies that identified teachers as effective in promoting sanitation
and hygiene within schools .
open defecation decreased by 13.8 percentage points
during teacher - facilitated clts , but the conventional facilitation
approach in ethiopia was associated with an 8.2 percentage point greater
decrease in open defecation ( 22 pp ) .
the full sample included kebeles
in two dissimilar regions to study how effectiveness differed between
settings .
in oromia , open defecation decreased by 26.2 percentage
points during teacher - facilitated clts and 49.1 percentage points
during conventional clts . in the snnp region , there were no significant
changes in open defecation associated with either intervention .
the
difference in outcomes is larger between regions than between interventions ,
indicating that context may be the greater determinant of effectiveness ,
which is not surprising given the variation in sanitation outcomes
between settings in other studies .
the only published
impact evaluation of clts , from mali , reports
a 23 percentage point reduction in open defecation .
evaluations of india s total sanitation campaign
report a 10 to 29 percentage point increased ownership of latrines
with signs of use .
we found
an overall 15.3 percentage point reduction in open defecation over
a 12 month time frame during clts in ethiopia , which is comparable
to these figures .
the majority of households stopping open defecation
switched to private latrine use , which did not appear to be influenced
by economic status ( as indicated by household roofing material ) .
our study reveals the challenges that teachers face in leading
the promotion of community - wide sanitation behavior change .
teachers
did not engage kebele leaders as quickly as did health workers , who
drew on their prior relationship with kebele leaders .
interviews indicated
that kebele leaders carried the most authority and influence during
the clts interventions .
another study reported that health workers
were more effective when influential leaders were trained and supported
their messages .
odf certification dates
( table 3 ) , while not
a precise measure of open defecation levels , show that teachers clts
may have had continuing impact beyond follow - up surveys .
given that
trained local actors continued living and working in project kebeles
once the project had ended , it is plausible that sanitation activity
and behavior change would have continued after the follow - up survey .
plan was able to train teachers as facilitators because the school
catchment area aligned with the level of clts facilitation : the kebele .
we hypothesize that training teachers as facilitators would be less
effective in countries where the school catchment area does not align
with the level of clts facilitation .
we analyzed latrine materials ,
maintenance , cleanliness , and handwashing
material availability alongside sanitation practices , which revealed
the importance of careful consideration of target outcomes and data
validation .
self - reported and visually confirmed latrine ownership
were similar ( 0.2% difference ) .
self - reported latrine ownership
was a good proxy for latrine ownership in this setting ; however , both
were poor proxies for access to a latrine with stable and safe flooring ,
and their use in evaluations could lead to inaccurate results and
conclusions .
others
have found that this may mean cheap , nondurable latrines are built
and handwashing is not fully addressed .
open defecation decreased by 15.3 percentage points
through increased latrine sharing and unstable latrines being upgraded
or replaced .
ownership of improved latrines with more
durable flooring materials , the standard used in international monitoring , did not change significantly .
households may have prioritized
maintenance and care for facilities over investments in hardware ,
possibly due to a lack of market availability of construction materials
and latrine components .
decreases in open defecation and increases
in stable latrine ownership
were highest in the oromia region , where baseline open defecation
was highest .
this suggests that clts may be most appropriate where
there are high levels of open defecation .
further improvements in
sanitation and hygiene may require addressing supply and financing
issues , which , when addressed together , can increase ownership of
hygienic latrines .
this study has
four potential limitations : nonrandom assignment
of kebeles , a small number of study sites , effect estimates limited
to a comparison of interventions , and a study duration limited to
1 year .
uncertainty regarding internal validity can not be completely
dispelled with nonrandom assignment of the six study kebeles .
comparing
multiple regression models suggested that prematching kebeles was
successful at minimizing bias .
use of a difference - in - difference estimator
and the inclusion of covariates in regressions further decrease the
chance of bias in effect estimates .
the effect estimates for teacher - facilitated
clts compared to conventional clts are robust , as they vary little
across outcomes and regression models .
because no true control group
was included , this study compares the effectiveness of two interventions
but does not estimate the effectiveness of clts .
changes in outcomes
that occurred beyond the 1-year follow - up survey are not captured
in this paper .
teachers may be more valuable to wash interventions
by supporting
health workers and local leaders once a project is initiated rather
than leading the effort .
this could be an attractive option in areas
where health workers are overburdened , as is the case in ethiopia .
clts was associated with increased ownership of stable , well - maintained
latrines and handwashing stations ; however , there was no increase
in more durable improved latrines . advancing to more durable and sustainable
latrines may require clts in combination or series with programs that
target supply chains and financing .
an operational research methodology
enabled an assessment of the implementation process , multiple outcomes ,
and variation of outcomes by setting . | community - led
total sanitation ( clts ) is a participatory approach
to addressing open defecation that has demonstrated success in previous
studies , yet there is no research on how implementation arrangements
and context change effectiveness .
we used a quasi - experimental study
design to compare two interventions in ethiopia : conventional clts
in which health workers and local leaders provided facilitation and
an alternative approach in which teachers provided facilitation . in
2012 ,
plan international ethiopia trained teachers from 111 villages
and health workers and leaders from 54 villages in clts facilitation .
the trained facilitators then implemented clts in their respective
villages for a year .
latrine ownership , use , and quality were measured
with household surveys .
differences between interventions were explored
using surveys and interviews .
the decrease in open defecation associated
with teacher - facilitated clts was 8.2 percentage points smaller than
for conventional clts ( p = 0.048 ) .
teachers had competing
responsibilities and initially lacked support from local leaders ,
which may have lessened their success .
teachers may be more appropriate
for a supporting rather than leading role in sanitation promotion
because they did demonstrate ability and engagement .
open defecation
decreased by 15.3 percentage points overall but did not change where
baseline open defecation was below 30% .
ownership of a latrine with
stable flooring increased by 8.7 percentage points overall .
improved
latrine ownership did not change during the intervention .
clts is
most appropriate where open defecation is high because there were
no significant changes in sanitation practices or latrine upgrades
where baseline open defecation was low . |
we consider
a propeller to be a rigid structure rotating with frequency f around a fixed axis of rotation .
the propeller is actuated
by an external force f and an external torque .
because the reynolds number is low ( see the supporting
information for an estimate ) , the hydrodynamics of the propeller
motion is described by the stokes equations1and2where p is
the pressure , is the dynamic viscosity , and u is the speed of the moving fluid .
if the propeller is rotating in
bulk fluid , the effect of gravity is neglected and we are interested
in time scales (1/f ) , the hydrodynamic problem
is on average rotationally symmetric around the axis of rotation of
the propeller .
therefore , the translatory propeller movement will
be parallel to the axis of rotation and the propeller speed can be
described by the scalar quantity v. assuming a no - slip
boundary condition , it follows from the
stokes equations that the speed v and rotation frequency f of the propeller motion must be linearly related to the
applied external force and torque3l is a size parameter for the propeller
and a , c , and d are parameters that depend only on the shape of the propeller for
a fixed axis of rotation .
the symmetric and positive definite matrix p is called the resistance matrix . in the case of magnetic propellers ,
therefore , the speed of the propeller is related
to the frequency of rotation in the following way4 in order to
compare the propulsion properties of propellers of different shapes ,
it is therefore useful to define a dimensionless speed that depends only on the shape of the propeller
and the axis of rotation5where v is the component of the propeller speed parallel to the
vector of rotation of the rotating magnetic field that actuates the
propeller .
this dimensionless speed corresponds to the number of body
lengths a propeller moves during one rotation and can be thought of
as an effective screw pitch ( see figure 1a , b ) . in our experiments
, we took care to
ensure that the magnetic torque was always sufficient to rotate the
propellers with the frequency of the rotating magnetic field .
thus , uy characterizes the hydrodynamic
coupling between rotation and translation for a particular propeller
shape and axis of rotation . in our experiments ,
the axis of
rotation is set by the magnetization state of the propeller and the
hydrodynamic forces acting on the propeller .
typically , the rotation
axis will be perpendicular to the net magnetic moment of the propeller
and perpendicular to the plane in which the external magnetic field
is rotating .
this leaves two degrees of freedom for the position of
the propeller relative to the axis of rotation .
we observe that the
axis of rotation passes through a point in the propeller that seems
to be typically close to its center of mass ( see figure 1c ) . in principle , the net magnetic
moment of the propeller does not need to rotate in the plane in which
the external magnetic field is rotating .
one could imagine that the
net magnetic moment rotates ( precesses ) on a cone around the axis
of rotation ( see figure 1d ) .
therefore , we take the axis of rotation as given and do not make
connections to the likely magnetization state of the propeller . the rotational friction constant cf of
the propeller , defined by the relationship = cff , is given by cf = l(d c / a ) . in
the supporting information , we show that cf is always positive , based on the fact that
the resistance matrix p is positive definite .
interestingly ,
the rotational friction constant cf is
reduced when the coupling between rotation and translation is strong
( large c ) .
this suggests that arbitrarily shaped
structures might have a tendency to rotate around an axis with strong
rotation translation coupling .
( a ) the red shape is an example of a 3d reconstruction of a
propeller .
( b )
after n ( here n = 79 to suit the
illustration ) rotations , the propeller has moved a certain distance
due to rolling ( x ) and a certain distance
due to propulsion ( y ) . the propeller speed v is the speed parallel to the rotation axis .
the length
scale l is chosen to be the size of the propeller
in the projection in which it appears largest ( see methods in supporting information ) .
the dimensionless speed
is thus the number of body lengths moved per rotation ( times 1000 )
and can be thought of as an effective screw pitch .
( c ) the net magnetic
moment ( yellow arrow ) of the propeller is likely rotating in the same
plane ( indicated by the blue shape ) as the external magnetic field ,
perpendicular to the axis of rotation .
this corresponds to a precession
angle of 90. ( d ) the precession angle does not necessarily have
to be equal to 90. because of the interplay between magnetic
and hydrodynamic forces , other precession angles are possible as well .
their dimensionless
speeds were measured ( 512 structures ) and their shapes were reconstructed
( 47 structures , see methods in the supporting
information ) .
the reconstructed shapes were compared to simulated
shapes generated in silico by a random process ( spheres are joined
in random directions without overlap , see supporting
information for details ) to assess whether the synthesized
structures are similarly random . in order to allow a quantitative
comparison ,
several geometric parameters were extracted from the reconstructed ,
as well as the simulated shapes ( see supporting
information ) .
the general accuracy of the reconstruction method
was assessed by comparing a size measure of the reconstructed shape
( lmax ) , to the size of the microstructures
as measured in the original video frames ( figure 2a ) .
lmax is the
largest distance ( in m ) between a pair of voxels belonging
to ( and forming ) the reconstructed shape .
the two measures are generally
in agreement , although the maximum voxel to voxel distance tends to
be a little larger .
this was expected , because only five different
2d projections were recorded and the 2d projection in which the propeller
would appear largest might not have been one of them . also , the maximum
voxel to voxel distance in the reconstruction might be larger than
the true size of the propeller due to overshadowing
. the compactness
of the reconstructed shapes as well as the artificially generated
shapes is characterized by plotting the surface area of the volume
equivalent sphere against the actual surface area of the shape ( figure 2c ) .
similarly , the
sphericity is defined as the ratio between the surface area of the
volume equivalent sphere to the actual surface area of a shape .
the
distribution of this dimensionless geometric parameter is broad for
both reconstructed as well as generated shapes , indicating that the
reconstructed shapes are similarly random ( figure 2b ) .
a shape is said to be chiral if it
can not be superposed on its mirror image by rotations and translations .
chirality can be quantified by6where a defines the reconstructed shape and a its mirror image , v ( ... ) is the
volume and the minimization is performed over all rotations and translations .
the pseudochirality is defined similarly ; however ,
the minimization is done only for one axis of rotation after moving
the center of mass ( assuming constant density ) of the shape and its
mirror image to the origin of the coordinate system . for the reconstructed
shapes ,
the rotation is around an axis parallel to the actual axis
of rotation of the experimental microstructure .
this pseudochirality
is easier to calculate and seemingly more useful for the characterization
of the propulsion properties of magnetic microstructures .
chirality
is independent of the axis of rotation of a shape , whereas pseudochirality
is not .
it has recently been observed that also achiral shapes can
propel , depending on their axis of rotation .
the distributions of pseudochirality are broad for both reconstructed
as well as generated shapes ( figure 2d ) .
we thus conclude that the synthesized structures
are indeed random , in the sense that their distributions of geometric
parameters are similar to those of shapes generated by a random process .
( a ) plot
of the microstructure size measured in video frames ( l ) against the maximum voxel to voxel distance in the reconstructed
shapes ( lmax ) .
the reconstructed shape
consists of 3d voxels , thus lmax is a
measure of the microstructure size .
the good correspondence between the two
measures of propeller size is an indication of the quality of the
3d reconstruction .
( b ) normalized histograms of the geometric parameter
sphericity extracted from reconstructed ( blue ) as well as artificially
generated ( red ) shapes .
( c ) the surface area of spheres , equivalent
in volume to the reconstructed ( blue ) or artificially generated ( red )
shapes , is plotted against the measured surface area of the shapes .
this plot is used to characterize the compactness of shapes and again
indicates the similarity of reconstructed and generated shapes .
( d )
same as ( b ) but for pseudochirality instead of sphericity .
the distribution of the measured dimensionless speed values is
shown in figure 3a .
we arbitrarily set the dimensionless speed to be negative for propellers
moving away from the observer when rotating clockwise , being viewed
along the axis of rotation .
a gaussian fit to the data yields a width
of 46.5 , which coincides with the standard deviation of the sample .
the measured
distribution of propeller speeds is lower than expected for a gaussian
at |uy| 25 and
higher than expected at uy 0 .
whether these deviations from a gaussian distribution
are statistically significant ( at the 5% level ) depends on the statistical
test that is used . while the lilliefors test does reject the null
hypothesis that the measured distribution stems from the normal family ,
the anderson - darling and the jarque - bera test can not reject this null
hypothesis
thus , we do not rule out the possibility of deviations
from a gaussian distribution but use the gaussian as a phenomenological
description . as discussed in the introduction , arbitrarily shaped
propellers might have a tendency to rotate around an axis for which
the rotation translation coupling is strong .
thus , the distribution
of dimensionless speeds we measure might not be identical to the dimensionless
speed distribution of randomly shaped structures rotating around random
axes of rotation in bulk fluid .
the dimensionless speeds we measure
might be increased by the dependence of the rotational friction constant
( cf ) on the rotation translation ( c ) coupling discussed above , and surface effects might have
an influence as well . measuring
the speed close to a surface probably
decreases the speed with respect to the bulk propulsion speed ( see supporting information ) .
however , the propulsion
speeds of previously published designs for magnetically actuated propellers
have almost always also been measured near a solid surface . thus
, although the dimensionless speeds reported here might not
correspond with an idealized theoretical situation , they lend themselves
perfectly to comparisons with published propeller designs .
figure 3b e shows
four examples of reconstructed shapes and their measured dimensionless
speeds .
the structure in figure 3c has a dimensionless speed of 190 , which is faster
than all previously reported magnetic micropropellers moving in low
reynolds number liquid .
interestingly , the shape of this propeller
is similar to optimized shapes reported in a theoretical study by
keaveny et al .
. arrives at a similar shape as well and they also
experimentally verify that this shape moves particularly fast . however , the dimensionless speed of their optimal
propeller ( 92 ) is significantly below the highest dimensionless speed
we find here ( 190 ) .
( a ) the measured values
for the dimensionless propulsion speed uy are displayed as a histogram .
however , lilliefors test rejects the null
hypothesis that the data comes from a normal distribution at the 5%
significance level .
the pronounced maximum at 0 and the minima at
about 25 are thus potentially no statistical flukes and in fact
the distribution might be bimodal . although a model with one central
gaussian and two symmetrically shifted gaussians is ( trivially ) better ,
we do not include it because we lack a physical argument motivating
such a bimodal distribution .
the standard deviation of the data and
the width of the gaussian fit is 46.5 .
the mean of the distribution
is 0.1 and thus close to zero as expected for symmetry reasons .
( b e ) from all 47 propellers for which the 3d shape was successfully
reconstructed , 4 examples were selected according to the following
criteria : the absolute value of the dimensionless speed was lowest
in ( b ) ( uy 1 ) ,
highest in ( c ) ( uy
190 ) , and close to the standard deviation in ( d ) ( uy 47 ) and ( e ) ( uy 50 ) . the
dashed black lines in ( b e )
the movement is upward if the dimensionless speed is negative
and vice versa ( see supporting information figure s1 ) .
scale bars are 1 m . in contrast , most previous designs for magnetic micropropellers
have used screw - like shapes , consisting of many helical turns , a design
originally inspired by the shape of bacterial flagella.figure 4 compares the dimensionless speeds of all previously reported
rigid magnetic micropropeller designs , now with the absolute value
of the dimensionless speeds of the random shapes reported here .
this
comparison shows that the observed standard deviation of =
46.5 in the dimensionless speed of random shapes is unexpectedly high .
the only propeller reported to have a significantly
higher dimensionless speed , moved in a high density solution of microbeads ,
instead of pure water .
this structure
might thus have moved more like a screw turning in a solid medium ,
rather than a propeller moving in low reynolds number liquid .
some nanofabricated propellers have dimensionless
speeds , which are smaller than 46.5 ( 1 ) .
the dimensionless
speed of the artificial bacterial flagella , used successfully in many
studies , is 33 and thus 49% of the observed
random shapes have a higher dimensionless speed .
comparison of randomly
shaped propellers from solution synthesis with previously published
nanofabricated propellers .
the dimensionless speeds of nanofabricated
propellers are shown in increasing order as in supporting information figure s3 ( zhang et al .
, li et al . , schamel
et al . , schuerle et al .
, gao et al . , walker et al . , tottori et al . , peters et al . , ghosh et al . , zhang et al . ) .
( a ) the absolute value of dimensionless speeds of the random shapes
and the dimensionless speed values from the literature are assigned
a propeller number , by sorting the values in ascending order . the
dimensionless speed of both random shapes and nanofabricated propellers
is then plotted against the propeller number .
the propeller number
of a nanofabricated propeller is thus an indication of how many random
shapes ( from the set of the 512 ) have a lower dimensionless speed .
( b ) the distribution of absolute dimensionless speed values is displayed
as a histogram .
the multiples of the standard deviation
of uy are displayed as
horizontal green lines . assuming a gaussian distribution , 68% of random
shapes have dimensionless speeds lower than the one standard deviation ,
95% lower than two , and 99.7% lower than three standard deviations . in the literature , there are also
flexible magnetic structures that can be actuated by rotating magnetic
fields ( for example , ref ( 39 ) ) .
we exclude these from our comparison , as their swimming
mechanism is different and does not allow the definition of a dimensionless
speed
. an exception could be the micropropellers reported by cheang
and kim , which consist of chains of flexibly
linked magnetic nanoparticles .
at least for a fixed actuation frequency ,
the shape of the structures is constant and a dimensionless speed
can be calculated .
they report swimming speeds for two micrometer - sized
structures moving in salt solution far from a solid interface .
the
corresponding dimensionless speeds are about 43 and 18 for the bigger
and smaller structure , respectively , both below the standard deviation
of random shapes we report here .
we extracted nine geometric parameters
from the reconstructed 3d shapes ( see supporting
information for parameter definitions ) .
the only geometric
parameter that correlated significantly with the dimensionless speed
is the dimensionless pseudochirality introduced above .
the pearson
correlation coefficient is 0.49 , with a p - value of 4 10 , meaning that the null - hypothesis of the pseudochirality
being uncorrelated to the dimensionless can be rejected with high
confidence .
the predictive power of the pseudochirality is limited ,
but it is already remarkable that a simple geometric parameter can
characterize the rotation translation coupling of a particular shape
to some extent , the theoretical calculation of which is challenging
and involves complex hydrodynamic simulations . if more such geometric
parameters could be found , the search for optimal designs of magnetically
actuated propellers could be greatly simplified .
the absolute value of
the dimensionless speed is plotted against the pseudochirality in
panel ( blue and red circles ) ( c ) .
the data points corresponding to
propellers displayed in figure 3 are marked in red .
( a ) this structure has a high dimensionless speed , despite having
a relatively low pseudochirality .
this might be because some small
but important geometric features could not be resolved in optical
microscopy .
( b ) this structure has a low pseudochirality and also
a low dimensionless speed .
( d ) this structure has a high pseudochirality
and also a high dimensionless - speed .
( e ) this relatively large structure
has a very low dimensionless speed , despite having a high pseudochirality .
the rotation axes of the propellers are marked by dashed lines , scale
bars are 1 m .
the
observation that helical propeller designs are mostly only average
when compared to randomly shaped propellers is very surprising .
however ,
randomness per se is not an advantage ; we simply use random shape
generation to sample a space of possible shapes .
the high speeds with
which some of our synthesized propellers move depends critically on
their specific geometry .
the discovery that randomly shaped magnetic
micropropellers have high dimensionless speeds has two main implications .
first , it shows that randomly shaped magnetic micropropellers could
be used in applications , which do not require particularly high dimensionless
speeds .
randomly shaped magnetic nanostructures can be produced cheaply
in very large quantities and we have shown previously that propellers
can be selected from such nanostructures .
the present work shows that such selection is probably not necessary
for certain applications , like micromixing , environmental remediation tasks , or
catalyst recovery .
the majority of the produced structures have dimensionless
speeds comparable to those of nanofabricated propellers and samples
of nanofabricated ( designed ) propellers always contain defective ( malformed )
structures with reduced speeds as well .
second , new micropropeller
designs could accommodate other design objectives , like ease of fabrication
or the incorporation of additional functionalities , without compromising
their ability to be effectively actuated by rotating magnetic fields .
the range of shapes for which such effective actuation is possible
seems to be much larger than was previously assumed .
the predominant
helical , flagella - like designs are certainly not the only option and
might be suboptimal for certain applications , such as particular microassembly
tasks or specific envisioned medical procedures .
nonetheless , helical propellers
with many turns might have specific advantages for certain applications ,
like pick and place devices . in
addition
, we identify a structure that has a higher dimensionless
speed than any propeller reported previously , showing that random
shape generation can be a viable optimization method .
the shape of
this fastest propeller is similar to theoretically optimized propeller
shapes , considering slender bodies with a single centerline .
future theoretical studies and simulations could
be tremendously helpful to better understand the relationship between
propeller shape and dimensionless speed .
which geometric shape has
the highest possible dimensionless speed remains an intriguing unsolved
problem in hydrodynamics , although in practice additional constraints
( minimum material thickness , magnetization state , brownian motion ,
and so forth ) and trade - offs ( for example , achievable magnetic moment
is also shape - dependent ) would need to be considered as well . for
biological applications ,
nonetheless ,
the fastest propeller we observe sets a lower bound for the highest
physically possible dimensionless speed . | studying propulsion mechanisms in
low reynolds number fluid has implications for many fields , ranging
from the biology of motile microorganisms and the physics of active
matter to micromixing in catalysis and micro- and nanorobotics .
the
propulsion of magnetic micropropellers can be characterized by a dimensionless
speed , which solely depends on the propeller geometry for a given
axis of rotation . however ,
this dependence has so far been only investigated
for helical propeller shapes , which were assumed to be optimal . in
order to explore a larger variety of shapes
, we experimentally studied
the propulsion properties of randomly shaped magnetic micropropellers .
surprisingly , we found that their dimensionless speeds are high on
average , comparable to previously reported nanofabricated helical
micropropellers .
the highest dimensionless speed we observed is higher
than that of any previously reported propeller moving in a low reynolds
number fluid , proving that physical random shape generation can be
a viable optimization strategy . |
the physician is often faced with treating an infection caused by specific micro - organisms not normally found at that site , or by microbes that have colonised a site that is normally sterile . in contrast , the mouth has a microbiome that is natural and is essential for the normal development of the physiology of the host ( 2 ) .
some of the beneficial aspects of the oral microbiota include :
- colonisation resistance , for example , exclusion of exogenous micro - organisms ( 3);- immunomodulatory activity , for example , down - regulation of excessive pro - inflammatory responses
to the resident microbiota on mucosal surfaces ( 4 ) ; and- enhancement of host defences and host physiology , for example , the metabolism of dietary nitrate
to nitrite leads to a reduction in blood pressure , a stimulation of mucous production , and the generation of antibacterial nitric oxide ( 5).therefore , the eradication of the oral microbiome should not be attempted as these organisms have evolved to have a synergistic relationship with the host . rather , the beneficial members of the oral microbiome should be nurtured and their relative proportions maintained , although research is needed to more fully characterise the composition and metabolic functions of the resident oral
microbiota .
micro - organisms grow on oral surfaces in structurally and metabolically organised communities of interacting species termed biofilms ( 6 )
. the properties of these microbial communities are more than the sum of the component organisms .
the combination of the oral microbiome being both a biofilm and a microbial community makes it particularly difficult to treat with antimicrobial agents ( 7 ) .
there are a number of mechanisms whereby these complex structures are more tolerant of inhibitors , including restricted penetration of agents into the depths of the biofilm , the slow growth rate and an altered phenotype of surface - associated microbes , and cross - protection by neighbouring cells.traditionally , microbiologists have focussed on the
selected representative species are then grown planktonically in pure culture to evaluate the spectrum of activity of antimicrobial agents using conventional mic / mbc read - outs as applied routinely in medical microbiology .
the application of contemporary molecular approaches to understand and define the diversity and metabolism of the oral microbiota is starting to challenge this approach .
the human microbiome project has used metagenomics to characterise the microbiota at several sites around the body .
when the microbial profiles of several hundred subjects were compared , there was a degree of heterogeneity in the individual taxa that were recovered from any one body site , such as the mouth , but when the samples were analysed in terms of biochemical functions ( e.g. atp synthesis , central carbohydrate metabolism ) then a huge degree of consistency was observed ( 8) .
therefore , a way forward could be to consider developing assays to screen for ( a ) antimicrobial agents that inhibit key metabolic functions rather than targeting specific bacterial species , and/or ( b ) prebiotics that promote the growth of resident microbial populations with beneficial functions .
there is a shift in the composition and metabolism of the oral microbiome in disease .
numerous studies , using either traditional culture or contemporary molecular approaches to compare the microbiota in biofilms from healthy surfaces with that from sites with dental caries and periodontal diseases , have shown that there are substantial differences in the composition of the microbiota in disease ( 2 ) .
pathogens ) can be found in biofilms from healthy sites , but they are present in clinically irrelevant numbers and at a far lower frequency ( 9 ) . therefore , disease is due to a shift in the composition of the biofilm ( dysbiosis ) rather than as a result of exogenous
infection. ideally , when a patient presents with disease , a clinician should attempt to determine the factors responsible for driving dysbiosis ( e.g. impaired saliva flow ; poor oral hygiene ; inappropriate lifestyle , including dietary habits ; presence of other risk factors ) , while recognising that these could vary from patient to patient . unless there is an attempt to interfere with the factor(s ) driving the dysbiosis
then the patient is likely to return to the surgery suffering from further episodes of disease ( 10 ) .
oral health care products that contain antimicrobial agents to control plaque biofilms are required to deliver two apparently contradictory requirements in order to meet regulatory guidelines .
these are to deliver a relevant and measurable clinical and microbiological benefit , while at the same time not disrupting the natural microbial ecology of the mouth , for example , by permitting overgrowth by opportunistic pathogens ( e.g. yeasts ) or exogenous micro - organisms ( 11 ) .
most antimicrobial agents used in oral care products are described as being broad spectrum ( see earlier ) , but under the conditions of use in the mouth ( twice daily for brief periods against drug tolerant biofilms ) they are present at mic or mbc levels for a relatively short time , with some retained for many hours at sub - lethal concentrations ( e.g. metal salts , triclosan ) ( 12 ) . at these levels
, these agents may have an important mode of action which is consistent with this new paradigm for oral care . at sub - lethal concentrations
, these agents can target key virulence traits such as sugar transport / acid production and protease activity , while also generally slowing bacterial growth . in this way
, they may have a more selective mode of action in which they mainly inhibit the growth and metabolism of organisms implicated in disease while leaving those associated with oral health relatively unaffected ( 1 ) .
the use of any antimicrobial agent carries the potential for bacterial resistance to develop , especially if used at sub - lethal concentrations . however , there is no evidence for a change in mic to agents used in oral care products following long - term clinical use ( 1315 ) .
unlike the principles behind antibiotic therapy , oral care products could function prophylactically to stabilise the normal oral microbiota under conditions that may otherwise predispose a site to caries or gingivitis , thereby maintaining the benefits derived from the resident microbiome.in order to pursue these theories , we have initiated in silico modelling studies to model in a biofilm the behaviour of two distinct microbial populations that differ in their aciduricity .
this model is based on a standard hybrid algorithm that has been widely employed in studies of environmental biofilms , and couples individual cells to continuous dispersed phases within a defined environment .
our computational studies demonstrate how small changes to the local oral environment can have a major impact on the competitiveness of oral bacteria .
the findings can be extrapolated to show how sub - lethal interference with the fluctuations in ph in a biofilm following dietary sugar intake can alter the proportions of different groups of bacteria over time . as shown in fig .
1a , by varying the buffering capacity of the plaque fluid , it was possible to modulate the biofilm composition between a weakly acidogenic state with a low fraction of aciduric bacteria , to a dysbiotic state where the aciduric population dominated , resulting in a lower ph and an increased risk of enamel demineralisation .
a similar trend was observed when the frequency of sugar intake was increased , as shown in fig .
both of these measured trends were a sub - lethal consequence of differential growth between the two bacterial groups , and demonstrated that environmental alterations , such as those achieved by a putative external agent that reduces the rate or frequency of fall in environmental ph , can beneficially modulate biofilm community dynamics without requiring any form of direct lethal antimicrobial action .
full details of the model , including a range of other perturbations that similarly alter both biofilm composition and ph , can be found elsewhere ( 16 ) .
the physician is often faced with treating an infection caused by specific micro - organisms not normally found at that site , or by microbes that have colonised a site that is normally sterile . in contrast , the mouth has a microbiome that is natural and is essential for the normal development of the physiology of the host ( 2 ) .
some of the beneficial aspects of the oral microbiota include :
- colonisation resistance , for example , exclusion of exogenous micro - organisms ( 3);- immunomodulatory activity , for example , down - regulation of excessive pro - inflammatory responses
to the resident microbiota on mucosal surfaces ( 4 ) ; and- enhancement of host defences and host physiology , for example , the metabolism of dietary nitrate
to nitrite leads to a reduction in blood pressure , a stimulation of mucous production , and the generation of antibacterial nitric oxide ( 5).therefore , the eradication of the oral microbiome should not be attempted as these organisms have evolved to have a synergistic relationship with the host . rather , the beneficial members of the oral microbiome should be nurtured and their relative proportions maintained , although research is needed to more fully characterise the composition and metabolic functions of the resident oral
microbiota .
- colonisation resistance , for example , exclusion of exogenous micro - organisms ( 3 ) ; - immunomodulatory activity , for example , down - regulation of excessive pro - inflammatory responses
to the resident microbiota on mucosal surfaces ( 4 ) ; and - enhancement of host defences and host physiology , for example , the metabolism of dietary nitrate
to nitrite leads to a reduction in blood pressure , a stimulation of mucous production , and the generation of antibacterial nitric oxide ( 5 ) .
therefore , the eradication of the oral microbiome should not be attempted as these organisms have evolved to have a synergistic relationship with the host . rather ,
the beneficial members of the oral microbiome should be nurtured and their relative proportions maintained , although research is needed to more fully characterise the composition and metabolic functions of the resident oral
microbiota .
micro - organisms grow on oral surfaces in structurally and metabolically organised communities of interacting species termed biofilms ( 6 ) .
the properties of these microbial communities are more than the sum of the component organisms .
the combination of the oral microbiome being both a biofilm and a microbial community makes it particularly difficult to treat with antimicrobial agents ( 7 ) .
there are a number of mechanisms whereby these complex structures are more tolerant of inhibitors , including restricted penetration of agents into the depths of the biofilm , the slow growth rate and an altered phenotype of surface - associated microbes , and cross - protection by neighbouring cells .
traditionally , microbiologists have focussed on the names of species present in a sample such as dental plaque .
selected representative species are then grown planktonically in pure culture to evaluate the spectrum of activity of antimicrobial agents using conventional mic / mbc read - outs as applied routinely in medical microbiology .
the application of contemporary molecular approaches to understand and define the diversity and metabolism of the oral microbiota is starting to challenge this approach . the human microbiome project has used metagenomics to characterise the microbiota at several sites around the body .
when the microbial profiles of several hundred subjects were compared , there was a degree of heterogeneity in the individual taxa that were recovered from any one body site , such as the mouth , but when the samples were analysed in terms of biochemical functions ( e.g. atp synthesis , central carbohydrate metabolism ) then a huge degree of consistency was observed ( 8) .
therefore , a way forward could be to consider developing assays to screen for ( a ) antimicrobial agents that inhibit key metabolic
functions rather than targeting specific bacterial species , and/or ( b ) prebiotics that promote the growth of resident microbial populations with beneficial functions .
there is a shift in the composition and metabolism of the oral microbiome in disease .
numerous studies , using either traditional culture or contemporary molecular approaches to compare the microbiota in biofilms from healthy surfaces with that from sites with dental caries and periodontal diseases , have shown that there are substantial differences in the composition of the microbiota in disease ( 2 ) .
pathogens ) can be found in biofilms from healthy sites , but they are present in clinically irrelevant numbers and at a far lower frequency ( 9 ) . therefore , disease is due to a shift in the composition of the biofilm ( dysbiosis ) rather than as a result of exogenous
infection. ideally , when a patient presents with disease , a clinician should attempt to determine the factors responsible for driving dysbiosis ( e.g. impaired saliva flow ; poor oral hygiene ; inappropriate lifestyle , including dietary habits ; presence of other risk factors ) , while recognising that these could vary from patient to patient .
unless there is an attempt to interfere with the factor(s ) driving the dysbiosis then the patient is likely to return to the surgery suffering from further episodes of disease ( 10 ) .
oral health care products that contain antimicrobial agents to control plaque biofilms are required to deliver two apparently contradictory requirements in order to meet regulatory guidelines .
these are to deliver a relevant and measurable clinical and microbiological benefit , while at the same time not disrupting the natural microbial ecology of the mouth , for example , by permitting overgrowth by opportunistic pathogens ( e.g. yeasts ) or exogenous micro - organisms ( 11 ) .
most antimicrobial agents used in oral care products are described as being broad spectrum ( see earlier ) , but under the conditions of use in the mouth ( twice daily for brief periods against drug tolerant biofilms ) they are present at mic or mbc levels for a relatively short time , with some retained for many hours at sub - lethal concentrations ( e.g. metal salts , triclosan ) ( 12 ) . at these levels
, these agents may have an important mode of action which is consistent with this new paradigm for oral care . at sub - lethal concentrations
, these agents can target key virulence traits such as sugar transport / acid production and protease activity , while also generally slowing bacterial growth . in this way
, they may have a more selective mode of action in which they mainly inhibit the growth and metabolism of organisms implicated in disease while leaving those associated with oral health relatively unaffected ( 1 ) .
the use of any antimicrobial agent carries the potential for bacterial resistance to develop , especially if used at sub - lethal concentrations .
however , there is no evidence for a change in mic to agents used in oral care products following long - term clinical use ( 1315 ) . unlike the principles behind antibiotic therapy
, oral care products could function prophylactically to stabilise the normal oral microbiota under conditions that may otherwise predispose a site to caries or gingivitis , thereby maintaining the benefits derived from the resident microbiome . in order to pursue these theories ,
we have initiated in silico modelling studies to model in a biofilm the behaviour of two distinct microbial populations that differ in their aciduricity .
this model is based on a standard hybrid algorithm that has been widely employed in studies of environmental biofilms , and couples individual cells to continuous dispersed phases within a defined environment .
our computational studies demonstrate how small changes to the local oral environment can have a major impact on the competitiveness of oral bacteria .
the findings can be extrapolated to show how sub - lethal interference with the fluctuations in ph in a biofilm following dietary sugar intake can alter the proportions of different groups of bacteria over time . as shown in fig .
1a , by varying the buffering capacity of the plaque fluid , it was possible to modulate the biofilm composition between a weakly acidogenic state with a low fraction of aciduric bacteria , to a dysbiotic state where the aciduric population dominated , resulting in a lower ph and an increased risk of enamel demineralisation .
a similar trend was observed when the frequency of sugar intake was increased , as shown in fig .
both of these measured trends were a sub - lethal consequence of differential growth between the two bacterial groups , and demonstrated that environmental alterations , such as those achieved by a putative external agent that reduces the rate or frequency of fall in environmental ph , can beneficially modulate biofilm community dynamics without requiring any form of direct lethal antimicrobial action .
full details of the model , including a range of other perturbations that similarly alter both biofilm composition and ph , can be found elsewhere ( 16 ) .
the effect of varying ( a ) the buffering capacity of the plaque fluid , and ( b ) the frequency of dietary sugar intake , on the proportions of non - aciduric ( light grey discs ) and aciduric ( black discs ) populations in an in silico simulation of biofilm development .
( a ) increased buffering ( here achieved by lowering the effective dissociation constant from kaeff=10 to 10 ) resulted in a higher ph during sugar intake ( vertical axis ) , and reduced proportions of aciduric bacteria ( horizontal axis ) .
image insets give representative examples of the composition of simulated biofilms at each end of the range sampled , where light grey ( black ) discs correspond to non - aciduric ( aciduric ) bacteria , respectively .
( b ) increasing the frequency of sugar intake from once every 10 h to once every 2 h resulted in the opposite trend , i.e. an increase in the proportions of aciduric bacteria .
data and images were taken after 20 days of sugar pulsing , starting from an initial state with equal proportions of aciduric and non - aciduric bacteria .
it has been argued that advances in the control of oral diseases will require a move away from approaches that have proved successful in many areas of medicine .
this may require a more holistic approach in which we monitor and manipulate the composition and metabolism of the oral microbiome in order to maintain the beneficial activities we derive from their presence and activity , while minimising the impact of any environmental and lifestyle factors that might lead to dysbiosis in the future .
there is no conflict of interest in the present study for any of the authors . | the mouth supports a diverse microbiota which provides major benefits to the host . on occasions , this symbiotic relationship breaks down ( dysbiosis ) , and disease can be a consequence .
we argue that progress in the control of oral diseases will depend on a paradigm shift away from approaches that have proved successful in medicine for many diseases with a specific microbial aetiology .
factors that drive dysbiosis in the mouth should be identified and , where possible , negated , reduced or removed , while antimicrobial agents delivered by oral care products may function effectively , even at sub - lethal concentrations , by modulating the activity and growth of potentially pathogenic bacteria . in this way
, the beneficial activities of the resident oral microbiota will be retained and the risk of dysbiosis occurring will be reduced . |
extracranial carotid artery pseudoaneurysms are very rare and make less than 4% of total peripheral arterial pseudoaneurysms . among these , external carotid artery pseudoaneurysm is rare and the pseudoaneurysm with fistula is even rarer .
color doppler ultrasound and computed tomography ( ct ) angiography are the primary modalities used for diagnosis , and in most of the cases are sufficient for diagnosis .
however , digital subtraction ( dsa ) angiography remain the gold standard to establish the diagnosis , evaluate the angioarchitecture , and planning of endovascular management . in our case
, it was penetrating trauma to the artery as well as vein , which led to pseudoaneurysm formation from the external carotid artery , which was subsequently fistulizing with the retromandibular vein .
an 8-year - old child presented to our hospital with painful swelling in the right preauricular region following a penetrating glass shrapnel injury .
there were two episodes of bleeding from the site , which was treated conservatively outside in a peripheral hospital .
then , he started developing ipsilateral facial nerve palsy and enlargement of the swelling , for which he was referred to our center . on local examination , he had tender pulsatile swelling in the right preauricular region .
( b ) ct angiography of the neck vessels shows a narrow neck ( thin arrow ) pseudoaneurysm arising from the right distal eca .
fundus of the pseudoaneurysm ( thick arrow ) is lying just deep to the skin .
( c ) right eca angiogram shows pseudoaneurysm ( thick white arrow ) from the distal eca ( thick black arrow ) with rent ( thin arrow ) just at the origin of sta ( curved arrow ) .
( d ) delayed right common carotid angiogram shows the a - v fistula that is pseudoaneurysm ( thick arrow ) draining into the retromandibular vein ( thin arrow ) color doppler ultrasound showed a narrow neck pseudoaneurysm from the distal external carotid artery .
catheter angiography showed pseudoaneurysm from distal right external carotid artery close to the origin of superficial temporal artery ( sta ) .
there was a very small rent at the origin of sta with slow opacification of pseudoaneurysm , which was slowly draining out into the retromandibular vein [ figure 1c and d ] .
ct angiography did not show early draining vein as delayed venous phase was not performed .
attempts were made to enter the rent with marathon microcatheter and mirage microwire , however , it was unsuccessful due to the extremely small size of the rent , which was located just at the origin of the superficial temporal artery .
the distal external carotid artery at the site of the rent was occluded with free platinum hilal coils and subsequently dilute glue ( nbca ) was injected into the coil mesh .
the rationale for using coils with glue was that coils act as a meshwork for glue ( a liquid embolic agent ) in the region of interest , thus preventing non - target embolization .
final external carotid artery angiogram showed occlusion of distal external carotid artery with non - opacification of the pseudoaneurysm and good reformation of internal maxillary artery and sta through collaterals [ figure 2a and b ] .
( a ) post right common carotid angiogram shows occlusion of distal external carotid artery ( thin white arrow ) .
( b ) plain skull radiograph in anteroposterior view shows the coil mass and glue ( nbca ) cast ( thin white arrow ) .
( c ) ct angiography of the neck vessels after 4 weeks shows complete thrombosis of the pseudoaneurysm and reduction of its size with coil mass in the distal external carotid artery ( thin white arrow ) post procedure the swelling was non - pulsatile .
ct scan after 4 weeks showed thrombosis of pseudoaneurysm with reduction in its size [ figure 2c ] .
follow - up , there was complete resolution of the swelling with partial recovery from lower motor neuron facial palsy .
direct carotid artery injury with formation of pseudoaneurysm and arteriovenous fistula is not a common entity , and is usually associated with neurological symptoms and high morbidity due to risk of bleeding .
endovascular management is an effective therapy in the management of pseudoaneurysm as compared to surgery .
are significantly are significantly high at more than 20% for surgery as compared with 1% in endovascular treatment for carotid artery injuries . exact localization of the lesion is mandatory before planning an endovascular treatment .
it may show the site of injury as well the status of proximal and distal arterial system .
cerebral ct angiography is rapid , non - invasive , and very accurate in the diagnosis of neurovascular injuries .
digital subtraction angiography ( dsa ) provides most accurate details of parent artery , site of origin of pseudoaneurysm any associated complication such as dissection or thrombosis , and associated findings such as arteriovenous fistula , as in our case .
available options include occlusion by glue , fibered platinum , or detachable coils or a combination of these materials .
the arteriovenous fistula needs to be disconnected by occlusion of the communicating channel with coils or dye .
however , in difficult cases like this case where neck of the pseudoaneurysm is difficult to localize as the rent was very small and was located just at the origin of sta , occlusion of the parent artery can yield good success .
follow up imaging of the endovascular treatment can establish the durability of the results . to our knowledge , concurrent pseudoaneurysm and arteriovenous fistula has not been described yet in maxillofacial region in the indian literature .
posttraumatic external carotid artery pseudoaneurysm may cause catastrophic complications such as intractable bleeding and various neurological complications .
external carotid artery pseudoaneurysm with fistula is a very rare entity and endovascular management is a safe and durable option of treatment .
| posttraumatic external carotid artery pseudoaneurysm with arteriovenous fistula is a rare condition .
an 8-year - old child presented with painful pulsatile swelling in the preauricular region following a penetrating glass shrapnel injury .
detailed evaluation showed distal external carotid artery pseudoaneurysm with fistula , which was draining into the retromandibular vein .
endovascular treatment was performed .
this case highlights the role of endovascular intervention for such rare complicated vascular pathologies . |
kaufman described that the mean length of the styloid process on the right and left sides was 30 mm and 29.49 mm , respectively in panographic radiographs of 484 hospitalized patients .
he also reported that the mean length for elongated styloid processes was roughly 33 mm and 53 mm for left and right side .
therefore , a case of an elongated styloid process over 6 cm in length is very rare .
our case report presents the clinical findings , the diagnostic usefulness of 3d - computed tomography ( 3d - ct ) , and a comparison of treatment methods with reference to the existing literature , which could lead to better understanding of the pathophysiology and safer therapies for elongated styloid process .
a 68-year - old japanese man complained of repeated left - sided throat pain for 10 years and hard swallowing pain since 2 weeks before .
3d - ct showed bilateral elongated styloid processes ( lengths of left and right : 7.8 cm and 8.0 cm ) .
the right styloid process was longer and thicker than the left styloid process ( figure 1 ) .
we performed shortening of only the left - sided styloid process ( the same side of throat pain ) using by a transcervical approach ( figure 2 ) .
stimulation of the pharynx or cervical nerves from an elongated styloid process is known to cause pharyngeal and swallowing pain .
some institutions reported that no statistically significant correlation was found between the presence of elongated styloid process between the genders .
ghosh and corrella revealed that this disease was largest distribution of patients in age was seen in fifth decade of life .
vieira indicated that 36.28% of the elongated styloid process showed both right and left sides as our case .
kaufman , vieira and corrella demonstrated that there was no significant correlation of process elongation and calcification to age .
chief complaints of our patient were repeated left - sided throat pain for 10 years and swallowing pain .
some reported that pain in the throat was most frequently encountered as the presenting complaints , and majority of kind of complaints ( e.g. , pain in the throat , painful swallowing , foreign body sensation in the throat , ) had more than one complaint as our case .
such a rare case , maik introduced a patient as having glossopharyngeal neuralgia due to elongated styloid process could lead to syncope and seizures .
this cartilage consists of four highly variable parts ( tympanohyale , stylohyale , ceratohyale , hypohyale ) .
long process results from the ossification of the tympanohyale and the styohyle portions during the first 8 years of life , and short process encountered from the ossification for the tympanohyale segment only while stylo- and ceratohyale portions degenerate .
conventionally , postnatal growth of the styloid has been advanced as a possible mechanism for the elongated styloid process . but a correlation between an increase in styloid process length and age has not been substantiated .
beder recommended that 3d - ct was the radiologic investigation of choice for elongated styloid process because of the most advanced technique available for definitively measuring the length of the styloid process .
also , our elongated styloid process could be easily diagnosed by 3d - ct ( left and right lengths : 7.8 cm and 8.0 cm , respectively ) and safety treated with surgery , since it can fully visualize the anatomic relationships .
the management of this disease process has been known to be non - surgical or surgical treatment .
the nonsurgical treatment generally involves pharmacotherapy with anticonvulsants or antidepressants , but the results are short - lived .
therefore , many hospitals stated that the first choice for treatment of elongated styloid process was surgery because of the severity of symptoms .
ghosh insisted that surgical shortening of the elongated process was the only to give symptomatic relief to the patient .
this surgical shortening of an elongated styloid process is often performed using an intraoral or external approach .
it can often enable an intraoral approach requiring shorter surgical time and less surgical trauma without leaving visible scars on the neck .
the external approach to the styloid process involves a transcervical approach to the parapharyngeal space , and wider visualization in the operative field than an intraoral approach . in our cases ,
recently , it reported that endoscopic - assisted styliodectomy for this disease could safety resect stylohyoid ligament with a smaller portion remaining at the skull base . in our case , the right styloid process was longer and thicker than the left styloid process .
however , since the throat symptoms at swallowing were on the left side , appeared that the longer and thicker right styloid process would work as the pivot of a door and that the left styloid process had an abnormal orbit that stimulated the pharynx muscles and cervical nerves . | we experienced a case of an elongated styloid process that was 8 cm in length .
the patient was a 68-year - old japanese man with frequent episodes of left - sided throat pain for 10 years .
the elongated styloid process could be diagnosed by 3d - computed tomography ( left and right length : 7.8 cm and 8.0 cm , respectively ) and successfully treated with surgery , since the anatomic relationships could be fully visualized .
surgical methods for shortening an elongated styloid process involve an intraoral or external approach .
the external approach to the styloid process involves a trans - cervical approach to the parapharyngeal space , which enables wider visualization in the operative field than an intraoral approach . in the present case
, the styloid process reached the parapharyngal space .
therefore , we selected the external approach and shortened only the left - sided styloid process ( the same side as the throat pain ) .
the chief complaint disappeared immediately after the operation . |
huntington disease ( hd ) is a hereditary , progressive neurodegenerative disease clinically characterized by abnormal involuntary movements , behavioral disturbance , cognitive dysfunction , and psychiatric disease .
the disease is caused by a cag ( glutamine ) trinucleotide expansion in exon 1 of the huntingtin ( htt ) gene at the location 4p16.9.1 the function of htt is not known , but it may be involved in internal cell signaling , maintenance of cyclic adenosine monophosphate response element binding protein , and preventing neuronal toxicity.2 early evidences suggest that the binding of the ras homologue enriched in striatum ( rhes ) protein to htt may be necessary to cause cellular toxicity.3 however , why the protein causes cellular toxicity in adulthood is not well understood .
recent evidence suggests that the interaction of the group 1 metabotropic glutamate receptors and htt protein may be at the root of delayed onset.4 although there is no established treatment to delay the onset or forestall the progression of hd , symptomatic treatment of chorea may be beneficial in some individuals , as it may have a favorable effect on motor function , quality of life , and safety.57 pathologically , hd is associated with diffuse loss of neurons , particularly involving the cortex and the striatum .
medium spiny neurons in the striatum that contain -aminobutyric acid ( gaba ) and enkephalin are impacted early in the disease , and are the primary neurons targeted in hd .
. then , there is progression to the remainder of the basal ganglia with subsequent dissemination , including cortex and substantia nigra .
huntingtin is cross - linked with other soluble htt to form the inclusion bodies in neurons .
it is not known if the accumulation of htt conglomerate results in cell death , or if the soluble form of the protein is the toxic form.8,9 dopamine , glutamate , and gaba are thought to be the most involved neurotransmitters in hd and are targeted for treatment ( table 1).1023 there are multiple theories on the pathogenesis of hd .
it is likely that more than 1 process may be occurring at once , but there is evidence to support multiple individual mechanisms , including toxic neuronal aggregates , transcriptional dysregulation , excitotoxicity , mitochondrial dysfunction with altered energy metabolism , and changes in axonal transport and synaptic dysfunction ( table 2).2430,31 most european populations show a prevalence rate of 48 cases per 100,000 , and hd may be frequently seen in india and parts of central asia.32 more recent studies in other european nations have confirmed this prevalence rate.33,34 hd is notably rare in finland and japan , but data for eastern asia and africa are inadequate .
a recent study found a slightly higher prevalence rate than expected and higher proportion of juvenile cases in mexico.35 there are also well - known large populations of patients with hd in scotland and the lake maracaibo region of venezuela.36,37 the disorder may be underestimated in the black american population .
there have been no widespread epidemiological studies of hd in the united states since the wide availability of genetic testing , but it is estimated that approximately 25,00030,000 individuals have manifest hd and a further 150,000250,000 individuals are at risk for hd.38 men and women are affected equally and typically become symptomatic in the third and fourth decades .
the symptoms of hd can start at any age ranging from 1 to 90 years .
approximately 5%10% of cases are classified as juvenile onset , with symptoms starting before the age of 20 years .
the vast majority of juvenile cases are inherited paternally , and instead of chorea , patients may exhibit more parkinsonian signs of bradykinesia , dystonia , tremors , and cognitive deficit.39 when patients exhibit more hypokinetic features ( bradykinesia and dystonia ) vs hyperkinetic features ( chorea ) , they are said to have the westphal variant of hd .
hd is diagnosed based on the presence of typical motor findings as measured by the unified huntington s disease rating scale in the setting of a family history of the disease
. there may be other manifestations of hd at the time of presentation or prior to diagnosis based on behavioral and cognitive symptoms . a dna test showing abnormal cag expansion in the htt gene can be used to confirm the diagnosis in symptomatic individuals . with proper genetic counseling and at the patient s request ,
dna analysis can be performed in individuals at risk for developing hd under the care of experienced clinicians .
there are many reasons why patients request presymptomatic testing , including financial planning , family planning , insurance decisions , and the need to know .
some people at risk for hd are reluctant to undergo testing and are brought into the clinic by future spouse , family , or others who believe testing should be performed . under these circumstances or
if the at - risk individual is likely to harm themselves or others based on the outcome of the test , genetic testing should be reconsidered .
there are ongoing studies to examine individuals who are gene positive but not yet symptomatic by motor criteria ( track - hd , neurobiological predictors of huntington s disease trial [ predict - hd ] , prospective huntington at risk observational study [ pharos]).4043 there is also a study currently enrolling subjects with hd and their affected and unaffected family members to further understand biomarkers of disease and signs of onset ( cooperative huntington s observational research trial [ cohort]).44 the focus of the cohort study is not neuroimaging and anatomical measures but rather clinical measures and biological samples .
subtle motor abnormalities have been associated with a smaller striatal volume and higher probability of disease diagnosis.40 lower scores on the hopkins verbal learning test - revised were associated with closer proximity to diagnosis and smaller striatal volumes.44 subjects with an expanded repeat and preclinical diagnosis of hd also had less accurate recognition of negative emotions.45 in addition , motor exam score , striatal volume , speeded finger tapping , self - timed finger tapping , word - list learning , and odor identifications in subjects in the predict - hd study were all associated with the predicted time to diagnosis .
46 expansion - positive individuals reported more psychiatric symptoms ( depression , anxiety , obsessive compulsiveness ) than expansion - negative individuals.47 the track - hd study has confirmed some and expanded upon findings in predict - hd in that presymptomatic subjects had significant changes in whole - brain volume , regional grey and white matter differences , impairment in a range of motor tasks , oculomotor findings , and cognitive and neuropsychiatric dysfunctions .
the various motor and nonmotor measures on the neurological examination used to diagnose and track hd are included in the unified huntington s disease rating scale.48 the scale is divided into 6 sections : motor , cognitive , behavioral , and 3 functional scales ( total functional capacity , functional checklist , and the independence scale ) . based on the guidelines published by the american college of genetics , patients with 40 or more repeats have 100% penetrance.49 in other words , if patients have 40 or more copies of the gene , they will inevitably express the disease clinically . in patients with a cag repeat length in the range of 3639 ,
there is reduced penetrance with increased likelihood of expression occurring with longer length of repeats and with longer lifespan of the patient .
although there are case reports of patients who manifest hd in this range , patients with fewer than 36 repeats will generally not develop clinical disease.50,51 patients with an allele repeat size of 2735 have demonstrated meiotic instability , particularly in sperm , indicating that the following generation is at higher risk of inheriting an expanded number of repeats , increasing the risk of clinical disease .
the length of repeat size correlates generally with the age of onset , but not necessarily with the severity or duration of disease.52 the course of the disease is typically 1520 years , with dementia , mutism , dystonia , and bradykinesia predominating in end - stages .
patients with more dystonia and swallowing issues may experience accelerated complications and , therefore , shorter lifespan .
chorea may become a safety issue with larger amplitude movements causing injury or poor positioning .
frequent movements may result in skin injuries , infections , or even fractures and head trauma .
cause of death is typically related to complications of immobility , such as skin breakdown , pneumonia , cardiac disease , or infection .
however , 25% of patients attempt suicide , which is a cause of death in 8%9% of patients.53 behavioral dyscontrol can be a severely disabling symptom of hd causing distress to the patient , family , and caregivers .
environmental approaches and cognitive interventions are the mainstay of treatments , but pharmacological agents can augment addressing disruptive behaviors .
depression , anxiety , aggressive , impulsive , and obsessive compulsive behaviors are also frequently treated pharmacologically and require behavioral intervention , but caution should be used to avoid oversedation and apathy , already common in patients with hd .
although not well studied , cognitive approaches to treat behavior may be more effective than pharmacotherapy for some aspects of the disease.54
most european populations show a prevalence rate of 48 cases per 100,000 , and hd may be frequently seen in india and parts of central asia.32 more recent studies in other european nations have confirmed this prevalence rate.33,34 hd is notably rare in finland and japan , but data for eastern asia and africa are inadequate . outside of venezuela , there are little epidemiological studies of hd in hispanic populations .
a recent study found a slightly higher prevalence rate than expected and higher proportion of juvenile cases in mexico.35 there are also well - known large populations of patients with hd in scotland and the lake maracaibo region of venezuela.36,37 the disorder may be underestimated in the black american population .
there have been no widespread epidemiological studies of hd in the united states since the wide availability of genetic testing , but it is estimated that approximately 25,00030,000 individuals have manifest hd and a further 150,000250,000 individuals are at risk for hd.38 men and women are affected equally and typically become symptomatic in the third and fourth decades .
the symptoms of hd can start at any age ranging from 1 to 90 years .
approximately 5%10% of cases are classified as juvenile onset , with symptoms starting before the age of 20 years .
the vast majority of juvenile cases are inherited paternally , and instead of chorea , patients may exhibit more parkinsonian signs of bradykinesia , dystonia , tremors , and cognitive deficit.39 when patients exhibit more hypokinetic features ( bradykinesia and dystonia ) vs hyperkinetic features ( chorea ) , they are said to have the westphal variant of hd .
hd is diagnosed based on the presence of typical motor findings as measured by the unified huntington s disease rating scale in the setting of a family history of the disease
. there may be other manifestations of hd at the time of presentation or prior to diagnosis based on behavioral and cognitive symptoms . a dna test showing abnormal cag expansion in the htt gene can be used to confirm the diagnosis in symptomatic individuals . with proper genetic counseling and at the patient
s request , dna analysis can be performed in individuals at risk for developing hd under the care of experienced clinicians .
there are many reasons why patients request presymptomatic testing , including financial planning , family planning , insurance decisions , and the need to know .
some people at risk for hd are reluctant to undergo testing and are brought into the clinic by future spouse , family , or others who believe testing should be performed . under these circumstances or
if the at - risk individual is likely to harm themselves or others based on the outcome of the test , genetic testing should be reconsidered .
there are ongoing studies to examine individuals who are gene positive but not yet symptomatic by motor criteria ( track - hd , neurobiological predictors of huntington s disease trial [ predict - hd ] , prospective huntington at risk observational study [ pharos]).4043 there is also a study currently enrolling subjects with hd and their affected and unaffected family members to further understand biomarkers of disease and signs of onset ( cooperative huntington s observational research trial [ cohort]).44 the focus of the cohort study is not neuroimaging and anatomical measures but rather clinical measures and biological samples .
subtle motor abnormalities have been associated with a smaller striatal volume and higher probability of disease diagnosis.40 lower scores on the hopkins verbal learning test - revised were associated with closer proximity to diagnosis and smaller striatal volumes.44 subjects with an expanded repeat and preclinical diagnosis of hd also had less accurate recognition of negative emotions.45 in addition , motor exam score , striatal volume , speeded finger tapping , self - timed finger tapping , word - list learning , and odor identifications in subjects in the predict - hd study were all associated with the predicted time to diagnosis . 46 expansion - positive individuals reported more psychiatric symptoms ( depression , anxiety , obsessive compulsiveness ) than expansion - negative individuals.47 the track - hd study has confirmed some and expanded upon findings in predict - hd in that presymptomatic subjects had significant changes in whole - brain volume , regional grey and white matter differences , impairment in a range of motor tasks , oculomotor findings , and cognitive and neuropsychiatric dysfunctions .
the various motor and nonmotor measures on the neurological examination used to diagnose and track hd are included in the unified huntington s disease rating scale.48 the scale is divided into 6 sections : motor , cognitive , behavioral , and 3 functional scales ( total functional capacity , functional checklist , and the independence scale ) . based on the guidelines published by the american college of genetics , patients with 40 or more repeats have 100% penetrance.49 in other words , if patients have 40 or more copies of the gene , they will inevitably express the disease clinically . in patients with a cag repeat length in the range of 3639 ,
there is reduced penetrance with increased likelihood of expression occurring with longer length of repeats and with longer lifespan of the patient .
although there are case reports of patients who manifest hd in this range , patients with fewer than 36 repeats will generally not develop clinical disease.50,51 patients with an allele repeat size of 2735 have demonstrated meiotic instability , particularly in sperm , indicating that the following generation is at higher risk of inheriting an expanded number of repeats , increasing the risk of clinical disease .
the length of repeat size correlates generally with the age of onset , but not necessarily with the severity or duration of disease.52 the course of the disease is typically 1520 years , with dementia , mutism , dystonia , and bradykinesia predominating in end - stages .
patients with more dystonia and swallowing issues may experience accelerated complications and , therefore , shorter lifespan .
chorea may become a safety issue with larger amplitude movements causing injury or poor positioning .
frequent movements may result in skin injuries , infections , or even fractures and head trauma .
cause of death is typically related to complications of immobility , such as skin breakdown , pneumonia , cardiac disease , or infection .
however , 25% of patients attempt suicide , which is a cause of death in 8%9% of patients.53 behavioral dyscontrol can be a severely disabling symptom of hd causing distress to the patient , family , and caregivers .
environmental approaches and cognitive interventions are the mainstay of treatments , but pharmacological agents can augment addressing disruptive behaviors .
depression , anxiety , aggressive , impulsive , and obsessive compulsive behaviors are also frequently treated pharmacologically and require behavioral intervention , but caution should be used to avoid oversedation and apathy , already common in patients with hd .
although not well studied , cognitive approaches to treat behavior may be more effective than pharmacotherapy for some aspects of the disease.54
many agents and surgical procedures have been evaluated in hd for their efficacy on suppressing chorea , including dopamine - depleting agents , dopamine antagonists , benzodiazepines , glutamate antagonists , acetylcholinesterase inhibitors , dopamine agonists , antiseizure medications , cannabinoids , lithium , deep brain stimulation , and fetal cell transplantation.5558 pharmacological interventions typically address the hyperkinetic movement disorders that may be associated with hd , such as chorea , dystonia , ballism , myoclonus , and tics . with regards to choose the agent ,
providers need to consider if there will be a positive or negative impact of the agent on psychiatric issues associated with hd , such as irritability , depression , anxiety , mania , apathy , obsessive compulsive disorder , or cognitive decline associated with hd .
adjunctive therapies , alternative and complementary therapies , behavioral plans , and cognitive interventions also may play a role in addressing the symptoms of hd and need to be considered when choosing medications .
several excellent reviews have summarized the symptomatic treatment of hd.5567 overall , there is not enough evidence available to guide long - term symptomatic treatment in hd , and double - blind and long - term studies assessing various treatment strategies in hd are needed.61 a cochrane review of studies for the symptomatic treatment of hd examined 22 trials that involved 1,254 different participants.62 nine trials had a crossover design and 13 were conducted in parallel .
the studies examined were of relatively short duration , ranging from 2 to 80 weeks .
the number of trials examining various pharmacological interventions included antidopaminergic drugs ( n = 5 ) , glutamate receptor antagonists ( n = 5 ) , and energy metabolites ( n = 5 ) .
based on available evidence , the authors of the cochrane review concluded that only tetrabenazine ( tbz ) showed clear efficacy for the control of chorea , but no statement can be made regarding the best medical practice for the control of motor and non - motor symptoms in hd .
tbz is the only us food and drug administration ( fda)-approved drug for hd , indicated for the treatment of chorea associated with hd .
in addition to the united states and canada , tbz is marketed in australia , denmark , france , germany , ireland , israel , italy , new zealand , portugal , spain , switzerland , and united kingdom .
an excellent review of the chemistry , pharmacodynamics , pharmacokinetics , and mechanism of action is available in a previous issue of this journal.68 by reversibly inhibiting the central vesicular monoamine transporter type 2 ( vmat2 ) , tbz more selectively depletes dopamine than norepinephrine.69,70 the highest binding density for tbz is in the caudate nucleus , putamen , and nucleus accumbens , areas known to bear the brunt of pathology in hd.71,72 vmat2 binding and monoamine depletion by tbz are reversible , last hours , and are not modified by long - term treatment.73,74 these features of the drug differentiate it from the other dopamine - depleting agent , reserpine .
while vmat2 is located solely in the central nervous system , vmat1 is localized to the peripheral nervous system , accounting for some of the peripheral adverse effects , such as orthostatic hypotension and diarrhea .
in contrast to tbz , reserpine binds irreversibly to vmat1 and vmat2 , making the duration of action considerably longer ( hours vs days ) .
in addition to tbz , the 2 active metabolites , - and -dihydrotetrabenazine , have longer half - lives and are more highly bound to proteins than the parent compound.7577 the efficacy of tbz as an antichoreic drug was convincingly demonstrated in a double - blind , placebo - controlled trial conducted by the huntington study group ( hsg).78 subjects were randomized to receive either tbz ( n = 54 ) or placebo ( n = 30 ) .
tbz was titrated weekly in 12.5 mg increments to a maximum of 100 mg / d or to the development of intolerable adverse effects . the primary efficacy outcome was the change from baseline in the total maximal chorea score of the unified huntington s disease rating scale.48 on this scale , chorea is graded from 0 to 4 ( with 0 representing no chorea ) for 7 body regions for a range in total scores from 0 to 28 . compared with baseline ,
tbz treatment resulted in a reduction of 5.0 units in chorea compared with a reduction of 1.5 units in the placebo group .
about 50% of tbz - treated subjects had a 6-point or greater improvement compared with 7% of placebo recipients .
there is also evidence to suggest continuous long - term efficacy and tolerability of tbz in patients with hd.7982 in the same study , the adverse events that occurred significantly more frequently in the tbz group included drowsiness or somnolence , insomnia , depressed mood , agitation , akathisia , and hyperkinesia .
however , by the conclusion of the maintenance phase , when subjects were presumably on optimal dosage , there were no significant differences between tbz and placebo . among subjects completing the study ,
the most common adverse event at the end of tbz exposure was fatigue , reported by 7 subjects on tbz ( 14.3% ) and 2 on placebo ( 6.9% ) .
there was 1 suicide in the double - blind study in a subject taking tbz .
however , attempted or completed suicides in hd do not necessarily correlate with severity of depression and may be related to associated impulsiveness , obsessive compulsive behavior , and a variety of socioeconomic factors .
nevertheless , all patients taking tbz need to be monitored for signs of depression and suicidal ideation .
cognition in the tbz group , as assessed by the unified huntington s disease rating scale , did not differ from that in the placebo group statistically , although both groups declined as expected over the course of the study .
there were no hd - related quality - of - life measures included in the published tbz studies .
the same group of study participants was given the opportunity to participate in an open - label extension study.82 seventy - five subjects were elected to participate initially , and at the end of the 3 phases of the extension study , 45 subjects had completed 80 additional weeks of treatment with tbz . in the extension study , no new adverse effects were reported , and the drug was well tolerated , despite the attrition rate in the study ( mostly for logistical reasons ) .
there are a number of older open - label studies , primarily out of the baylor college of medicine database.80 the side effects and doses reported were fairly consistent with the double - blind study with the exception of insomnia .
chronic doses of the baylor group were comparable with that in the long - term hsg study of 62.5-mg total daily dose .
the safety of tbz was evaluated in these studies , and in an additional study , the safety of sudden withdrawal after long - term use was assessed .
the side effects are consistent among studies , and once the drug is titrated slowly to effect , the drug is well tolerated .
potential side effects include akathisia , depression , dizziness , fatigue , or parkinsonism ( table 3 ) . during titration
if at any time side effects do occur , the dose can be lowered to the previously well - tolerated dose .
withdrawal of tbz results in a recurrence of chorea , not worse than before starting the drug.83 there are no particular characteristics that may predispose patients to experience 1 or more side effects , but patients with a history of depression should be more closely monitored for changes in mood or impulsivity . although it is listed in the product information sheet , tbz does not cause and in fact may be an excellent treatment for tardive dyskinesia .
other medications that are commonly considered when treating chorea include dopamine antagonists , benzodiazepines , and glutamate antagonists .
dopamine antagonists ( neuroleptics ) are perhaps the most commonly considered agents in the management of chorea and psychosis in patients with hd , but few double - blind , placebo - controlled studies evaluating the efficacy and safety of these agents have been published.8486 none of the typical neuroleptics have been found to be effective in reducing chorea in placebo - controlled trials .
however , in a study of haloperidol in 10 subjects , oral doses of 1.510 mg / d corresponded with at least a 30% reduction in chorea compared with baseline.87 the quantity and quality of these efficacy data need to be taken into account when considering the risks of using typical neuroleptics , particularly tardive dyskinesia .
apathy and akathisia , other potential adverse effects of the dopamine receptor blockers , can be particularly problematic in patients with hd , as they may not have the insight to recognize these problems or may wrongly attribute the symptoms to hd . owing to their presumed better tolerability ,
olanzapine has been used in small open - label studies to treat the motor symptoms of hd.8892 the range of effect on chorea has been 0%66% reduction .
there are no clinical trials of risperidone for hd , but a few reports suggest a positive effect on the disease with a tolerable adverse - effect profile.9397 quetiapine has been tried in multiple , small , uncontrolled , nonrandomized trials for hd with some success on both motor and psychiatric symptoms of hd.9799 clozapine was studied in patients with hd up to a dose of 150 mg / d for up to 31 days , but many adverse events , such as drowsiness , fatigue , anticholinergic symptoms , and walking difficulties , occurred without beneficial effects.100 the newer atypical agent with multiple mechanisms of action , aripiprazole , has been found to be beneficial in a few small trials with a reduction in chorea equivalent to that with tbz.101103 although aripiprazole may have fewer adverse effects on mood than tbz , it is associated with tardive dyskinesia , similar to other typical and atypical neuroleptics .
the n - methyl d - aspartate - antagonist amantadine has been shown in controlled trials to significantly reduce chorea in patients with hd.104 doses in the range of 400 mg / d or higher may be required for symptomatic benefit , but even in doses used to treat influenza , amantadine may increase irritability and aggressiveness.105 because riluzole retards striatal glutamate release and the pathological consequences in neurotoxic animal models of hd , multiple large trials have been conducted to determine if there is a possible neuroprotective effect .
riluzole was found to reduce chorea at a dose of 200 mg / d , but not 100 mg / d.106,107 benzodiazepines are also frequently used clinically in patients with hd to treat anxiety and chorea , and there is limited evidence to suggest that higher doses of clonazepam ( up to 5.5 mg / d ) may be needed to suppress chorea.108 at this dose , sedation becomes a potential adverse effect . in a few short - term studies ( hours )
, there is evidence that dopamine agonists may reduce the motor signs of hd.109111 although the pharmacological rationale for using dopamine agonists in the treatment of chorea is not clear , presumably they act by activating the presynaptic dopamine receptors leading to decreased dopamine turnover .
more likely , however , is that the observed symptomatic effects are related to nonspecific or sedating effects . for patients with the akinetic form of hd ( westphal variant ) , antiparkinsonian medications , such as levodopa , dopamine agonists , and amantadine , may be beneficial.112115 botulinum toxin injections
tbz is the only us food and drug administration ( fda)-approved drug for hd , indicated for the treatment of chorea associated with hd .
in addition to the united states and canada , tbz is marketed in australia , denmark , france , germany , ireland , israel , italy , new zealand , portugal , spain , switzerland , and united kingdom .
an excellent review of the chemistry , pharmacodynamics , pharmacokinetics , and mechanism of action is available in a previous issue of this journal.68 by reversibly inhibiting the central vesicular monoamine transporter type 2 ( vmat2 ) , tbz more selectively depletes dopamine than norepinephrine.69,70 the highest binding density for tbz is in the caudate nucleus , putamen , and nucleus accumbens , areas known to bear the brunt of pathology in hd.71,72 vmat2 binding and monoamine depletion by tbz are reversible , last hours , and are not modified by long - term treatment.73,74 these features of the drug differentiate it from the other dopamine - depleting agent , reserpine .
while vmat2 is located solely in the central nervous system , vmat1 is localized to the peripheral nervous system , accounting for some of the peripheral adverse effects , such as orthostatic hypotension and diarrhea .
in contrast to tbz , reserpine binds irreversibly to vmat1 and vmat2 , making the duration of action considerably longer ( hours vs days ) .
in addition to tbz , the 2 active metabolites , - and -dihydrotetrabenazine , have longer half - lives and are more highly bound to proteins than the parent compound.7577 the efficacy of tbz as an antichoreic drug was convincingly demonstrated in a double - blind , placebo - controlled trial conducted by the huntington study group ( hsg).78 subjects were randomized to receive either tbz ( n = 54 ) or placebo ( n = 30 ) .
tbz was titrated weekly in 12.5 mg increments to a maximum of 100 mg / d or to the development of intolerable adverse effects .
the primary efficacy outcome was the change from baseline in the total maximal chorea score of the unified huntington s disease rating scale.48 on this scale , chorea is graded from 0 to 4 ( with 0 representing no chorea ) for 7 body regions for a range in total scores from 0 to 28 . compared with baseline , tbz treatment resulted in a reduction of 5.0 units in chorea compared with a reduction of 1.5 units in the placebo group .
about 50% of tbz - treated subjects had a 6-point or greater improvement compared with 7% of placebo recipients .
there is also evidence to suggest continuous long - term efficacy and tolerability of tbz in patients with hd.7982 in the same study , the adverse events that occurred significantly more frequently in the tbz group included drowsiness or somnolence , insomnia , depressed mood , agitation , akathisia , and hyperkinesia .
however , by the conclusion of the maintenance phase , when subjects were presumably on optimal dosage , there were no significant differences between tbz and placebo . among subjects completing the study ,
the most common adverse event at the end of tbz exposure was fatigue , reported by 7 subjects on tbz ( 14.3% ) and 2 on placebo ( 6.9% ) .
there was 1 suicide in the double - blind study in a subject taking tbz .
. however , attempted or completed suicides in hd do not necessarily correlate with severity of depression and may be related to associated impulsiveness , obsessive compulsive behavior , and a variety of socioeconomic factors .
nevertheless , all patients taking tbz need to be monitored for signs of depression and suicidal ideation .
cognition in the tbz group , as assessed by the unified huntington s disease rating scale , did not differ from that in the placebo group statistically , although both groups declined as expected over the course of the study .
there were no hd - related quality - of - life measures included in the published tbz studies .
the same group of study participants was given the opportunity to participate in an open - label extension study.82 seventy - five subjects were elected to participate initially , and at the end of the 3 phases of the extension study , 45 subjects had completed 80 additional weeks of treatment with tbz . in the extension study ,
no new adverse effects were reported , and the drug was well tolerated , despite the attrition rate in the study ( mostly for logistical reasons ) .
there are a number of older open - label studies , primarily out of the baylor college of medicine database.80 the side effects and doses reported were fairly consistent with the double - blind study with the exception of insomnia .
chronic doses of the baylor group were comparable with that in the long - term hsg study of 62.5-mg total daily dose .
the safety of tbz was evaluated in these studies , and in an additional study , the safety of sudden withdrawal after long - term use was assessed .
the side effects are consistent among studies , and once the drug is titrated slowly to effect , the drug is well tolerated .
potential side effects include akathisia , depression , dizziness , fatigue , or parkinsonism ( table 3 ) . during titration
if at any time side effects do occur , the dose can be lowered to the previously well - tolerated dose . since the half - life is short , side effects tend to wane quickly .
withdrawal of tbz results in a recurrence of chorea , not worse than before starting the drug.83 there are no particular characteristics that may predispose patients to experience 1 or more side effects , but patients with a history of depression should be more closely monitored for changes in mood or impulsivity . although it is listed in the product information sheet , tbz does not cause and in fact may be an excellent treatment for tardive dyskinesia .
other medications that are commonly considered when treating chorea include dopamine antagonists , benzodiazepines , and glutamate antagonists .
dopamine antagonists ( neuroleptics ) are perhaps the most commonly considered agents in the management of chorea and psychosis in patients with hd , but few double - blind , placebo - controlled studies evaluating the efficacy and safety of these agents have been published.8486 none of the typical neuroleptics have been found to be effective in reducing chorea in placebo - controlled trials . however , in a study of haloperidol in 10 subjects , oral doses of 1.510 mg / d corresponded with at least a 30% reduction in chorea compared with baseline.87 the quantity and quality of these efficacy data need to be taken into account when considering the risks of using typical neuroleptics , particularly tardive dyskinesia .
apathy and akathisia , other potential adverse effects of the dopamine receptor blockers , can be particularly problematic in patients with hd , as they may not have the insight to recognize these problems or may wrongly attribute the symptoms to hd . owing to their presumed better tolerability ,
olanzapine has been used in small open - label studies to treat the motor symptoms of hd.8892 the range of effect on chorea has been 0%66% reduction .
there are no clinical trials of risperidone for hd , but a few reports suggest a positive effect on the disease with a tolerable adverse - effect profile.9397 quetiapine has been tried in multiple , small , uncontrolled , nonrandomized trials for hd with some success on both motor and psychiatric symptoms of hd.9799 clozapine was studied in patients with hd up to a dose of 150 mg / d for up to 31 days , but many adverse events , such as drowsiness , fatigue , anticholinergic symptoms , and walking difficulties , occurred without beneficial effects.100 the newer atypical agent with multiple mechanisms of action , aripiprazole , has been found to be beneficial in a few small trials with a reduction in chorea equivalent to that with tbz.101103 although aripiprazole may have fewer adverse effects on mood than tbz , it is associated with tardive dyskinesia , similar to other typical and atypical neuroleptics .
the n - methyl d - aspartate - antagonist amantadine has been shown in controlled trials to significantly reduce chorea in patients with hd.104 doses in the range of 400 mg / d or higher may be required for symptomatic benefit , but even in doses used to treat influenza , amantadine may increase irritability and aggressiveness.105 because riluzole retards striatal glutamate release and the pathological consequences in neurotoxic animal models of hd , multiple large trials have been conducted to determine if there is a possible neuroprotective effect .
riluzole was found to reduce chorea at a dose of 200 mg / d , but not 100 mg / d.106,107 benzodiazepines are also frequently used clinically in patients with hd to treat anxiety and chorea , and there is limited evidence to suggest that higher doses of clonazepam ( up to 5.5 mg / d ) may be needed to suppress chorea.108 at this dose , sedation becomes a potential adverse effect . in a few short - term studies ( hours )
, there is evidence that dopamine agonists may reduce the motor signs of hd.109111 although the pharmacological rationale for using dopamine agonists in the treatment of chorea is not clear , presumably they act by activating the presynaptic dopamine receptors leading to decreased dopamine turnover .
more likely , however , is that the observed symptomatic effects are related to nonspecific or sedating effects . for patients with the akinetic form of hd ( westphal variant ) , antiparkinsonian medications , such as levodopa , dopamine agonists , and amantadine , may be beneficial.112115 botulinum toxin injections
the medication is not available at local pharmacies but must be ordered through a central distribution center .
prescription information can be found through the company s web site , including prescribing information and obtaining the prescription form.116 clinicians should note on the form that patients ( or their guardian ) must sign the release of information for the pharmacy to be able to communicate with the patient s insurance company .
tbz should be started at 12.5 mg / d , and every week , the dose should be increased by 12.5 mg / d , distributing the drug in 3-times daily dosing .
once patients reach twice - daily dose of 12.5 mg and daily dose of 25 mg ( total daily dose of 50 mg ) , as per the prescribing information , before patients are given a daily dose of greater than 50 mg , they should be tested for the cyp2d6 gene to determine whether they are poor , extensive , or intermediate metabolizers .
this test can be costly ( up to $ 3,000 ) and may or may not be covered by insurance .
in addition , the drug is titrated to clinical effect , and the genotyping has no impact on the clinical assessment of the patient on the drug . ultimately , it is up to the discretion of the provider with the patient to determine if this test should be performed .
the goal of the effect of tbz is to reduce chorea to a point that is acceptable to the patient .
the goal should not be to eliminate chorea completely or to reduce chorea to a level that is acceptable to the caregiver or even physician treating the patient .
the most common scenario is the titration of the drug to a side effect and then reducing the daily dose to a tolerated dose . because hd is a progressive disease that changes over time , the dose of tbz needs to be reassessed frequently ( every 23 months ) .
there is no evidence if tbz can be used safely or effectively with neuroleptics or other commonly used medications for chorea .
until clear neuroprotective strategies are found , clinicians can address the symptoms of patients with this devastating disease .
the most and best evidence for treating troublesome chorea leads toward tbz , but other abnormal involuntary movements , cognition , affective disorders , and behavioral dyscontrol all need to be considered when choosing therapies for patients .
further studies to determine interactions and combination therapy of tbz need to be completed , as most patients need multiple medications to adequately address symptoms . other issues related to hd also need to be studied more closely when treating patient with tbz .
although there is one fda - approved option at this time and other medications hopefully soon to be available , there are not enough effective , safe interventions that can be offered to our patients and their families with this invariably fatal disease . | huntington disease ( hd ) is a dominantly inherited progressive neurological disease characterized by chorea , an involuntary brief movement that tends to flow between body regions .
hd is typically diagnosed based on clinical findings in the setting of a family history and may be confirmed with genetic testing .
predictive testing is available to those at risk , but only experienced clinicians should perform the counseling and testing .
multiple areas of the brain degenerate mainly involving the neurotransmitters dopamine , glutamate , and -aminobutyric acid . although pharmacotherapies theoretically target these neurotransmitters , few well - conducted trials for symptomatic or neuroprotective interventions yielded positive results .
tetrabenazine ( tbz ) is a dopamine - depleting agent that may be one of the more effective agents for reducing chorea , although it has a risk of potentially serious adverse effects .
some newer antipsychotic agents , such as olanzapine and aripiprazole , may have adequate efficacy with a more favorable adverse - effect profile than older antipsychotic agents for treating chorea and psychosis .
this review will address the epidemiology and diagnosis of hd as background for understanding potential pharmacological treatment options .
because tbz is the only us food and drug administration - approved medication in the united states for hd , the focus of this review will be on its pharmacology , efficacy , safety , and practical uses .
there are no current treatments to change the course of hd , but education and symptomatic therapies can be effective tools for clinicians to use with patients and families affected by hd . |
chronic kidney disease ( ckd ) has become a public health problem due to its increasing prevalence globally and associated high morbidity and mortality.123 the burden of ckd is more felt in developing countries like nigeria where there is no health insurance to meet the huge financial demands the disease places on its sufferers and their families.4 majority of ckd patients in nigeria present late to nephrologists when they are already uremic and requiring renal replacement therapy ( rrt).3 this may be due to both patient and primary physician - related factors .
early referral of ckd patients to nephrologists is key to retarding progression to end - stage renal disease , reducing hospitalization , cost of health care , and improving patients ' survival before and eventually after commencement of rrt.567 prevention and early detection of ckd are the main instruments for combating ckd in the world today . to this effect , programs have been initiated to actualize this goal .
one of such is the world kidney day initiative established by the international society of nephrology and international federation of kidney foundation marked yearly worldwide for the past 10 years with the primary objective of educating and enlightening the public on prevention and early detection of kidney disease as well as giving support to kidney disease sufferers .
nigerian nephrologists have been actively involved in the world kidney day initiative , and there has been an increase in public enlightenment campaigns on ckd .
the goal of this study was to assess the sociodemographic , clinical characteristics , and referral pattern of ckd patients at the time of first presentation to a tertiary hospital in southwest nigeria .
this will help determine if existing preventive nephrology programs and strategies are achieving desired results and also to identify areas requiring improvement .
this study was a descriptive retrospective survey carried out in kidney care centre , ondo city , a tertiary health institution in southwest nigeria .
ckd patients who presented to the center over an 18 month period from july 2014 to december 2015 were recruited for the study .
the case records of the patients were retrieved , and the following information extracted : sociodemographic data , clinical characteristics at presentation and mode of referral to the nephrologist .
the modification of diet in renal disease ( mdrd ) equation was used to estimate glomerular filtration rate ( gfr ) .
ckd staging was done using estimated gfr as follows : stage 1 ( gfr of 90 ml / min with evidence of kidney damage ) , stage 2 ( gfr of 6089 ml / min with evidence of kidney damage ) , stage 3 ( gfr of 5930 ml / min with or without evidence of kidney damage ) , stage 4 ( gfr of 1529 ml / min with or without evidence of kidney damage ) , and stage 5 ( gfr < 15 ml / min with or without evidence of kidney damage).8 hypertension was graded according to the jnc7 guidelines as mild hypertension : blood pressure ( bp ) between 140159/9099 mmhg , moderate hypertension : bp between 160179/99109 mmhg and severe hypertension : bp 180/110 mmhg.9 anemia was defined as hematocrit < 36% in females and < 39% in males.10 anemia was graded as severe anemia ( hematocrit < 18% ) , moderate anemia ( hematocrit of 1825% ) , and mild anemia ( hematocrit of 2635% for females and 2638% for males ) .
data generated were analyzed using the statistical package for social sciences inc , chicago released 2008 , ibm corp , new york ) .
continuous variables were presented as mean and standard deviation for unskewed data and median , interquartile range for skewed data .
data generated were analyzed using the statistical package for social sciences inc , chicago released 2008 , ibm corp , new york ) .
continuous variables were presented as mean and standard deviation for unskewed data and median , interquartile range for skewed data .
a total of 202 ckd patients seen during the period reviewed , met the inclusion criteria .
the majority of the ckd subjects were married and below 65 years of age ; accounting for 169 ( 83.7% ) and 165 ( 81.7% ) of the ckd subjects respectively .
sociodemographic characteristics of study population the mean age , systolic bp , diastolic bp , hematocrit of the study population were 48.15 16.69 years , 154.66 35.20 mmhg , 91.56 23.13 mmhg and 24.44 7.08% , respectively .
the median serum creatinine and estimated gfr of the study population were 997 ( 764.6 ) mol / l and 3.17 ( 5.05 ) ml / min/1.73 m respectively [ table 2 ] .
physical , hematological and biochemical parameters of chronic kidney disease patients the common presenting complaints of the study patients were body swelling in 143 ( 70.8% ) , malaise in 100 ( 49.5% ) , nausea and vomiting in 104 ( 51.5% ) , and oliguria in 95 ( 47% ) [ table 3 ] .
forty - three ( 21.3% ) had mild hypertension , 46 ( 22.8% ) had moderate hypertension , and 65 ( 32.2% ) had severe hypertension at presentation [ figure 1 ] .
severe , moderate , and mild anemia were present in 30 ( 14.9% ) , 68 ( 33.7% ) , and 43 ( 21.3% ) respectively at presentation .
seven ( 3.5% ) were in ckd stage 3 , 22 ( 10.9% ) in stage 4 and 173 ( 85.6% ) in stage 5 . at the time of presentation ,
123 ( 60.9 ) required in - patient 's care and 101 ( 50% ) required urgent rrt .
clinical characteristics of study population degree of hypertension and anemia among chronic kidney disease patients at first presentation the common etiologies of ckd were chronic glomerulonephritis , hypertension , diabetes mellitus , obstructive nephropathy in 69 ( 34.2% ) , 47 ( 23.3% ) , 38 ( 18.8% ) , and 21 ( 10.4% ) respectively [ figure 2 ] .
etiology of chronic kidney disease only ( 18 ) 9% of these ckd patients presented by self - referral while ( 103 ) 51% were referred from secondary and private health facilities [ figure 3 ] .
this study showed that most ckd patients still present very late , requiring in - patient care and emergency rrt despite present efforts geared towards prevention and early detection of ckd .
there were more male ckd patients in this study which is similar to previous studies.3111213 this may be because ckd is more common in males and men tend to have more financial power to access health care services .
more than 80% of these patients were young and middle aged which also agreed with previous reports from nigeria and other parts of sub - saharan africa31112 unlike in developed countries where the elderly are more commonly affected by ckd.114 common etiologies of ckd in this study are chronic glomerulonephritis , hypertension , diabetes mellitus , and obstructive uropathy similar to the findings in previous studies.13111215 therefore , persons with risk factors of the aforementioned conditions should be identified early and treated to therapeutic targets as recommended by standard guidelines , to reduce the incidence of ckd .
most of the patients were in ckd stage 5 at the time of first presentation similar to findings from previous reports.312 a major proportion of the patients already had uremic symptoms at the time of first presentation to the nephrologist and required emergency rrt .
about half of the ckd patients in this study were referred from private hospitals and secondary health facilities where there were no nephrologists .
this reiterates the need for health workers at this level of care to be familiar with ckd management guidelines to reduce the burden of the disease through early risk identification , prevention , and early referral to the nephrologist .
this could be achieved by incorporating such guidelines into the continuing medical education programs organized for health workers .
although factors associated with late presentation were not assessed in this study , both physician and patient - related factors have been reported to be responsible in previous studies .
patient - related factors include poor health seeking behavior , erroneous belief that their health problems resulted from spiritual attack and lack of funds to seek proper health care while physician - related factors include perception preferences and knowledge deficits.161718 an increase in public awareness and education on early features of kidney disease geared toward improving appropriate health - seeking attitude among nigerians is necessary .
this can be achieved by collaborating with religious institutions since some of these patients present initially to spiritual homes because of the erroneous belief that spiritual forces are the source of their ill - health .
a significant proportion of these patients presented with moderate to severe hypertension and anemia , both of which are cardiovascular risk factors that contribute to left ventricular hypertrophy and subsequent mortality in ckd patients.192021 anemia has been reported to be associated with increased expenditure and hospitalization rates in ckd patients.2223 those with severe anemia had to receive blood transfusions thereby increasing the cost of care .
none of the patients had received vaccination against hepatitis b , which is part of the predialysis care in ckd patients because of the increased risk of hepatitis b virus infection during hemodialysis and blood transfusion .
it is recommended that hepatitis b vaccination be administered early in ckd for it to be effective.24 furthermore , due to the late presentation of these patients , they do not have the opportunity to benefit from interventions such as early correction and treatment of anemia , hypertension , proteinuria , calcium , and phosphate abnormalities with appropriate and recommended therapeutic agents which have been reported to be cost - effective and also have indirect effects in reducing disability and improving productivity in early stages of ckd.2526 sixty - one percent of the patients in this study required in - patient 's care at the time of first presentation due to the fact that they were quite ill .
the time spent by these patients on admission has adverse effects on the overall nation 's economic activities and their families because most of these patients were gainfully employed and constituted the economically productive age group in our nation .
furthermore , hospitalization of ckd patients has been associated with increased morbidity and mortality.27 this is avoidable if patients are seen earlier before reaching advanced stages of ckd .
all the patients in this study who required dialysis used temporary vascular access due to their poor clinical state at the time of presentation .
this is more expensive compared to native arteriovenous fistula which offers higher blood flow rates and better dialysis efficiency . initiating hemodialysis with temporary vascular access
is also associated with infection , hospitalization , poor patient survival , increased cost and mortality in ckd patients compared to the use of arteriovenous fistula.2829 the limitation of this study is that we depended on the accuracy of previous documentation in the patients ' records .
the strength of this study , however , lies in the fact that it showed a cogent need to re - strategize efforts toward prevention and early detection of ckd as the present strategies have not achieved the desired results .
the following recommendations are proposed :
regular health education of nigerians aimed toward improving their health seeking attitude and the adoption of lifestyles that supports healthy kidneysreligious institutions should be involved in educating and encouraging their followers to improve their health seeking attitudes while yet seeking spiritual intervention and healingthere should be continuous medical education for health workers , especially those in primary and secondary health institutions aimed at early diagnosis , treatment , and referral of ckd patients to nephrologiststhere is also need to adequately upgrade more health institutions to train more nephrologists in nigeria .
regular health education of nigerians aimed toward improving their health seeking attitude and the adoption of lifestyles that supports healthy kidneys religious institutions should be involved in educating and encouraging their followers to improve their health seeking attitudes while yet seeking spiritual intervention and healing there should be continuous medical education for health workers , especially those in primary and secondary health institutions aimed at early diagnosis , treatment , and referral of ckd patients to nephrologists there is also need to adequately upgrade more health institutions to train more nephrologists in nigeria .
this study concludes that ckd is more common in males and the economically productive age group in nigeria , with majority of them presenting very late to nephrologists in uremic state and commencing rrt without adequate predialysis care .
the present strategies toward prevention and early detection of ckd have not yet yielded the desired results . furthermore , most of the causes of ckd in nigeria are preventable if at - risk individuals are identified early and effectively managed .
| background : chronic kidney disease ( ckd ) has become a public health problem in nigeria .
efforts are being geared toward early diagnosis and prevention of ckd .
this study involved the evaluation of the referral pattern and mode of presentation of ckd patients at first contact in a tertiary health institution.patients and methods : patients ' records over an 18 month period were retrieved and the following information extracted : sociodemographic data , referral hospital , mode of presentation , etiology of ckd , packed cell volume , blood pressure , and estimated glomerular filtration rate ( gfr ) at first presentation.results:there were 202 ckd patients with a male : female ratio of 1.7:1 and a mean age of 48.15 16.69 years .
the median estimated gfr of the patients at presentation was 3.17 ml / min/1.73 m2 .
the common etiologies of ckd were chronic glomerulonephritis , hypertension , diabetes mellitus , obstructive nephropathy in 69 ( 34.2% ) , 47 ( 23.3% ) , 38 ( 18.8% ) , and 21 ( 10.4% ) respectively . among these patients ,
111 ( 55% ) and 98 ( 48.6% ) had moderate to severe hypertension and anemia , respectively , 173 ( 85.6% ) presented in ckd stage 5 , 101 ( 50% ) required urgent hemodialysis whereas 123 ( 60.9% ) required in - hospital admission . only ( 18 ) 9% of these ckd patients presented by self - referral while ( 103 ) 51% were referred from secondary and private health facilities.conclusion:most nigerian ckd patients still present very late to nephrologists implying that the present preventive strategies have not yielded desired results .
early diagnosis and referral of ckd patients could be better achieved through regular education of the public and retraining of health workers especially those in primary and secondary health institutions . |
calcium channel blocker ( ccb ) toxicity is associated with refractory hypotension and myocardial depression which can be fatal .
amlodipine is a dihydropyridine ccb that inhibits calcium entry into slow l - type calcium channels in vascular smooth muscle , the myocardium , and pancreatic beta cells .
the hemodynamic consequences of ccb toxicity are peripheral vasodilation , decreased contractility , and bradycardia .
suppression of insulin release from the pancreas leads to hyperglycemia and to decreased free fatty acid utilization by the myocardium .
ccb 's undergo extensive first pass metabolism and 90% of the parent drug is metabolized in the liver .
amlodipine is 95% protein bound and so can not be dialyzed . in therapeutic concentrations , amlodipine has a half - life of 30 50hrs .
after ccb overdose , however , hepatic enzymes become saturated and so the half - life of amlodipine can be even longer .
a thirteen year old , 61 kg young woman intentionally ingested thirty 10 mg tablets of amlodipine , six bottles of beer , and an unknown quantity of barbiturates .
she presented to the emergency department ( ed ) six hours after the ingestion complaining of tiredness and increased heart rate .
she had a blood pressure of 73/34 ( mean 47 ) mmhg , heart rate of 103/min , and respiratory rate of 16/minute .
her blood pressure did not improve despite the administration of multiple boluses of normal saline , two grams of calcium chloride , and infusion of norepinephrine ( 0.1 mcg / kg / min ) .
the patient was transferred to the pediatric intensive care unit ( picu ) . on admission to the picu she was hypotensive and drowsy , but would awaken and respond to questions .
, the patient had worsening metabolic and lactic acidosis [ table 1 ] , increased creatinine concentration , oliguria , and decreased mental status .
the norepinephrine infusion was increased ( 0.4 mcg/ kg/ min ) and epinephrine infusion ( 0.1 mcg / kg / min ) was added .
insulin ( 0.1 unit/ kg/ hour ) and dextrose ( d12.5 at 100 cc / hr ) were then infused . due to evidence of decreased end organ perfusion and persistent refractory hypotension , which was expected to last at least 48 hrs after the ingestion of amlodipine , the patient was electively placed on veno - arterial extracorporeal life support ( va ecls ) 15 hrs after the ingestion and 9 hrs after presentation to the ed .
the patient was induced with fentanyl 10 mcg/ kg iv , paralyzed with rocuronium and intubated a 17 french ( fr ) arterial cannula was placed in the right femoral artery .
a 12 fr arterial cannula was placed distal to this site to maintain perfusion to the right lower extremity .
a goretex graft was used during the insertion of the arterial cannulas to facilitate subsequent decannulation .
ecls flow was initiated slowly and increased to 70 cc/ kg / min as tolerated .
heparin was infused to maintain the activated clotting time 160 - 180 seconds . over the subsequent 24 hrs
the ecls flows were decreased and the patient was successfully decannulated after 57 hrs on ecls .
the cardiovascular symptoms of ccb overdose are hypotension , due to peripheral vasodilation , myocardial depression , and bradycardia .
intentional ccb overdose is often exacerbated by the ingestion of other drugs , e.g. , tricyclic antidepressants , or - blockers , which exacerbate ccb toxicity . in the case
treatment of ccb overdose includes initial stabilization and resuscitation . gastric decontamination with activated charcoal and polyethylene glycol
is indicated if the patient presents within two hours of ingestion or if a delayed release preparation has been ingested , supportive therapy initially includes the administration of iv crystalloid .
the antidote for ccb overdose is iv calcium chloride ( 20mg / kg , up to 1 gram ) or iv calcium gluconate ( 50 mg / kg , up to 1 gram ) in an attempt to increase circulating calcium and overcome blockade of calcium channels .
insulin resistance occurs after ccb overdose and decreases intracellular glucose stores and increases fatty acid oxidation .
insulin activates calcium and potassium channels and increases intracellular atp , which improves myocardial contractility , and inhibits the release of pro - inflammatory mediators . in our patient
, there was no hemodynamic improvement within two hours of starting insulin therefore the patient was placed on ecls .
glucagon has been used to treat ccb overdose , although this is an unlabelled use of the medication .
glucagon bolus followed by an infusion has been used with favorable response by doyon et al and helps to improve the metabolism of cardiac myocytes and thus improves contractility .
levosimendan sensitizes myocytes to calcium by binding to troponin c and increasing the availability of calcium to actin and myosin fibers .
varpula et al , reported the administration of levosimendan in two patients with ccb toxicity and refractory hypotension .
both patients had in improvement in their mean arterial pressure within 60 to 90 minutes and were weaned off inotropic support within 12 hours .
ecls has been reported twice in cases of refractory ccb overdose , once due to diltiazem overdose and once due to verapamil and propranolol overdose .
both cases involved 16 yr old females with ccb overdoses who were emergently cannulated onto ecls due to cardiac arrest and subsequently decannulated with no evidence of neurologic deficit .
the patient outlined in this report was electively cannulated and subsequently decannulated in less than 72 hrs .
ecls is useful because it restores end organ perfusion and allows metabolism and excretion of the ccb .
experimental therapies such as the administration of glucagon , insulin and levosimendan , have also been reported .
ecls may be indicated if a patient with ccb overdose continues to have refractory and severe hypotension despite conventional and experimental therapies . | calcium channel blocker ( ccb ) toxicity is associated with refractory hypotension and can be fatal . a 13 year old young woman presented to the emergency department(ed ) six hours after an intentional overdose of amlodipine , barbiturates , and alcohol .
she remained extremely hypotensive despite the administration of normal saline and calcium chloride and despite infusions of norepinephrine , epinephrine , insulin , and dextrose .
due to increasing evidence of end organ dysfunction , extracorporeal life support ( ecls ) was initiated 9 hours after presentation to the ed . the patient 's blood pressure and end organ function immediately improved after cannulation .
she was successfully decannulated after 57 hours of ecls and was neurologically intact .
patients with calcium channel blocker overdose who are resistant to medical interventions may respond favorably to early ecls . |
macrodystrophia lipomatosa is a congenital , progressive , nerve territory oriented , localized gigantism . there is proliferation of all mesenchymal elements , but especially adipose tissue .
the abnormal growth ceases at puberty , but the overgrown part is prone to joint degeneration and compression of the neurovascular bundle in some patients .
it needs to be differentiated from other conditions such as hemangioma , lymphangioma , or neurofibromatoses .
a 20 year old female presented with painless enlargement of the lateral aspect of the left hand , left thumb , the index and middle fingers , and also of the left shoulder , which had been present since childhood .
there were no associated nodules , cafe - au - lait spots , or pitting edema , and no audible bruits or thrills .
plain radiograph showed enlargement of the phalanges of the thumb , index , and middle fingers , along with soft tissue hypertrophy ( fig . 1 ) and soft tissue enlargement about the left shoulder .
mri examination revealed hypertrophy of soft tissue ( mainly fat ) of the first , second and third digits , mainly on the volar aspect , along with fusiform enlargement of the median nerve just distal to the carpel tunnel ( fig .
there was hypertrophy of soft tissue ( mainly fat ) in the region of the shoulder , along with fatty infiltration of the supraspinatus , infraspinatus , and teres minor muscles , and the posterior half of the deltoid muscle .
the axillary nerve was enlarged and showed fatty infiltration in the quadrilateral space ( fig .
3 ) , the left brachial plexus showed similar masses involving all ( upper , middle and lower ) trunks , along with hypertrophy of soft tissue ( mainly fat ) in this area ( fig .
our patient appeared to have a combination of fibrolipomatous hamartoma ( flh ) and macrodystrophia lipomatosa , though it is very difficult to separate these two entities , as detailed in the underlying discussion .
the soft tissue bulk was reduced and partial amputation of the overgrown digits was done .
the middle phalanges of the second and third digits were removed , while the distal phalanges were retained ( to preserve nails ) ( fig .
1plain radiograph ( ap view ) of the left hand showing enlargement of the phalanges of the thumb and the index and middle fingers , along with soft tissue hypertrophyfig . 2t1 w coronal ( a ) and axial ( b ) images of the left hand , showing soft tissue hypertrophy and an enlarged median nerve , with fat infiltrating the nervefig . 3t1 w coronal oblique image of the left shoulder , showing enlargement of the axillary nerve in the quadrilateral space ( arrow ) with fatty infiltration of the nervefig .
4t2 w coronal ( a ) and t1 w sagittal ( b ) images of the left brachial plexus , showing enlargement of all three trunks ( arrows ) along with fatty infiltrationfig .
5postoperative plain radiograph ( ap view ) of the left hand showing partial amputation of the overgrown digits ( middle phalanges of the second and third digits have been removed ) and reduced soft tissue bulk plain radiograph ( ap view ) of the left hand showing enlargement of the phalanges of the thumb and the index and middle fingers , along with soft tissue hypertrophy t1 w coronal ( a ) and axial ( b ) images of the left hand , showing soft tissue hypertrophy and an enlarged median nerve , with fat infiltrating the nerve t1 w coronal oblique image of the left shoulder , showing enlargement of the axillary nerve in the quadrilateral space ( arrow ) with fatty infiltration of the nerve t2 w coronal ( a ) and t1 w sagittal ( b ) images of the left brachial plexus , showing enlargement of all three trunks ( arrows ) along with fatty infiltration postoperative plain radiograph ( ap view ) of the left hand showing partial amputation of the overgrown digits ( middle phalanges of the second and third digits have been removed ) and reduced soft tissue bulk
first described by feriz in 1925 , macrodystrophia lipomatosa is an unusual form of local hypertrophy , congenital in origin but not hereditary [ 13 ] , and characterized by proliferation of all mesenchymal elements but with a disproportionate increase in adipose tissue .
it is a form of true macrodactyly where the degree of overgrowth of the affected digits / limb is faster than the normal growth pattern , and such abnormal growth ceases at puberty .
involvement of a lower limb is more frequent than an upper limb , and the most common sites are the second and third digits .
flh of the nerve is a rare tumor - like condition that consists of an infiltration of connective tissue and fatty elements of the nerve .
flh of the nerve may occur without macrodactyly , but those with macrodactyly are the same as mdl .
it usually presents as an isolated nerve lesion and associated intramuscular fat deposition , while in mdl , fat deposition also occurs in periosteum , leading to bony changes in addition to fat deposition in nerve sheath , subcutaneous , and muscle compartments .
the enlargement of the axillary nerve and all of the cords of the brachial plexus in our patient likely represents typical flh according to the above description , while enlargement of the hand and three digits represents mdl , because there is no bony overgrowth of the humerus , while there is bony overgrowth of the second and third metatarsals and phalanges .
moreover , a combination of flh and mdl of the median nerve is reported to exist , as in our case .
the flhs involving three cords of the brachial plexus , axillary nerve , median nerve , and digital nerves in our case may also represent such skip lesions .
various hypotheses include lipomatous degeneration , disturbed fetal circulation , erroneous segmentation , the trophic influence of a tumified nerve , and the in utero disturbance of a growth - limiting factor .
the condition is largely asymptomatic , but localized overgrowth can lead to deformities that can hinder function .
moreover , due to deformities secondary to the localized overgrowth , there are premature degenerative changes in joints . there may be compression of a neurovascular bundle .
the patient seeks consultation for either cosmetic reasons or due to functional impairment due to deformity or degenerative changes .
the corrective surgery aims to reduce the bulk of the overgrown fibrofatty tissue and to partially amputate the overgrown digits .
our patient underwent surgery of the hand , which reduced the bulk of the soft tissue , and the overgrown digits were partially amputated ( middle phalanges of the second and third digits were removed ) .
histology of the lesion shows a massive increase in adipose tissue interspersed with a fine mesh of fibrous tissue that involves subcutaneous tissue , bone marrow , periosteum , muscle , and nerve sheaths .
the soft tissue growth is more marked on the volar aspect , leading to dorsal deviation of the involved digits , as in our case .
macrodystrophia lipomatosa should be distinguished from other congenital anamolies such as hemangioma , lymphangioma , ollier s disease , klippel
hemangioma , lymphangioma , and klippel trenaunay weber syndrome can be easily distinguished based on ultrasound and signal characteristics on mri .
hemangiomas show a septate configuration of high signal intensity channels on t2 w images corresponding to the vascular channels and fibrous strands .
lymphangiomas are hyperintense to muscle on t1 w and hyperintense to fat on t2 w images .
mr images show malformed venous and lymphatic lesions as areas of high signal intensity on t2 w images in klippel
trenaunay weber syndrome , and depict the deep extension of low - flow vascular malformations into muscular compartments and the pelvis .
ollier s disease can also be easily made out on x - rays of the affected part .
plexiform neurofibromatosis is most difficult to distinguish from macrodystrophia lipomatosa , but unlike nf , macrodystrophia lipomatosa does not show familial occurance or cutaneous stigmata .
in addition , there are thinned bone shafts and waxy cortices in nf , while in mdl there is bony enlargement .
as mentioned previously , mdl is a nonhereditary congenital localized gigantism with skeletal abnormalities and a marked disappropriate increase in fibroadipose tissue .
some authors suggest that mdl is a localized form of proteus syndrome [ 11 , 12 ] .
though the exact etiology is unknown , the disease is believed to be due to the alteration of somatic cells during limb bud development and disturbed fetal circulation .
recent genetic studies have indicated involvement of the pten ( phosphatase and tensin homolog deleted on chromosome 10 ) gene , which is primarily a tumor suppressor gene and substantially influences cell / tissue growth .
pten mutations and pten nullizygosity have already been reported in proteus and proteus - like syndromes , which in addition to hemihypertrophy , lower limb asymmetry , and arteriovenous malformations also presents with lipomatosis [ 15 , 16 ] . although the regulatory function of the pten gene in cell growth is well established , which possibly accounts for the marked increase in adipose tissue mass , more evidence on the molecular genetic side is needed to categorically prove the role of the pten gene in mdl , and more specifically the mechanism of disproportionate lipomatous formation [ 17 , 18 , 19 ] .
macrodystrophia lipomatosa may be considered a part of proteus syndrome , a generalized hamartomatous disorder involving hemihypertrophy consisting of lipomatous tumor in addition to other features , such as skull anamolies , pigmented naevi , lung cyst , and intra - abdominal lipomas [ 2022 ] .
our case corroborates this fact , as all the hypertrophied parts and nerve hamartomas are on the same side of the body ( i.e. , the left side ) , thus pointing to localized hemihypertrophy along with lipomatous tumors .
mdl and flh are two entities that are closely related and difficult to dissociate , as in our case .
it is important to diagnose macrodystrophia lipomatosa as the condition is initially progressive but stabilizes at puberty .
correct diagnosis obviates unnecessary anxiety , and corrective surgery at the appropriate stage may help the patient .
the involvement of the pten gene in the etiology of both mdl and proteus syndrome suggests that mdl is a localized form of proteus syndrome .
the unilateral involvement of all nerve territories in our patient provides physical evidence of this fact , as it suggests localized hemihypertrophy , a component of proteus syndrome . | macrodystrophia lipomatosa ( mdl ) , a rare congenital disorder , is considered by some to be a localized form of proteus syndrome .
the implication of the pten ( phosphatase and tensin homolog deleted on chromosome 10 ) gene in both strengthens this belief .
we present a case who had mdl in multiple nerve territories
all on the same side of the body with hypertrophy of mainly fibroadipose tissue throughout their distribution , thus pointing to a form of localized hemihypertrophy ; both hemihypertrophy and lipomatous tumors are components of proteus syndrome . |
human lipoxygenases
( lox )
are nonheme iron - containing enzymes that catalyze the dioxygenation
of 1,4-cis , cis - pentadiene - containing
polyunsaturated fatty acids ( e.g. , linoleic acid ( la ) and arachidonic
acid ( aa ) ) to form hydroperoxy fatty acids . the
nomenclature of the lox isozymes is loosely based on the carbon position
( e.g. , 5 , 12 , or 15 ) at which they oxidize arachidonic acid to form
the corresponding hydroperoxyeicosatetraenoic acid ( hpete ) , which is reduced to the hydroxyeicosatetraenoic
acid ( hete ) by intracellular glutathione peroxidases .
5-lox has been implicated in cancer , asthma , copd , allergic rhinitis , osteoarthritis , and atherosclerosis , whereas platelet - type 12-lox has been implicated
in diabetes , blood coagulation , psoriasis , and
cancer .
human reticulocyte 15-lipoxygenase-1 ( 12/15-lox ,
aka 15-lox-1 ) is also an attractive therapeutic target , particularly
for its role in atherogenesis , diabetes , alzheimer s disease , newborn periventricular leukomalacia , breast cancer , and
stroke , with the latter being the focus of this
work .
stroke is the fourth - leading cause of death in the united
states
and the leading cause of disability .
however , only
tissue plasminogen activator ( tpa ) is an fda - approved drug treatment ,
and it is used in less than 5% of stroke patients .
the first reports of
the contributions of 12/15-lox to neuronal cell death in oxidative
stress models were published starting in the late 1990s . because oxidative stress is a major neurodegenerative process in
ischemic diseases such as stroke , a role for 12/15-lox in stroke - induced
brain injury seemed reasonable and , indeed , was found .
later work showed that 12/15-lox was detrimental not only to neurons
but also to the brain vasculature after stroke via a mechanism that involves an intracellular attack on
mitochondria and translocation of the apoptosis - inducing factor ( aif )
to the nucleus .
recent studies have shown that 12/15-lox is also
increased in human stroke . in line with
these findings
, several lox inhibitors have been found to reduce infarct
size , leakage of
the blood brain barrier and edema formation , and even hemorrhagic transformation following infusion of tpa .
consequently , these broad implications
of 12/15-lox in stroke regulation
emphasize the need for small molecule inhibitors that effectively
cross the blood brain barrier and target affected tissue .
however ,
an additional aspect that must be considered in discovering human
12/15-lox inhibitors is interspecies variability .
the gene for 12/15-lox
for both humans and mice is termed alox15 , reflecting the close homology
( around 78% ) and functional equivalence in both species .
12/15-lox
isozymes can oxidize both c12 and c15 , forming 12-hpete or 15-hpete ,
respectively ; however , the dominant product varies between species ,
with several isozymes oxidizing aa at c12 , leading to a somewhat confusing
nomenclature .
the 12/15-lox isozymes targeted here have historically
been called 15-lox , 15-lox-1 , or 15-lo-1 in humans and l-12-lox , leukocyte - type
12-lo , or l-12-lo in mice .
the number prefix reflects the preference
for 15-hete generation by human 12/15-lox , whereas the preference
is mostly for 12-hete production for mouse 12/15-lox .
unfortunately ,
this difference in substrate specificity could mean that an isoform - specific
inhibitor that is selective toward human 12/15-lox may not target
the orthologous mouse 12/15-lox , which would preclude the use of small
animal stroke models .
considering that drug discovery relies to a
large extent on rodent models of disease because of availability and
cost , which is compounded by ethical considerations , it is therefore
of great importance to identify bioactive compounds that target 12/15-lox
in both rodents and humans . over the last 12 years
, our laboratory and others have attempted to identify potent and selective human 12/15-lox
inhibitors , which has been met with limited success .
unfortunately ,
many of these inhibitors are reductive and/or promiscuous , such as
boswellic acid ( ic50 = 1 m ) , nor - dihydroguaiaretic acid ( ndga ) ( ic50 = 0.11
m ) , and baicalein ( ic50 = 1.6 m ) , as shown in figure 1 .
computational docking methods have been used to
identify novel , nonreductive inhibitors , but the potency and selectivity
of these compounds is limited ( loxblock-1 , figure 1 ) .
the most drug - like 12/15-lox
inhibitors that had been published prior to our investigations were
those reported by researchers at bristol - myers squibb ( bms ) as well
as parke - davis ( now pfizer ) compound pd146176 .
the bms team identified tryptamine - based compounds ( 37l ) , imidazole - based compounds
( 21n ) , and pyrazole - based
compounds ( 15i ) , as shown
in figure 1 .
these inhibitors exhibited low
nanomolar potency versus rabbit 12/15-lox ( r12/15-lox ) and modest
selectivity against both 5-lox and 12-lox , but they had generally
unfavorable physical properties ( e.g. , solubility and logp ) and unacceptable pk properties for use in vivo .
most importantly ,
none of the above inhibitors , with the exception of loxblock-1 , were
screened in parallel against mouse 12/15-lox , so it is unlikely that
any would be amenable to stroke drug development .
examples of previously
reported human 12/15-lox ( 12/15-lox ) and
rabbit 12/15-lox ( r12/15-lox ) inhibitors .
our previously discovered inhibitor , loxblock-1 ( figure 1 ) , demonstrated good activity in mouse models for
stroke ; however , it is nonselective , suggesting that the identification of novel
selective inhibitors to interrogate 12/15-lox biology in vivo is desirable .
from our original quantitative high - throughput screen ( qhts ) of 74 290
small molecules ,
the 1,3,4-oxadiazole-2-thiol chemotype ( ml094 , figure 1 ) was
optimized for nanomolar potency and selectivity ; however , we were
unable to observe activity of ml094 in a cellular context . therefore , in this work , we present our efforts
to identify a specific 12/15-lox inhibitor that is potent against
both the human and the mouse homologues of 12/15-lox .
as such , we
rescreened our top hits from the original hts with our cellular mouse
model of oxidative stress - related cell death and discovered a novel oxazole-4-carbonitrile core scaffold
. we
discuss the sar and biological activity of this novel scaffold , which
has nanomolar potency against human 12/15-lox , selectivity over related
eicosanoid producing enzymes , and potency against mouse 12/15-lox
in both a cellular and an in vivo model of stroke .
intermediates 2i18i and 19 were obtained by propylphosphonic anhydride ( t3p)-assisted coupling of the corresponding commercially available
carboxylic acids with 2-aminomalanonitrile followed by cyclization
in one step .
this modified synthesis of 5-amino-2-substituted-1,3-oxazole-4-carbonitriles
allows facile access to a variety of compounds via a simple workup
and purification from commercially available carboxylic acids . because
of the low nucleophilicity of the amine group ,
our initial attempts
to monomethylate the 5-amino-2-substituted-1,3-oxazole-4-carbonitrile
intermediate with several known methylation methods including methyl
iodide , formic acid , or methyl boronic acid were met with limited
success .
however , condensation with paraformaldehyde in the presence
of sodium methoxide in methanol followed by in situ reduction with
sodium borohydride afforded required products 118 in modest yields .
reagents
and conditions : ( a )
t3p , net3 , etoac , rt , 12 h ; ( b ) paraformaldehyde ,
naome , meoh , 65 c , 1 h , then nabh4 , rt , 1 h. scheme 2 represents the general
methodology
utilized for the synthesis of compounds listed in table 2 . toward this end , 5-amino-2-(naphthalen-1-yl)oxazole-4-carbonitrile
19 was converted to an advanced intermediate ( 19a ) via sandmeyer reaction by treating with t - butyl
nitrite in the presence of copper(ii ) chloride .
heating the various
amines to reflux with intermediate 19a in thf afforded
required analogues 2026 and 2831 .
compound 27 was obtained
via acetylation of intermediate 19 with acetic anhydride
and cat .
reagents and conditions : ( a )
cucl2 , t - buno2 , mecn , 25 c ,
3 h , 35% ; ( b ) thf , reflux , 112 h. the synthesis of compounds 3240 ( table 3 ) is outlined in scheme 3 .
analogues 3234 were prepared via chlorination of intermediates 32a34a with hexachloroethane in the presence of
lda followed by condensation with methylamine .
amination of 5-chloro-2-(naphthalen-1-yl)thiazole-4-carbonitrile
( 32b ) required microwave heating in a polar solvent ,
such as 2-propanol , for complete conversion of the starting material .
the details of the synthesis of intermediate 32a(63,64 ) are described in the supporting information .
intermediates 33a and 34a were synthesized
following the literature protocols described for similar compounds .
the reaction of 4-methyl-3-thiosemicarbazide with 1-naphthoic acid
in the presence of edc furnished oxadiazole analogue 35 , but , surprisingly , when t3p coupling conditions were
used , only thiadiazole analogue 36 was formed .
synthesis
of compound 37 was accomplished utilizing a cornforth
rearrangement of intermediate 37b , which was prepared
from intermediate 37a by coupling with methylamine , as
shown in scheme 3 .
intermediate 37a was prepared according to a known protocol reported to similar compounds
in the literature .
the synthesis of analogue 38 from 1-naphthyl boronic acid and intermediate 38a using classical suzuki conditions was met with limited success .
gratifyingly , the suzuki coupling reaction was
moderately successful using a silica - bound dpp - pd catalyst ( siliacat - dpp - pd )
under reflux conditions over 24 h. synthesis of intermediates 39a40a was accomplished using a pd(pph3)4-catalyzed microwave - assisted regioselective
suzuki coupling between 1-naphthyl boronic acid and 2,6-dichloro pyridine
or pyrimidine derivative , respectively .
subsequent condensation of
these intermediates with methylamine furnished desired analogues 3940 following hplc purification .
reagents and conditions : ( a )
c2cl6 , lda , thf , 78 c , 24
h ; ( b ) ch3nh2 , i - proh , microwave ,
120 c , 0.5 h ; ( c ) ch3nh2 , thf , 65 c ,
12 h ; ( d ) edc , dmf , 25 c , 12 h ; ( e ) t3p ,
net3 , dmf , 25 c , 6 h ; ( f ) ( 1 ) ( cocl)2 ,
ch2cl2 , ( 2 ) ch3nh2 , net3 , ch2cl2 , 25 c , 1 h ; ( g ) toluene ,
110 c , 12 h ; ( h ) siliacat - dpp - pd , na2co3 , dme , 105 c , 24 h ; ( i ) pd(pph3)4 , na2co3 , dme , microwave , 150 c , 45 min .
to investigate structural requirements
for optimal 12/15-lox inhibition ,
we conducted a sar study of the lead molecule , as shown in tables 13 ( 140 ) .
initially , the 1-naphthyl
group on the left side of the molecule was replaced with various aryl
and heterocyclic groups , as shown in table 1 .
the bioisosteric replacement of 1-naphthyl with 2,3-dichlorophenyl
( 7 ) or 3,4-dichlorophenyl ( 8) groups showed
comparable , albeit slightly lower , potencies than 1 ( ic50 = 0.46 , 0.81 , and 0.20 m , respectively ) .
replacement
with a 2-naphthyl group showed reduced potency ( 2 , ic50 > 30 m ) compared to the 1-naphthyl substitution ,
which , on the basis of space - filling analysis , agreed with the findings
we observed for dichloro analogues 7 and 8 .
several other modifications , including saturated rings or heterocyclic
rings at this region ( 36 and 918 ) , also resulted in reduced potency .
thus , in general , the 1-naphthyl group appeared to be optimal for
12/15-lox inhibition , as variations in size and electrostatics in
this region were not well tolerated .
accordingly , the 1-naphthyl group
was held constant while other regions of the molecule were explored
for further sar ( tables 2 and 3 ) .
ic50 values represent
the half - maximal ( 50% ) inhibitory concentration , as determined in
the uv vis cuvette - based assay in triplicate .
compound possessed low efficacy ,
with less than 50% maximal inhibition at 25 m inhibitor .
ic50 values represent
the half - maximal ( 50% ) inhibitory concentration , as determined in
the uv vis cuvette - based assay in triplicate .
compound possessed low efficacy ,
with less than 50% maximal inhibition at 25 m inhibitor .
ic50 values represent
the half - maximal ( 50% ) inhibitory concentration , as determined in
the uv vis cuvette - based assay in triplicate .
compound possessed low efficacy ,
with less than 50% maximal inhibition at 25 m inhibitor .
having explored modifications to
the 1-naphthyl moiety , our next
focus was to explore modifications of the n - methyl
side chain at the 5-position , as shown in table 2 ( 1931 ) .
removal of the methyl
group drastically reduced potency ( 19 , ic50 = 25 m ) , and dimethylation reduced potency as well ( 20 , ic50 > 30 m ) .
modifications of the
methyl
group with other alkyl substitutions , such as ethyl , n - propyl , n - butyl , and n - pentyl
groups , were tolerated ( 2124 ) , with
comparable or even improved potency being observed . however , replacing
the methyl group with a branched alkyl group ( 26 ) or
heterocyclic groups ( 3031 ) significantly
reduced the potency .
larger groups , such as benzyl- ( 28 , ic50 > 30 m ) or phenyl - substituted analogues
( 29 , ic50 > 30 m ) , also showed diminished
12/15-lox activity .
overall , these data suggest that the monoalkylation
with straight - chain alkyl groups is critical for optimal 12/15-lox
inhibition .
lastly , we turned our attention to optimization
of the 1,3-oxazole
core and its various substituents ( table 3 ) .
replacing the 1,3-oxazole core with a 1,3-thiazole ring ( 32 ,
this result could be partly attributed to the difference
in size and hardness / softness of the sulfur and oxygen atoms in the
thiazole and oxazole rings , respectively .
the diminished potency trend
continued further for the pyridine ( 39 , ic50 > 40 m ) and pyrimidine analogues ( 40 , ic50 > 40 m ) .
attempts to mimic the interaction of
the
oxazole core were largely unsuccessful with the n - methyl / oxadiazole derivative ( 35 , ic50 =
5.4 m ) or the thiadiazole analogue ( 36 , ic50 > 40 m ) .
interchanging the positions of the 1-naphthyl
group and the -nhme group ( analogues 3334 ) also led to complete loss of activity . in summary , this
chemotype showed very tight sar , and our efforts to make several changes
around the lead molecule resulted in similar or lower potency , with
the exception of analogues 2123 ,
which showed marginally improved potency . concurrently
with the sar investigations against human 12/15-lox in vitro , we investigated
the potency of 1 against mouse 12/15-lox , utilizing a
neuronal cell - based assay .
glutamate - induced oxidative stress leads to time - dependent cell death mediated by 12/15-lox in both
immature primary neurons and mouse hippocampal ht22 cells .
primary neurons require a lengthy
isolation procedure and also show relatively modest increases in cell
death in this assay , indicating that they are not suited for a high - throughput
testing approach .
in contrast , ht22 cells grow quickly and consistently
feature low cell death under control conditions , but a majority of
cells die after glutamate treatment ( 50100% ) .
we have now
adapted this assay to 96-well plates , allowing for moderate - to - high
throughput against the screened compounds .
the characteristic z score used to evaluate the technical quality and
suitability of this approach as a screening tool averaged 0.77 over
10 experiments ( sd = 0.13 ; range = 0.490.94 ) , where values
above 0.5 indicate an excellent assay . to verify that inhibition of oxidative stress - related cell
death in ht22 cells is indeed a good measure of the efficacy of a
given inhibitor against the mouse 12/15-lox
levels of the mouse
12/15-lox product , 12-hete , are known to be increased in the mouse
brain following ischemia , and they are
also elevated by glutamate treatment of ht22 cells .
we used hplc / ms to measure 12-hete secreted to the medium from cells
incubated under control conditions or treated with glutamate as well
as cotreated with several compounds with or without inhibitory activity
for mouse 12/15-lox .
following exclusion of one outlier with extremely
high 12-hete ( but also high levels of cell death ) , there was a good
correlation between cell death and levels of 12-hete secreted : when
12-hete levels were high , there was increased cell death , and when
12-hete levels were low , there was little cell death ( figure 2a ) .
interestingly , levels of 15-hete were also increased
in the medium of glutamate - treated ht22 cells to a similar extent
as 12-hete ( figure 2b ) . this was surprising
because mouse 12/15-lox is generally thought to make only minor amounts
of 15-hete , typically less than 20% of the 12-hete generated .
nonetheless ,
15-hete production was also blocked by 12/15-lox inhibitors that protected
ht22 cells and reduced 12-hete levels , indicating that both arachidonic
acid metabolites were made by the same enzyme .
it is unclear why this
large amount of 15-hete was generated , and we are currently working
on a different line of research to address this important result .
( a ) 12-hete , which is graphed as the percent of 12-hete secreted from
glutamate - treated cells vs the percent of cell death in glutamate - treated
samples .
( b ) positive correlation between 15-hete and cell death . upon testing of compound 1 in ht22 cells
, we found
a dose - dependent protection against oxidative glutamate toxicity ( figure 3a ) . to verify the on - target efficacy of 1 , we also measured 12-hete secreted from the cells .
the increase
in 12-hete following glutamate treatment was completely reversed by
incubation in the presence of 10 m of 1 ( figure 3b ) .
these results suggest that 1 is
capable of reaching its target in the cell and effectively inhibiting
mouse 12/15-lox , albeit with lower affinity than seen against human
12/15-lox in vitro ( ic50 = 200 nm ) .
( a ) protection against
glutamate - induced ht22 death by increasing
amounts of 1 ( * * p < 0.01 and * * * p < 0.001 vs glutamate only ) .
( b ) inhibition of 12-hete
in ht22 cells by 10 m of 1 following treatment
with 5 mm glutamate ( * p < 0.05 vs glutamate only ) .
in addition to analogues that
were active in vitro against human
12/15-lox , we also tested several inactive analogues ( 11 , 31 , and 34 ) in ht22 cells , with the goal
of identifying a good negative control ( figure 4 ) .
surprisingly , 11 and 31 featured similar
protective qualities at 5 m , suggesting that they are able
to inhibit the mouse homologue of 12/15-lox even though they are inactive
against human 12/15-lox . in contrast , 5 m of 34 did not protect ht22 cells , suggesting that it is inactive against
both human and mouse 12/15-lox and is a suitable negative control
for 1 .
these data re - emphasize the importance of screening
12/15-lox inhibitors against both human and mouse 12/15-lox because
activity against one species does not guarantee activity or inactivity
against the other .
cellular protection at 5 m of 1 and
some analogues
in ht22 cells . despite not inhibiting human 12/15-lox , 11 and 31 show similar protection to 1 , indicating
that they inhibit the mouse enzyme , whereas 34 does not
protect ht22 cells . upon the determination
that 1 was potent against both
in vitro human 12/15-lox and ex vivo mouse 12/15-lox ( ht22 cell assay ) ,
we then investigated the selectivity of a few of our top analogues
against related human lox isozymes ( 5-lox , 12-lox , and 15-lox-2 ) .
of the four 12/15-lox inhibitors tested , 1 , 7 , 8 , and 32 , all displayed excellent selectivity
against all three isozymes ( ic50 > 50 m , supporting information , table
s1 ) .
we were encouraged by these findings because few compounds
reported in the literature have achieved nanomolar potency toward
12/15-lox while maintaining excellent selectivity toward other isozymes .
moreover , we investigated whether these analogues inhibited cyclooxygenase-1
( cox-1 ) and/or cox-2 and determined that none of them displayed inhibition
( < 10% at 15 m ) .
lox inhibitors are known to exhibit a variety of
inhibitory mechanisms ,
such as chelative , reductive , or competitive .
the uv vis pseudoperoxidase
activity assay was therefore performed on four selected analogues
( 1 , 7 , 8 , and 32 ; supporting information , table s1 ) to determine if the mechanism was reductive
in nature . it was observed that the hydroperoxide product was not
degraded , 12/15-lox was not irreversibly inhibited , and there was
no elongation of the enzymatic lag phase ( data not shown ) .
these data
are consistent with a nonreductive inhibitory mechanism . to investigate
the nature of inhibition further
, steady - state kinetics were performed
using compound 1 by monitoring the formation of 15-hpete
as a function of substrate and inhibitor concentration in the presence
of 0.01% triton x-100 .
replots of km / vmax and 1/vmax versus
inhibitor concentration yielded linear plots ( supporting information , figure s2 ) , with ki equaling 0.1 0.002
m and ki equaling 1.2
0.02 m . these parameters are defined as the equilibrium constants
of dissociation from the catalytic ( ki ) and secondary sites ( ki ) , respectively .
the ki value is in good agreement with
the ic50 value , and because of the greater than 10-fold
difference between ki and ki , we assume the secondary site to be the allosteric
site , which is consistent with our previous studies
of 12/15-lox inhibition . our previously
reported small molecule inhibitor for 12/15-lox inhibition , ml094 , demonstrated excellent potency and selectivity but
lacked sufficient solubility , cell permeability , and microsomal stability
( t1/2 < 2 min ) .
moreover , this compound possessed an essential ester moiety
that could be susceptible to intracellular and plasma esterases , rendering
it inactive and possibly limiting its utility in advanced biological
models .
thus , we were encouraged by the in vitro adme ( table 4 ) and in vivo pk properties ( table 5 ) of 1 , as they represent a vast improvement
over the majority of compounds reported previously ( vide infra ) .
the
majority of the compounds possess a low molecular weight ( e.g. , 249
da ) and a favorable log d ( ph 7.4 ) between 2 and 3 , which was obtained
by analiza inc .
using their scaled - down shake flask lipophilicity
method , yet most analogues have poor solubility .
the aqueous kinetic
solubility in pbs buffer ( ph 7.4 ) was determined to be 1.2 m ,
which is about 7 times the in vitro ic50 value .
empirically ,
we did observe a vast improvement in the solubility in the 15-lox
assay buffer ( data not shown ) , which was encouraging and suggests
that solubility was not a detrimental factor in the biochemical studies .
importantly , the compound demonstrated favorable pampa permeability
( passive ) and acceptable caco-2 permeability of > 1 ( 1.5 cm / s ) , with no evidence of efflux ( efflux ratio : 0.7 ) ,
indicating that
the compound is not susceptible to the action of p - glycoprotein 1
( pgp ) , a well - characterized abc transporter .
moreover , 1 was stable in various aqueous solutions ( ph 2 , 7.4 , and 9 ) ( see supporting information ) and mouse plasma ( table 4 ) .
in addition , 1 exhibited minimal
cyp inhibition of the 2d6 and 3a4 isoforms at 10.3 and 3.5% inhibition ,
respectively .
microsomal stability appears to be species - dependent ,
with 1 possessing modest stability to rat liver microsomes
( 18 min ) while being less stable to mouse liver microsomes ( 5.5 min ) .
the compounds were completely stable in the absence of nadph , suggesting
cyp - mediated degradation . given our plan to test compound 1 in proof - of - concept ( poc ) mouse models of stroke , we also obtained
in vivo pk data on 1 ( table 5 )
and found a suitable formulation for 1 ( 10% solutol ,
10% cremophor el , and 20% peg400 in saline ) .
as anticipated from the
microsomal stability studies , 1 has a fast half - life
in both plasma and brain ( t1/2
1 h ) , with a cmax of 13.8 m in
plasma and 28.8 m in brain .
encouragingly , 1 has
a brain / plasma ratio of 2.8 , which demonstrates favorable blood
brain
barrier permeability and suggests that this compound is suitable for
in vivo poc models of ischemic stroke ( vide infra ) .
all experiments were conducted
at pharmaron inc . in pbs
buffer ( ph 7.4 ) .
the probe compound showed no degradation without
nadph present over a 1 h period .
data
was collected in triplicate at eight time points over a 24 h period . formulated as a solution ( 10%
solutol ,
10% cremophor el , and 20% peg 400 in saline )
. brain / plasma ratio [ auclast(brain)/auclast(plasma ) ] . after establishing
the sar profile against recombinant human 12/15-lox
, determining the
efficacy of 1 in our cellular mouse 12/15-lox assay ,
and measuring its in vitro adme / in vivo pk properties , we then sought
to determine its efficacy in mouse models of ischemic stroke . for
these initial studies
, we chose the permanent focal ischemia model
in mice , which has been shown to mimic the pathophysiological mechanisms
following ischemic injury .
the advantage
of this model is that it does not inflict surgical trauma and has
a low mortality compared to other methods .
the thrombosis event in
the middle cerebral artery ( mca ) is induced by topical application
of fecl3 to the intact dura mater , leading to a cortical
infarct .
laser doppler flowmetry was used to monitor blood flow reduction ,
and infarct size was measured following sacrifice at 24 h by staining
of 1 mm thick brain slices with 2,3,5-triphenylterazolium hydrochloride
( ttc ) .
ip administration of 1 given 2 h after the induced
ischemia resulted in a 30% reduction in infarct size ( p < 0.01 ) , demonstrating efficient neuroprotection in
this mouse model of permanent focal ischemia ( figure 5 ) . ( a ) activity of compound 1 ( 50 mg / kg ) administered
ip in a mouse distal middle cerebral artery occlusion ( mcao ) model
of permanent focal ischemia ( * * p < 0.01 ) .
( b )
typical examples of sequential ( top to bottom represents front to
back sections of a single brain , respectively ) ttc - stained brain sections
from vehicle- and compound 1-treated mice .
the white
areas in the cortex ( top right ) indicate nonviable infarcted tissue .
12/15-lox
contributes to neuronal cell death in oxidative stress
models and is also detrimental not only to neurons but also to the brain
vasculature after stroke via intracellular
attack on mitochondria and translocation of aif to the nucleus .
moreover , the protein level of 12/15-lox gradually increases over
time after experimental stroke , providing
an opportunity for therapeutic intervention .
our initial efforts to
identify a stroke - relevant 12/15-lox inhibitor proved to be unsuccessful
because of its inactivity in our mouse ex vivo model , resulting from
either cellular inactivation or inactivity against mouse 12/15-lox .
fortunately , reinterrogation of the hts data
along with simultaneous cellular mouse 12/15-lox screening revealed
another 12/15-lox inhibitor ( 1 ) with better drug - like
properties and , more importantly , mouse ex vivo activity .
our initial
medicinal chemistry efforts , described herein , provided important
sar to guide future investigations , but we were unable to improve
potency significantly , and the tolerability for the structural changes
is limited .
the most significant loss of inhibition was observed at
the 1-naphthyl position , where almost all substitutions resulted in
significant loss in activity , with the exception of its bioisosteric
dichloro analogues ( 7 and 8) , which only
showed a slight decrease in activity .
modifications of the methyl
group on the n - methyl side chain were not well tolerated
either , indicating that the straight - chain monoalkylated amine was
critical for potency . moreover , the only substitution tolerated for
1,3 oxazole core ring was the replacement with a 1,3-thiazole ( 32 ) , which resulted in a 2-fold decrease in the potency .
the
cyanide group on the oxazole core was found to be essential , with
modifications to this group completely abolishing activity .
subsequent
selectivity studies of 1 and related analogues revealed
that these compounds have minimal activity toward 5-lox , 12-lox , 15-lox-2 ,
cox-1 , and cox-2 .
most importantly , 1 was active
in both protecting
mouse neuronal cells ( ht22 ) and in reducing the infarct size of stroke - induced
mice .
this is a significant discovery because identifying therapeutically
useful specific inhibitors of 12/15-lox is not trivial due to the
differences between the human and mouse 12/15-lox isozymes .
interestingly ,
we have identified analogues of compound 1 ( 11 and 31 ) that did not inhibit human 12/15-lox but protected
ht22 cells against oxidative stress .
these might work in mice to protect
against experimental stroke , yet in human stroke , they would most
likely fail because of their inability to target human 12/15-lox .
the lesson to be drawn from this is that simply because a given inhibitor
is selective for a lox isoform in one species its efficacy can not
be readily extrapolated to other species .
moreover , the lox isotype
involved in a given process can not be reliably inferred from inhibitor
experiments without taking into account the species from which the
cells were derived . in conclusion ,
compound 1 is
the first highly selective
12/15-lox inhibitor active against human 12/15-lox and mouse 12/15-lox ,
as seen by its effectiveness in our mouse stroke model .
this attribute
of 1 is significant because it maintains the human 12/15-lox
selectivity , which is beneficial to a human therapeutic , but is also
functional in a mouse stroke model , which is critical to developing
its biological efficacy .
therefore , using our pipeline approach , we
have identified a potent and selective inhibitor of human 12/15-lox
that is neuroprotective in a mouse model of stroke and therefore can
be utilized in preclinical studies to develop it as a potential first - line
stroke therapy .
all air- or moisture - sensitive
reactions were performed under positive pressure of nitrogen with
oven - dried glassware .
anhydrous solvents such as dichloromethane , n , n - dimethylforamide ( dmf ) ,
acetonitrile , dioxan , dimethoxyethane , methanol , and triethylamine
were purchased from sigma - aldrich .
tetrakis and a 50% of solution
of t3p in dmf or ethyl acetate were purchased from strem chemicals
and used as such .
preparative purification
was performed on a waters semipreparative hplc system using a phenomenex
luna c18 column ( 5 m , 30 75 mm ) at a flow rate of 45
ml / min . the mobile phase consisted of acetonitrile and water ( each
containing 0.1% trifluoroacetic acid ) .
analytical analysis
was performed on an agilent lc / ms ( agilent technologies , santa clara ,
ca ) .
method 1 : a 7 min gradient of 4100% acetonitrile ( containing
0.025% trifluoroacetic acid ) in water ( containing 0.05% trifluoroacetic
acid ) was used with an 8 min run time at a flow rate of 1 ml / min .
a phenomenex luna c18 column (
3 m , 3 75 mm ) was used
at a temperature of 50 c .
method 2 : a 3 min gradient of 4100%
acetonitrile ( containing 0.025% trifluoroacetic acid ) in water ( containing
0.05% trifluoroacetic acid ) was used with a 4.5 min run time at a
flow rate of 1 ml / min . a phenomenex gemini phenyl column ( 3 m ,
3 100 mm )
uhplc
long gradient equivalent 4100% acetonitrile ( 0.05% trifluoroacetic
acid ) in water was used over 3.5 min with a run time of 4 min and
a flow rate of 0.8 ml / min .
a phenomenex kinetex 1.7 m c18 column
( 2.1 100 mm ) was used at a temperature of 50 c .
purity
determination was performed using an agilent diode array detector
for methods 13 .
mass determination was performed using an
agilent 6130 mass spectrometer with electrospray ionization in the
positive mode .
chemical shifts are reported in ppm , with undeuterated
solvent ( dmso - d6 at 2.49 ppm ) as internal
standard for dmso - d6 solutions .
all of
the analogues tested in the biological assays have a purity greater
than 95% on the basis of both analytical methods .
high - resolution
mass spectrometry was recorded on agilent 6210 time - of - flight lc / ms
system .
confirmation of molecular formula was accomplished using electrospray
ionization in the positive mode with the agilent masshunter software
( version b.02 ) .
a mixture of carboxylic
acid ( 5.77 mmol , 1 equiv ) and 2-aminomalononitriletsoh ( 5.77
mmol , 1 equiv ) in ethyl acetate ( 15 ml ) was added net3 ( 17.32
mmol , 3 equiv ) followed by a 50% solution of t3p in ethyl
acetate ( 14.44 mmol , 2.5 equiv ) .
the reaction was allowed to stir
at room temperature for 12 h and was then diluted with ethyl acetate .
the organic layer was successively washed with water , saturated bicarbonate
solution , and brine .
the crude product
was purified on a biotage flash system , eluting with 50100%
ethyl acetate in hexanes containing 0.1% triethylamine to provide
pure products . a mixture of 5-amino-2-substituted
oxazole-4-carbonitrile ( 1.79 mmol , 1 equiv ) , paraformaldehyde ( 0.11
g , 3.57 mmol , 2 equiv ) , and sodium methoxide ( 0.096 g , 1.79 mmol ,
1 equiv ) in methanol ( 10 ml ) was stirred at 65 c for 1 h until
a clear mixture was obtained .
the reaction mixture was cooled to room
temperature , sodium borohydride ( 3.57 mmol , 2 equiv ) was added slowly ,
and the mixture was stirred further at room temperature for 1 h. the
crude product was extracted with ethyl acetate and successively washed
with water and brine .
the crude product
was taken up in dmso and purified on a reversed - phase hplc system .
characterization data for key compounds is given below ( see supporting information for details and characterization
data for all other compounds ) this compound was prepared
starting from 1-naphthoic acid and aminomalononitrile via intermediate 19 following the above general procedure .
lc - ms retention
time : ( method 1 ) 6.011 and ( method 2 ) 2.42 min .
h nmr
( 400 mhz , dmso - d6 ) 9.15 ( dq , j = 8.7 and 0.9 hz , 1h ) , 8.44 ( brs , 1h ) , 8.107.99
( m , 3h ) , 7.747.57 ( m , 3h ) , 3.073.01 ( m , 3h ) .
c nmr ( 400 mhz , dmso - d6 )
161.9 , 161.9 , 149.5 , 134.1 , 134.0 , 131.2 , 129.2 , 128.3 , 127.2 , 127.2 ,
127.0 , 126.9 , 125.8 , 125.8 , 125.8 , 125.7 , 122.1 , 116.5 , 116.5 , 84.1 ,
84.1 , 84.1 , 29.7 , 29.7 , 29.6 . hrms ( esi ) : m / z
this compound was prepared starting
from 2,3-benzoic acid and aminomalononitrile via intermediate 7i following the above general procedure .
lc - ms retention
time : ( method 3 ) 2.639 and ( method 2 ) = 3.559 min .
h nmr
( 400 mhz , dmso - d6 ) 8.50 ( q , j = 4.9 hz , 1h ) , 7.83 ( ddd , j = 7.9 , 1.5 ,
0.4 hz , 1h ) , 7.78 ( ddd , j = 8.1 , 1.6 , 0.4 hz , 1h ) ,
7.547.46 ( m , 1h ) , 2.99 ( d , j = 4.9 hz , 3h ) .
hrms ( esi ) : m / z ( m + h ) calcd for c11h8cl2n3o , 269.0039 ; found , 269.0031 .
this compound was prepared starting
from 3,4-benzoic acid and aminomalononitrile via intermediate 8i following the above general procedure .
h nmr ( 400 mhz , dmso - d6 ) 8.48 ( q , j = 4.9
hz , 1h ) , 7.95 ( td , j = 1.8 , 0.7 hz , 1h ) , 7.807.72
( m , 2h ) , 3.092.91 ( m , 3h ) .
hrms ( esi ) : m / z ( m + h ) calcd for c11h8cl2n3o , 269.0039 ; found , 269.0043 .
this compound was prepared starting
from 4-phenylbenzoic acid and aminomalononitrile via intermediate 11i following the above general procedure .
lc - ms retention
time : ( method 1 ) 6.215 and ( method 2 ) 3.708 min , h nmr
( 400 mhz , dmso - d6 ) 8.38 ( q , j = 4.9 hz , 1h ) , 7.927.79 ( m , 4h ) , 7.767.68
( m , 2h ) , 7.537.46 ( m , 2h ) , 7.437.37 ( m , 1h ) , 3.01
( dd , j = 4.8 , 0.6 hz , 3h ) .
hrms ( esi ) : m / z ( m + h ) calcd for c17h14n3o , 276.1131 ; found , 276.1143 . a mixture of 5-chloro-2-(naphthalen-1-yl)oxazole-4-carbonitrile
( 19a )
( details of the synthesis are provided in the supporting information ) ( 0.25 mmol , 1 equiv )
and 1-boc-3-(amino)azetidine ( 1 mmol , 4 equiv ) in thf was heated to
reflux for 0.5 h. the reaction mixture was then cooled , and the solvent
was removed by forced air .
the crude product was taken up in dmso
and purified via reversed - phase preparative hplc .
this pure product
was suspended in dichloromethane ( 2 ml ) and treated with tfa ( 0.5
ml ) . after stirring for 0.5 h at room temperature ,
the crude product was taken up in dmso and
purified on a preparative hplc to give pure product ( 31 ) as its tfa salt . lc - ms retention time : ( method 1 ) 4.078 and ( method
2 ) 3.812 min , h nmr ( 400 mhz , dmso - d6 ) 9.29 ( s , 1h ) , 9.12 ( d , j = 8.6
hz , 1h ) , 8.90 ( s , 1h ) , 8.148.01 ( m , 2h ) , 7.757.59
( m , 3h ) , 4.77 ( p , j = 7.0 , 6.0 hz , 1h ) , 4.324.18
( m , 2h ) , 4.184.08 ( m , 2h ) , hrms ( esi ) : m / z ( m + h ) calcd for c17h15n4o , 291.1252 ; found , 291.125 . a mixture of 2-bromothiazole-4-carbonitrile
( 1.85 g , 9.79 mmol , 1 equiv ) , naphthalen-1-ylboronic acid ( 2.52 g ,
14.68 mmol , 1.5 equiv ) , 2 m sodium carbonate ( 12.23 ml , 24.47 mmol ,
2.5 equiv ) , and pd(pph3)4 ( 1.11 g , 0.979 mmol ,
10 mol % ) in dimethoxy ethane ( 20 ml ) was degassed with argon and
then heated under microwave for 45 min at 150 c .
the reaction
mixture was concentrated , taken up in dichloromethane , stirred with
palladium scavenger resin , and filtered through celite .
the crude
product obtained after evaporating the solvent was purified on a biotage
flash system , eluting with 10% ethyl acetate in hexanes to obtain
1.52 g ( yield : 66% ) of 2-(naphthalen-1-yl)thiazole-4-carbonitrile
( 32a ) as a white solid .
a solution of the above
2-(naphthalen-1-yl)thiazole-4-carbonitrile 32a ( 0.5 g ,
2.12 mmol , 1 equiv ) in thf ( 10 ml ) was added a 2 m thf solution of
lda ( 1.16 ml , 2.33 mmol , 1.1 equiv ) dropwise at 78 c .
after stirring for 30 min at 78 c , perchloroethane
( 0.551
g , 2.33 mmol , 1.1 equiv ) was added in one portion and allowed to warm
to room temperature over 4 h. the reaction was then quenched with
saturated ammonium chloride and extracted with ethyl acetate .
the ethyl acetate layer
was subsequently dried over mgso4 and filtered . the crude
product obtained after concentrating under diminished pressure was
purified on a biotage flash system , eluting with 50% ch2cl2 in hexanes to furnish 4.5 g ( yield : 79% ) of 5-chloro-2-(naphthalen-1-yl)thiazole-4-carbonitrile 32b as a white solid .
h nmr ( 400 mhz , dmso - d6 ) 8.67 ( d , j = 8.4
hz , 1h ) , 8.20 ( d , j = 8.3 hz , 1h ) , 8.138.05
( m , 1h ) , 7.94 ( dd , j = 7.3 , 1.3 hz , 1h ) , 7.69 ( dddd , j = 14.7 , 9.3 , 6.9 , 1.7 hz , 3h ) .
a mixture of 5-chloro-2-(naphthalen-1-yl)thiazole-4-carbonitrile 32b ( 0.2 g , 0.739 mmol , 1 equiv ) and a 2 m thf solution of
methylamine ( 1.9 ml , 3.69 mmol , 5 equiv ) in 2-propanol ( 1 ml ) was
heated via microwave for 30 min at 120 c .
the crude product
obtained after evaporation of the solvent was taken up in dmso and
purified by reversed - phase preparative hplc to obtain 5-(methylamino)-2-(naphthalen-1-yl)thiazole-4-carbonitrile
( 32 ) as its tfa salt .
lc - ms retention time : ( method 3 )
2.814 and ( method 2 ) 3.756 min , h nmr ( 400 mhz , dmso - d6 ) 8.88 ( ddt , j = 8.5 ,
1.4 , 0.8 hz , 1h ) , 8.17 ( q , j = 4.7 hz , 1h ) , 8.077.98
( m , 2h ) , 7.77 ( dd , j = 7.2 , 1.2 hz , 1h ) , 7.717.53
( m , 3h ) , 3.01 ( d , j = 4.7 hz , 3h ) , hrms ( esi ) : m / z ( m + h ) calcd for c15h12n3s , 266.0754 ; found , 266.0746 . a solution of 5-(naphthalen-1-yl)oxazole-4-carbonitrile 34a ( 0.9 g , 4.09 mmol , 1 equiv ) in thf ( 15 ml ) was added a
1 m thf solution of lhmds ( 4.5 ml , 4.5 mmol , 1.1 equiv ) dropwise at
78 c .
the reaction mixture was stirred for 30 min at
78 c , perchloroethane ( 1.064 g , 4.5 mmol , 1.1 equiv )
was added in one portion , and the mixture was allowed to reach room
temperature over 4 h. the reaction mixture was quenched with saturated
ammonium chloride and extracted with ethyl acetate .
the organic layer
was washed with water and brine , dried over magnesium sulfate , filtered ,
and concentrated under diminished pressure .
the crude product was
purified on a biotage flash system , eluting with 10% ethyl acetate
in hexanes to furnish 0.75 g ( yield : 72% ) of 2-chloro-5-(naphthalen-1-yl)oxazole-4-carbonitrile 34b as a white solid .
a solution of 2-chloro-5-(naphthalen-1-yl)oxazole-4-carbonitrile 34b ( 0.1 g , 0.393 mmol , 1 equiv ) in thf ( 1 ml ) was added a
2 m solution of methanamine in thf ( 1.0 ml , 1.98 mmol , 5 equiv ) and
stirred at 65 c for 1 h in a sealed tube .
this crude product was taken
up in dmso and purified on a preparative hplc to obtain pure product
( 34 ) as its tfa salt .
lc - ms retention time : ( method 1 )
5.509 and ( method 2 ) 3.498 min , h nmr ( 400 mhz , dmso - d6 ) 8.177.96 ( m , 4h ) , 7.81 ( dd , j = 7.3 , 1.3 hz , 1h ) , 7.717.59 ( m , 3h ) , 2.88 ( d , j = 4.8 hz , 3h ) , hrms ( esi ) : m / z ( m + h ) calcd for c15h12n3o , 250.0975 ; found , 250.0979 .
different commercial fatty acids as lipoxygenase
substrates , were purchased from nu chek prep , inc .
the
fatty acids were further repurified using a higgins haisil ( 5 m ,
250 10 mm ) c-18 column .
an isocratic elution of 85% solvent
a ( 99.9% methanol and 0.1% acetic acid)/15% solvent b ( 99.9% water
and 0.1% acetic acid ) was used to purify all the fatty acids .
post
purification , the fatty acids were stored at 80 c for
a maximum of 6 months .
the product
generation protocol involved reacting 50 m substrate in 500
ml of 100 mm borate buffer , ph 9.2 , with soybean lox-1 .
a small sample
from the larger reaction was monitored on the uv vis spectrometer
until complete turnover was observed .
the products were then extracted
using dichloromethane , reduced with trimethylphosphite , evaporated
to dryness , and reconstituted in methanol .
the products were hplc - purified
using an isocratic elution of 75% a ( 99.9% methanol and 0.1% acetic
acid)/25% b ( 99.9% water and 0.1% acetic acid ) .
different
lipoxygenases , such as human reticulocyte 15-lipoxygenase-1 ( 12/15-lox ) ,
human epithelial 15-lipoxygenase-2 ( 15-lox-2 ) , and human platelet
12-lipoxygenase ( 12-lox ) , were expressed as n - terminal his6-tagged proteins and were purified via immobilized metal - affinity
chromatography using ni - nta resins for 12/15-lox and 15-lox-2 , whereas
ni - ida resin was used for 12-lox .
the protein
purity was evaluated by sds - page analysis and was found to be greater
than 90% .
human 5-lipoxygenase ( 5-lox ) was expressed as a nontagged
protein and used as a crude ammonium sulfate protein fraction , as
published previously .
the inhibitor compounds
were screened initially using one concentration point on a perkinelmer
lambda 40 uv vis spectrometer .
the percent inhibition was determined
by comparing the enzyme rates of the control ( dmso solvent ) and the
inhibitor sample by following the formation of the conjugated diene
product at 234 nm ( = 25 000 m cm ) .
the reactions were initiated by adding either 40
nm 12-lox , 40 nm 12/15-lox , 0.5 m 15-lox-2 , or 200
nm 5-lox ( ammonium sulfate suspension ) to a cuvette with a 2 ml of
reaction buffer that was constantly stirred using a magnetic stirrer
bar at room temperature ( 22 c ) . it should be noted that lox
isozymes are often expressed in the inactive demetalated form , so
it is best to utilize activity to determine the optimal lox concentration
for the assay ( optimal rate of approximately 0.001 abs / s at 10 m
aa ) .
reaction buffers used for various lipoxygenase were as follows :
25 mm hepes ( ph 7.3 ) , 0.3 mm cacl2 , 0.1 mm edta , 0.2 mm
atp , 0.01% triton x-100 , and 10 m aa for the crude , ammonium
sulfate - precipitated 5-lox ; 25 mm hepes ( ph 8) , 0.01% triton x-100 ,
and 10 m aa for 12-lox , and 25 mm hepes buffer ( ph 7.5 ) , 0.01%
triton x-100 , and 10 m aa for 12/15-lox and 15-lox-2 . the substrate
concentration was quantitatively determined by allowing the enzymatic
reaction to go to completion in the presence of 15-lox-2 . for the
inhibitors that showed more than 50% inhibition at the one - point screens
( 25 m inhibitor ) ,
ic50 values were obtained by determining
the enzymatic rate at various inhibitor concentrations and were plotted
against inhibitor concentration ( approximate range : 0.125
m inhibitor ) followed by a hyperbolic saturation curve fit
( assuming total enzyme concentration [ e ] ki , so ic50 ki ) .
it should be noted that all of the potent
inhibitors displayed greater than 80% maximal inhibition unless it
is otherwise stated in the tables .
the pseudoperoxidase
activity
rates were determined with bwb70c as the positive control , 13-(s)-hpode as the oxidizing product . and 12/15-lox on a perkinelmer
lambda 40 uv vis spectrometer , as described previously .
briefly , activity was determined by monitoring
the decrease at 234 nm ( product degradation ) in buffer ( 50 mm sodium
phosphate ( ph 7.4 ) , 0.3 mm cacl2 , 0.1 mm edta , 0.01% triton
x100 , and 20 m 13-(s)-hpode ) .
12/15-lox was
added to buffer ( 22 c ) , and the reaction initiated by addition
of 20 m inhibitor ( 1:1 ratio to product ) .
the percent consumption
of 13-(s)-hpode was recorded , with individual controls
being conducted with inhibitor alone with product and enzyme alone
with product .
the steady - state kinetics
experiments were performed with the parent analogue , compound 1 ( ml351 ) , to determine the mode of inhibition ,
as described previously .
the inhibitor concentrations
of 0 , 0.05 , 2 , and 5 m were used .
reactions were initiated
by adding approximately 4060 nm 12/15-lox to a constantly
stirring 2 ml cuvette containing 120 m aa in 25 mm
hepes buffer ( ph 7.5 ) in the presence of 0.01% triton x-100 .
lipoxygenase
rates were determined by monitoring the formation of the conjugated
product , 15-hpete , at 234 nm ( = 25 000 m cm ) with a perkinelmer lambda 40 uv vis
spectrometer . the substrate concentration was quantitatively determined
by allowing the enzymatic reaction to proceed to completion using
15-lox-2 .
kinetic data were obtained by recording initial enzymatic
rates at varied substrate and inhibitor concentrations , and the data
were subsequently fitted to the henri michaelis
menten
equation using kaleidagraph ( synergy ) to determine the microscopic
rate constants , vmax ( mol / min / mg )
and vmax / km ( mol / min / mg/m ) .
these rate constants were subsequently
replotted with 1/vmax and km / vmax versus inhibitor concentration ,
yielding ki and ki , respectively .
about 25
g of either
cox-1 or cox-2 were added to buffer containing 0.1 m tris - hcl buffer
( ph 8.0 ) , 5 mm edta , 2 mm phenol , and 1 m hematin at 37 c .
the inhibitors were added to the reaction cell followed by an incubation
of 5 min with either of the cox enzymes .
the reaction was then initiated
by adding 100 m aa in the reaction cell .
data was collected
using a hansatech dw1 oxygen electrode , and the consumption of oxygen
was recorded .
indomethacin and the solvent , dmso , were used as positive
and negative controls , respectively , and the percent inhibition of
the enzyme was calculated by comparing the rates from samples and
the controls .
glutathione
depletion was induced
in ht22 cells by glutamate treatment , and ldh release into the medium
was measured to detect cell death , as described previously .
briefly , ht22 cells were cultured in dmem containing
10% fetal bovine serum and penicillin / streptomycin ( all media from
invitrogen ) . for viability experiments ,
cells were seeded at 1
10 cells / well in 96-well plates ( corning ) and were treated
18 h later when the cells were approximately 5070% confluent .
treatment consisted of exchanging the medium to 100 l of fresh
culturing medium and adding 5 mm glutamate ( stock solution 0.5 m in
pbs ) in the presence or absence of dmso ( maximum 0.1% final concentration )
as control or the indicated concentrations of 1 .
ldh
content was determined separately for the cell extracts and corresponding
media using a cytotoxicity detection kit ( roche ) , and the percentage
of ldh released to the medium was calculated after subtracting the
corresponding background value . to determine levels of the 12/15-lox
metabolite , 12-hete , we cultured ht22 cells in 75 cm flasks
in dmem medium without phenol red that was supplemented with 5% fbs ,
and the cells were treated the next day when they were 5070%
confluent .
twenty - four hours later , the eicosanoid - containing fraction
was isolated via sep - pak c-18 column , and 12-hete was detected with
a 12-hete elisa kit ( enzo life sciences ) , which was used according
to the manufacturer s instructions .
the eicosanoids containing fractions were transferred to
scintillation vials followed by addition of perdeuterated 13-hode
( 13-d31-hode ) as an internal control for
extraction and 1.5% glacial acetic acid for protein precipitation .
the samples were extracted with methylene chloride , reduced with trimethylphosphite ,
and evaporated to dryness .
the dry samples were then reconstituted
in methanol , and an internal control 12-deuterated ( d8)-hete ( 12-d8-hete ) was added
to each sample for detector response variation ; it was assumed that
the change in detector response for 12-hete and 12-d8-hete would be similar .
the samples were run on and analyzed
by a finnigan ltq liquid chromatography tandem mass spectrometry
( lc ms / ms ) system . a thermo electron corp .
aquasil ( 3 m ,
100 mm 2.1 mm ) c-18 column was used to detect the hetes with
an elution protocol consisting of a 0.2 ml / min flow rate and a linear
gradient from 54.9% acn , 45% h2o , and 0.1% thf to 69.9%
acn , 30% h2o , and 0.1% thf .
the electrospray voltage was
set to 5.0 kv and a global acquisition ms mode was used . the ms ms
scan was performed for the five most abundant precursor ions .
the
collision - induced dissociation was used for ms ms with a collision
energy of 35 ev .
the corresponding 12-hete , 12-d8-hete , and 13-d31-hode compounds
were detected using selective ion monitoring analysis ( m / z = 318.7319.7 , 326.8327.7 , and
325.8326.8 , respectively ) in negative ion mode and then identified
by fragmentation pattern ( 12-hete , parent ion at m / z 319 and fragments at m / z 179 and 163 ; 12-d8-hete , parent
ion at m / z 327 and fragments at m / z 184 ; and 13-d31-hode , parent ion m / z 326
and fragments at m / z 213 ) from ms ms .
the peak area of 12-d8-hete for each sample
was normalized to the area of 13-d31-hode .
the amount of
12-hete in samples was estimated from the pure 12-(s)-hete standard curve . to study 1 in a model of distal mcao ,
c57bl6j mice were treated with ferric chloride
( fecl3 ) to cause occlusion of the distal middle cerebral
artery .
mice were kept under anesthesia with 1.5% isoflurane in a nitrous
oxide / oxygen mixture via facemask .
the body temperature was monitored
by a rectal probe and maintained at 37 0.3 c by a homeothermic
blanket control unit .
briefly , mice were placed in a stereotaxic frame ,
the scalp was opened , and the right temporal muscle was dissected .
the area between zygomatic arch and squamous bone was thinned by a
high - speed drill and cooled with saline .
the trace of mca was visualized ,
and the thin bony film was lifted up by forceps .
after that , a laser
doppler flowmetry probe was placed 2 mm posterior and 6 mm lateral
to the bregma to monitor the regional cerebral blood flow ( rcbf ) .
after obtaining a stable epoch of the preischemic rcbf ,
a piece of
10% fecl3-saturated filter paper was placed over the intact
dura mater along the trace of mca , and the rcbf was continuously monitored
during the next 3 h. after 2 h of ischemia , either 50 mg / kg of 1 or dmso vehicle was injected intraperitoneally . following
sacrifice at 24 h
, brains were sectioned into 1 mm slices , and infarct
sizes were determined by staining with ttc using the indirect method
( infarct volume = contralateral volume minus uninfarcted ipsilateral
volume ) .
both the surgeon carrying out the procedure and the researcher
determining infarct volumes were blinded as to which treatment the
mice received .
different commercial fatty acids as lipoxygenase
substrates , were purchased from nu chek prep , inc .
the
fatty acids were further repurified using a higgins haisil ( 5 m ,
250 10 mm ) c-18 column .
an isocratic elution of 85% solvent
a ( 99.9% methanol and 0.1% acetic acid)/15% solvent b ( 99.9% water
and 0.1% acetic acid ) was used to purify all the fatty acids .
post
purification , the fatty acids were stored at 80 c for
a maximum of 6 months .
the product
generation protocol involved reacting 50 m substrate in 500
ml of 100 mm borate buffer , ph 9.2 , with soybean lox-1 .
a small sample
from the larger reaction was monitored on the uv vis spectrometer
until complete turnover was observed .
the products were then extracted
using dichloromethane , reduced with trimethylphosphite , evaporated
to dryness , and reconstituted in methanol .
the products were hplc - purified
using an isocratic elution of 75% a ( 99.9% methanol and 0.1% acetic
acid)/25% b ( 99.9% water and 0.1% acetic acid ) .
different
lipoxygenases , such as human reticulocyte 15-lipoxygenase-1 ( 12/15-lox ) ,
human epithelial 15-lipoxygenase-2 ( 15-lox-2 ) , and human platelet
12-lipoxygenase ( 12-lox ) , were expressed as n - terminal his6-tagged proteins and were purified via immobilized metal - affinity
chromatography using ni - nta resins for 12/15-lox and 15-lox-2 , whereas
ni - ida resin was used for 12-lox .
the protein
purity was evaluated by sds - page analysis and was found to be greater
than 90% .
human 5-lipoxygenase ( 5-lox ) was expressed as a nontagged
protein and used as a crude ammonium sulfate protein fraction , as
published previously .
the inhibitor compounds
were screened initially using one concentration point on a perkinelmer
lambda 40 uv vis spectrometer .
the percent inhibition was determined
by comparing the enzyme rates of the control ( dmso solvent ) and the
inhibitor sample by following the formation of the conjugated diene
product at 234 nm ( = 25 000 m cm ) .
the reactions were initiated by adding either 40
nm 12-lox , 40 nm 12/15-lox , 0.5 m 15-lox-2 , or 200
nm 5-lox ( ammonium sulfate suspension ) to a cuvette with a 2 ml of
reaction buffer that was constantly stirred using a magnetic stirrer
bar at room temperature ( 22 c ) . it should be noted that lox
isozymes are often expressed in the inactive demetalated form , so
it is best to utilize activity to determine the optimal lox concentration
for the assay ( optimal rate of approximately 0.001 abs / s at 10 m
aa ) .
reaction buffers used for various lipoxygenase were as follows :
25 mm hepes ( ph 7.3 ) , 0.3 mm cacl2 , 0.1 mm edta , 0.2 mm
atp , 0.01% triton x-100 , and 10 m aa for the crude , ammonium
sulfate - precipitated 5-lox ; 25 mm hepes ( ph 8) , 0.01% triton x-100 ,
and 10 m aa for 12-lox , and 25 mm hepes buffer ( ph 7.5 ) , 0.01%
triton x-100 , and 10 m aa for 12/15-lox and 15-lox-2 . the substrate
concentration was quantitatively determined by allowing the enzymatic
reaction to go to completion in the presence of 15-lox-2 . for the
inhibitors that showed more than 50% inhibition at the one - point screens
( 25 m inhibitor ) ,
ic50 values were obtained by determining
the enzymatic rate at various inhibitor concentrations and were plotted
against inhibitor concentration ( approximate range : 0.125
m inhibitor ) followed by a hyperbolic saturation curve fit
( assuming total enzyme concentration [ e ] ki , so ic50 ki ) .
it should be noted that all of the potent
inhibitors displayed greater than 80% maximal inhibition unless it
is otherwise stated in the tables .
the pseudoperoxidase
activity
rates were determined with bwb70c as the positive control , 13-(s)-hpode as the oxidizing product .
and 12/15-lox on a perkinelmer
lambda 40 uv vis spectrometer , as described previously .
briefly , activity was determined by monitoring
the decrease at 234 nm ( product degradation ) in buffer ( 50 mm sodium
phosphate ( ph 7.4 ) , 0.3 mm cacl2 , 0.1 mm edta , 0.01% triton
x100 , and 20 m 13-(s)-hpode ) .
12/15-lox was
added to buffer ( 22 c ) , and the reaction initiated by addition
of 20 m inhibitor ( 1:1 ratio to product ) .
the percent consumption
of 13-(s)-hpode was recorded , with individual controls
being conducted with inhibitor alone with product and enzyme alone
with product .
the steady - state kinetics
experiments were performed with the parent analogue , compound 1 ( ml351 ) , to determine the mode of inhibition ,
as described previously .
the inhibitor concentrations
of 0 , 0.05 , 2 , and 5 m were used .
reactions were initiated
by adding approximately 4060 nm 12/15-lox to a constantly
stirring 2 ml cuvette containing 120 m aa in 25 mm
hepes buffer ( ph 7.5 ) in the presence of 0.01% triton x-100 .
lipoxygenase
rates were determined by monitoring the formation of the conjugated
product , 15-hpete , at 234 nm ( = 25 000 m cm ) with a perkinelmer lambda 40 uv vis
spectrometer . the substrate concentration was quantitatively determined
by allowing the enzymatic reaction to proceed to completion using
15-lox-2 .
kinetic data were obtained by recording initial enzymatic
rates at varied substrate and inhibitor concentrations , and the data
were subsequently fitted to the henri michaelis
menten
equation using kaleidagraph ( synergy ) to determine the microscopic
rate constants , vmax ( mol / min / mg )
and vmax / km ( mol / min / mg/m ) .
these rate constants were subsequently
replotted with 1/vmax and km / vmax versus inhibitor concentration ,
yielding ki and ki , respectively .
about 25
g of either
cox-1 or cox-2 were added to buffer containing 0.1 m tris - hcl buffer
( ph 8.0 ) , 5 mm edta , 2 mm phenol , and 1 m hematin at 37 c .
the inhibitors were added to the reaction cell followed by an incubation
of 5 min with either of the cox enzymes .
the reaction was then initiated
by adding 100 m aa in the reaction cell .
data was collected
using a hansatech dw1 oxygen electrode , and the consumption of oxygen
was recorded .
indomethacin and the solvent , dmso , were used as positive
and negative controls , respectively , and the percent inhibition of
the enzyme was calculated by comparing the rates from samples and
the controls .
glutathione
depletion was induced
in ht22 cells by glutamate treatment , and ldh release into the medium
was measured to detect cell death , as described previously .
briefly , ht22 cells were cultured in dmem containing
10% fetal bovine serum and penicillin / streptomycin ( all media from
invitrogen ) . for viability experiments ,
cells were seeded at 1
10 cells / well in 96-well plates ( corning ) and were treated
18 h later when the cells were approximately 5070% confluent .
treatment consisted of exchanging the medium to 100 l of fresh
culturing medium and adding 5 mm glutamate ( stock solution 0.5 m in
pbs ) in the presence or absence of dmso ( maximum 0.1% final concentration )
as control or the indicated concentrations of 1 .
ldh
content was determined separately for the cell extracts and corresponding
media using a cytotoxicity detection kit ( roche ) , and the percentage
of ldh released to the medium was calculated after subtracting the
corresponding background value . to determine levels of the 12/15-lox
metabolite , 12-hete ,
we cultured ht22 cells in 75 cm flasks
in dmem medium without phenol red that was supplemented with 5% fbs ,
and the cells were treated the next day when they were 5070%
confluent .
twenty - four hours later , the eicosanoid - containing fraction
was isolated via sep - pak c-18 column , and 12-hete was detected with
a 12-hete elisa kit ( enzo life sciences ) , which was used according
to the manufacturer s instructions .
the eicosanoids containing fractions were transferred to
scintillation vials followed by addition of perdeuterated 13-hode
( 13-d31-hode ) as an internal control for
extraction and 1.5% glacial acetic acid for protein precipitation .
the samples were extracted with methylene chloride , reduced with trimethylphosphite ,
and evaporated to dryness .
the dry samples were then reconstituted
in methanol , and an internal control 12-deuterated ( d8)-hete ( 12-d8-hete ) was added
to each sample for detector response variation ; it was assumed that
the change in detector response for 12-hete and 12-d8-hete would be similar .
the samples were run on and analyzed
by a finnigan ltq liquid chromatography tandem mass spectrometry
( lc ms / ms ) system . a thermo electron corp .
aquasil ( 3 m ,
100 mm 2.1 mm ) c-18 column was used to detect the hetes with
an elution protocol consisting of a 0.2 ml / min flow rate and a linear
gradient from 54.9% acn , 45% h2o , and 0.1% thf to 69.9%
acn , 30% h2o , and 0.1% thf .
the electrospray voltage was
set to 5.0 kv and a global acquisition ms mode was used . the ms ms
scan was performed for the five most abundant precursor ions .
the
collision - induced dissociation was used for ms ms with a collision
energy of 35 ev .
the corresponding 12-hete , 12-d8-hete , and 13-d31-hode compounds
were detected using selective ion monitoring analysis ( m / z = 318.7319.7 , 326.8327.7 , and
325.8326.8 , respectively ) in negative ion mode and then identified
by fragmentation pattern ( 12-hete , parent ion at m / z 319 and fragments at m / z 179 and 163 ; 12-d8-hete , parent
ion at m / z 327 and fragments at m / z 184 ; and 13-d31-hode , parent ion m / z 326
and fragments at m / z 213 ) from ms ms .
the peak area of 12-d8-hete for each sample
was normalized to the area of 13-d31-hode .
the amount of
12-hete in samples was estimated from the pure 12-(s)-hete standard curve . to study 1 in a model of distal mcao ,
c57bl6j mice were treated with ferric chloride
( fecl3 ) to cause occlusion of the distal middle cerebral
artery .
mice were kept under anesthesia with 1.5% isoflurane in a nitrous
oxide / oxygen mixture via facemask .
the body temperature was monitored
by a rectal probe and maintained at 37 0.3 c by a homeothermic
blanket control unit .
briefly , mice were placed in a stereotaxic frame ,
the scalp was opened , and the right temporal muscle was dissected .
the area between zygomatic arch and squamous bone was thinned by a
high - speed drill and cooled with saline .
the trace of mca was visualized ,
and the thin bony film was lifted up by forceps .
after that , a laser
doppler flowmetry probe was placed 2 mm posterior and 6 mm lateral
to the bregma to monitor the regional cerebral blood flow ( rcbf ) .
after obtaining a stable epoch of the preischemic rcbf , a piece of
10% fecl3-saturated filter paper was placed over the intact
dura mater along the trace of mca , and the rcbf was continuously monitored
during the next 3 h. after 2 h of ischemia , either 50 mg / kg of 1 or dmso vehicle was injected intraperitoneally . following
sacrifice at 24 h , brains were sectioned into 1 mm slices , and infarct
sizes were determined by staining with ttc using the indirect method
( infarct volume = contralateral volume minus uninfarcted ipsilateral
volume ) .
both the surgeon carrying out the procedure and the researcher
determining infarct volumes were blinded as to which treatment the
mice received . | a key challenge facing drug discovery
today is variability of the
drug target between species , such as with 12/15-lipoxygenase ( 12/15-lox ) ,
which contributes to ischemic brain injury , but its human and rodent
isozymes have different inhibitor specificities . in the current work ,
we have utilized a quantitative high - throughput ( qhts ) screen to identify
compound 1 ( ml351 ) , a novel chemotype for
12/15-lox inhibition that has nanomolar potency ( ic50 =
200 nm ) against human 12/15-lox and is protective against oxidative
glutamate toxicity in mouse neuronal ht22 cells .
in addition , it exhibited
greater than 250-fold selectivity versus related lox isozymes , was
a mixed inhibitor , and did not reduce the active - site ferric ion .
lastly , 1 significantly reduced infarct size following
permanent focal ischemia in a mouse model of ischemic stroke . as such
,
this represents the first report of a selective inhibitor of human
12/15-lox with demonstrated in vivo activity in proof - of - concept mouse
models of stroke . |
subdural hematoma is a neurosurgical condition resulting from the collection of blood or remnants of blood elements in the subdural space following bleeding .
it can be acute , subacute , or chronic depending on the duration since bleeding .
chronic subdural hematoma is characterized by the persistence of liquefied hematoma in the subdural space for more than 3 weeks by encapsulation .
craniocerebral disproportion is a potential risk factor for subdural hematoma formation . in the elderly , due to cerebral
atrophy the disproportion between the cranial cavity and cerebral cortex is more that gives enough space for expansion of the hematoma .
currently , it is believed that recurrent bleeding in the subdural space due to series of events - local angiogenesis , inflammation , continuing coagulation , and fibrinolysis - results in persistence of the hematoma .
chronic subdural hematoma commonly present with features like alteration of the sensorium raised intracranial tension and motor weakness .
common psychiatric manifestation in patients with chronic subdural hematoma is cognitive impairment which may mimic delirium or dementia .
trauma is an important cause of subdural hematoma and in elderly population even a trivial trauma is enough in causing subdural hematoma .
spontaneous subdural hematoma usually occurs in the presence of risk factors such as vasculopathy , coagulopathy , and concurrent anticoagulation treatment .
chronic alcoholism and postoperative pneumocephalus are poor prognostic factors in chronic subdural hematoma . computed tomography of the brain is diagnostic of chronic subdural hematoma ; however , magnetic resonance imaging of the brain gives more structural details of hematoma .
an adult , homeless male of approximately 40 years age was brought to psychiatric emergency by a voluntary organization .
the patient was also not able to give any information related to his current and past psychiatric condition .
he was also unable provide any history of seizure disorder , trauma , or any other physical illness . at the time of first consultation to the psychiatrist at the emergency
he was conscious but his orientation could not be checked as he was not speaking at all .
in inpatient evaluation , it was found that he used to remain withdrawn to self .
he had little interest in the interaction with others , play activities , television , music , and things happening around .
though , he was attending the call of nature but most of the time he used to do it in clothes .
respiratory system , cardiovascular system , and preabdominal examination revealed no abnormality . in neurological examination
due to speech impairment and uncooperativeness , his higher mental functions and some of the cranial nerve function , sensory function , motor function , as well as cerebellar functions could not be tested .
on the basis of clinical evaluation and ward behavior observation a diagnosis of psychosis not otherwise specified is kept with differential diagnosis of undifferentiated schizophrenia .
risperidone and the dose was escalated to 6 mg per day over a period of 4 days along with anticholinergic tab .
trihexyphenidyl 2 mg per day , but as the patient had developed extrapyramidal side effects in the form of tremor , rigidity , and excessive salivation .
the dose of trihexyphenidyl was escalated up to 6 mg per day without much benefit in the extrapyramidal side effects .
this warranted withdrawal of antipsychotics and review of diagnosis . to rule out any possible organicity , neuroimaging ( initially ct scan of head and subsequently magnetic resonance imaging of brain )
the figures 1 and 2 shows the neuroimaging findings on preoperative mri and postoperative ct scan of the brain , respectively .
after evacuation of the hematoma in the first setting , the patient had improvement in his speech and emotional responsiveness .
, there was improvement in involvement in group activity , initiative as well as self - care .
he was involved in play activities like indoor games ( carom ) , which he was enjoying a lot .
magnetic resonance imaging of brain ( pre - operative ) showing bilateral subdural hematoma and diffuse cerebral atrophy ct scan of head ( post - operative ) showing the burr - hole , postoperative collection of exudate in the subdural space , pneumocephalus changes , subdural hematoma on the opposite side ( nonoperated side - left side ) , and diffuse cerebral atrophy
in this case , there was delay in detection of subdural hematoma . as the patient was not having any neurological deficit and his biological functions are little affected ,
the possibility of organicity was not considered initially . however , as the patient had developed neurological side effects to antipsychotics like risperidoneand olanzapine at lower doses and did not responded to the treatment , a possibility of organicity ( central nervous system pathology ) was suspected .
after neuroimaging ( noncontrast ct scan of head ) , bilateral subdural hematoma was detected .
there was large bilateral subdural hematoma without any midline shift or compression of brain parenchyma .
the architecture of sulci and gyri were intact despite of two large space occupying subdural hematomas on both sides .
it suggests that there was some pre - existing potential space ( resulting from diffuse cerebral atrophy ) due to which , the hematoma had accommodated without any pressure effect .
as craniocerebral disproportion is a major risk factor for chronic subdural hematoma and in our patient there seems to be significant cranial
cerebral disproportion due to diffuse cerebral atrophy which might have been attributed to the subdural hematoma .
patient 's withdrawn behavior , absence of speech , apathy , blunted affect , and poor self - care was initially thought to be the manifestation of psychosis ( possibly psychosis not otherwise specified or undifferentiated schizophrenia ) and treatment had been started in that line , till the subdural hematomas are detected .
patient 's clinical presentation could be due to subdural hematoma or existing before subdural hematoma and might got distorted due to subdural hematoma .
the patient had diffuse cerebral atrophy at the age of 40 years which is an unusual phenomenon in an otherwise normal individual .
so there is every possibility , that the patient might have psychiatric illness ( long standing psychosis ) or some nutritional or neurological illness before the development of hematoma which might have attributed to diffuse cerebral atrophy .
the patient had shown clinical improvement , immediately following the evacuation of the subdural hematoma by burr - hole surgery .
chronic subdural hematoma may result in dementia like presentation and often needs evacuation by neurosurgical intervention which may reverse the dementia process .
the subdural hematoma may have exclusively psychiatric manifestation and the treatment nonresponders or those who develop neurological adverse drug reaction in a low dose of psychotropic medications , needs to be evaluated rigorously .
these groups of patients should undergo neuroimaging for ruling out possible brain pathology . in homeless patients , where no clinical history is available and
mental status examination is not possible , reaching at a diagnosis is a big challenge . due to lack of clinical information , misdiagnosis and wrong treatment
may occur . but frequent review of diagnosis , utilization of clinical clues ( adverse effects , nonresponse ) and appropriate investigation ( psychometric assessment or neuroimaging ) may guide in reaching a diagnosis . | the clinical manifestation of chronic subdural hematoma is not limited to neurological deficits or cognitive impairment .
it may present with behavioral abnormalities .
when the behavioral abnormalities present without obvious neurological deficits and in the absence of trauma , it leads to misdiagnosis . a trivial trauma may cause intracranial bleed that is either missed or ignored in the clinical history .
this case report highlights the clinical picture of a homeless patient who presented with withdrawn and disorganized behavior , apathy and poverty of speech in the absence of any neurological deficit .
his clinical presentation led to a possibility of psychosis and he was started on antipsychotics .
he had developed extrapyramidal side effects in low dose of antipsychotic without any clinical benefit in his clinical picture .
neuroimaging done to rule out any possible organicity - revealed bilateral subdural hematoma , which was later evacuated by neurosurgical intervention in multiple settings and the patient had improved .
this case report unfolds the mystery behind the psychotic presentation in a homeless adult . |
abiotrophia species have been referred to as nutritionally variant streptococci because of their fastidious nutritional requirements for growth .
abiotrophia species sometimes cause sepsis and bacteremia , and are especially known as one of the major pathogens of infective endocarditis .
a 48-year - old unemployed woman was admitted to our hospital with a two - week history of severe low back pain .
she suffered from parkinson 's disease , but she was not a compromised host . on admission , she was unable to sit up because of the pain .
her vital signs were as follows : temperature 38.2 c , pulse 80 beats / min , and blood pressure 100/68 mmhg .
physical examination revealed moderate tenderness to palpation on the spinous processes of lower lumbar spine .
the peripheral white blood cell count was 11,600/mm , and her c - reactive protein level was 9.9 mg / dl .
plain x - ray showed degenerative changes of the lumbar vertebrae with narrowing of the disk spaces at l3 - 4 , and l4 - 5 .
magnetic resonance imaging revealed edema and contrast enhancement of the l4 and l5 vertebral bodies with high signal intensity in the l3 - 4 and l4 - 5 intervertebral discs on the t2-weighted images ( fig .
cultures of disk biopsy samples and blood yielded gram positive cocci in short chains with scanty growth on chocolate agar . further subculture with supplemented medium and subsequent 16s ribosomal rna gene sequencing identified the pathogen as abiotrhophia adiacens . as a result of the survey regarding bacteremia origin , we could detect the dental caries .
the patient was treated with intravenous ampicillin ( 8 g / day ) for 6 weeks .
the clinical and laboratory test abnormalities improved within 2 weeks . at 6-month follow - up ,
spinal infections can be devastating and result in significant pain , deformity , and neurologic deterioration .
staphylococcus aureus is the most common etiologic organism of spinal infections , followed by escherichia coli , streptococcus species , s. epidermidis , and pseudomonas species ; however , causative microorganism remain unidentified in 2540% of the cases .
abiotrophia species ( a. adiacens and a. defectiva ) have formerly been referred to as nutritionally variant streptococci because of their fastidious nutritional growth requirements .
the organisms grow poorly on conventional solid media , and require pyridoxal or thiol group supplementation .
use of brucella hk agar or gam agar plate is helpful for detection of abiotrophia species .
prolonged incubation ( at least 72 h ) is also recommended . spinal infection due to abiotrophia species is extremely rare , and only four cases have been reported to date ( table 1 ) . in the previous reports ,
this organism was identified only from blood sample by using columbia blood and chocolate agar plates . in the present case , we first confirmed the diagnosis by direct identification of abiotrophia adiacens from infected disk .
abiotrophia species are one of the major pathogens of infective endocarditis accounting for 5% of cases .
abiotrophia endocarditis carries higher morbidity and mortality than endocarditis caused by other streptococci , reflecting their strong virulence .
abiotrophia discitis reported previously occurred concomitantly with infective endocarditis although repeated echocardiography showed normal study in our case .
patients with history of infective endocarditis or valve disease may be candidate for exploration of abiotrophia discitis .
we were able to detect her dental cavity , and it could be a predisposing factor of bacteremia .
considering their fastidious nature , it is likely that most cases of abiotrophia discitis are falsely classified as culture - negative discitis ; therefore , their role in pyogenic discitis may be underestimated .
adequate diagnosis and treatment of this entity requires a high index of suspicion for the organism , even if cultures are negative .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
k.u . assisted in the surgery , was involved in the preoperative work up and postoperative care of the patient , and wrote the paper .
performed the surgery , was involved in the preoperative work up and postoperative care of the patient , and helped in the preparation of the write up . k.t .
were involved in the preoperative work up and postoperative care of the patient , and revised the final article . y.h . and k.o . | introductionabiotrophia species have been referred to as nutritionally variant streptococci because of their fastidious nutritional requirements for growth .
abiotrophia species are difficult to identify with conventional solid culture.presentation of casea 48-year - old woman was admitted to our hospital with severe low back pain and body temperature of 38.2 c .
magnetic resonance imaging revealed edema and contrast enhancement of the l4 and l5 vertebral bodies with high signal intensity in the l3 - 4 and l4 - 5 intervertebral discs on the t2-weighted images .
the patient underwent needle biopsy of the l3 - 4 disk .
cultures of disk biopsy samples and blood yielded gram positive cocci in short chains with scanty growth on chocolate agar .
further subculture with supplemented medium and subsequent 16s ribosomal rna gene sequencing identified the pathogen as abiotrhophia adiacens .
the patient was treated with intravenous ampicillin . at 6-month follow - up ,
the patient was free of symptoms.discussioncausative microorganisms remain unidentified in 2540% of spinal infection cases .
abiotrophia species grow poorly on conventional solid media , and require pyridoxal or thiol group supplementation .
use of brucella hk agar or gam agar plate is helpful for detection of abiotrophia species .
we first confirmed the diagnosis by direct identification of abiotrophia adiacens from infected disk .
abiotrophia species are one of the major pathogens of infective endocarditis accounting for 5% of cases . considering their fastidious nature ,
it is likely that most cases of abiotrophia discitis are falsely classified as culture - negative discitis ; therefore , their role in pyogenic discitis may be underestimated.conclusionsubculture using nutritionally supplemented media is crucial for their identification . |
initially described after treatment with flutamide , responses to withdrawal of hormonal therapies in prostate cancer patients have been documented after cessation of other antiandrogens such as nilutamide , bicalutamide , or cyproterone acetate and of megestrol acetate , diethylstilbestrol or estramustine . in the southwest oncology group trial 9426 ,
21% of 210 patients had confirmed psa decreases of 50% after treatment with nonsteroidal antiandrogens with a median progression - free survival of 3 months .
it has been proposed that withdrawal responses could result from mutations in the androgen receptor ( ar ) .
transfection experiments revealed that ar point mutations in the hormone - binding domain allow activation by ligands other than dihydrotestosterone .
mutated ar from clinical samples was shown to be activated by progesterone , estradiol , adrenal androgens , hydrocortisone or hydroxyflutamide . however , a prospective study failed to demonstrate a clear association between detectability of ar mutations and antiandrogen withdrawal responses .
an improvement in overall survival has been reported with abiraterone acetate ( aa ) , a selective inhibitor of cytochrome p450c17 ( cyp17 ) , in castrate - resistant prostate cancer patients who have failed docetaxel chemotherapy .
the addition of low - dose corticoids to aa is mandatory in daily practice since the combination has been shown to minimize the syndrome of secondary mineralocorticoid excess related to cyp17 inhibition . here ,
we report the case of a patient who had a confirmed psa decrease of 50% after stopping aa , mimicking a withdrawal syndrome .
a 60-year - old patient was diagnosed with a prostate adenocarcinoma ( gleason score 8 , 4 + 4 ) with synchronous bone metastases in april 2007 .
he successively received an lhrh agonist , a combined androgen blockade with lhrh agonist and bicalutamide , without subsequent response to bicalutamide withdrawal , docetaxel ( 10 cycles ) , then mitoxantrone ( 3 cycles ) and diethylstilbestrol . in april 2011 ,
aa was started at the standard dose of 1,000 mg daily in combination with prednisone ( 5 mg twice daily ) .
psa slightly decreased to a nadir of 68 ng / ml one month after the start of treatment . in parallel , a metabolic partial response according to consensus criteria on 18-fluorodeoxyglucose positron emission tomography ( pet ) and choline pet ( suvmax = 37% ) was observed .
however , aa and prednisone were stopped in august 2011 because of back pain related to vertebral metastases and concomitant psa progression ( psa 128 ng / ml ) .
one month later , a confirmed psa decrease to a nadir of 62 ng / ml occurred , defining a partial biological response according to standard criteria , with a concomitant improvement in back pain .
to the best of our knowledge , this is the first case ever reported of a withdrawal response with aa .
currently proposed mechanisms of resistance to aa include ligandless activation of ar by constitutively active variants lacking the ligand - binding domain , cross - talk with relative signaling pathways and/or activation of amplified or promiscuous ar by nonandrogenic ligands such as corticosterone or other steroids .
following the latter hypothesis , the withdrawal response observed in the present case could be related rather to prednisone than to aa itself as it was stopped at the same time .
| in 2011 abiraterone acetate ( aa ) was approved for the treatment of castrate - resistant metastatic prostate cancer patients who have failed docetaxel chemotherapy .
we report the case of a patient who experienced a confirmed psa decrease of 50% after stopping aa , mimicking an antiandrogen withdrawal syndrome . |
its chronic nature and the limitations it imposes contribute significantly to the increase in hospitalization , disability , and mortality rates .
the occurrence of diabetes in population groups is linked mainly to socioeconomic factors , cultural factors , stress , and family predisposition . in the year 2000 , the prevalence of diabetes in the world was 171 million , and it is estimated that this number will reach 366 million by 2030 . in brazil , there are approximately 10 million people with diabetes .
studies suggest that diabetes is a risk factor for periodontal disease , pointing out that the prevalence , incidence , and severity of periodontal disease are higher among individuals with diabetes in comparison to healthy individuals [ 10 , 11 ] . the mechanisms by which diabetes influences periodontal disease include vascular abnormalities , neutrophil dysfunction , abnormalities in collagen synthesis , and genetic predisposition .
the most commonly employed clinical parameters for the diagnosis and classification of periodontal disease are measurements of the depth of periodontal pockets , clinical insertion loss , and bleeding upon probing , which often generate different information regarding the prevalence of the disease .
there is no consensus in the scientific literature regarding a gold standard clinical diagnostic criterion for periodontal disease , which limits comparison among different studies .
the issue of accuracy of periodontal protocols of the national health and nutrition examination survey ( nhanes ) was discussed .
the authors verified that the periodontal disease case used could lead to an underestimate of the prevalence of periodontal disease by 50% or more .
the change in the medical paradigm from focus on the presence or absence of disease to a broader view that considers the subjective experience of individuals regarding physical , social , and psychological wellbeing has allowed more detailed knowledge of the living conditions of the population . beside the need for the convergence of clinical diagnostic criteria ,
the concomitant use of nonnormative measures is important to determining whether periodontal disease causes an impact on people 's daily lives .
one of the first efforts to document this issue in brazil was made by leo et al .
, who used the dental impact on daily living index to measure periodontal health impacts on daily living.we made the highlighted change according to the list of references . please check roughly 60% of interviewed individuals reported some dissatisfaction with their quality of life .
it has been observed that studies relating the impact of periodontal disease on quality of life of diabetics are very scarce . among these few studies , drumond - santana et al . evaluated the potential impact of periodontal disease on quality of life in diabetics .
they found that diabetics with mild - to - moderate and advanced periodontal disease suffered a more negative impact on quality of life than those who were periodontally healthy or had gingivitis .
the oral health impact profile ( ohip ) and its shortened version , the ohip-14 , are indexes that can be used for this purpose [ 18 , 19 ] .
therefore , based on the hypothesis that the impact of periodontal disease on quality of life may vary depending on the clinical diagnostic criteria used for its diagnosis , the aim of this study was to test associations between different clinical diagnostic criteria for the determination of periodontal disease and its impact on the daily life of individuals with diabetes .
a cross - sectional study was carried out in 2005 in the city of belo horizonte , state capital of minas gerais , brazil .
the city has 2,412,937 inhabitants and occupies a strategic position on the geopolitical maps of brazil and latin america .
the study was approved by the ethics committee of the federal university of minas gerais ( protocol no .
sample calculation considered both type i and ii errors . for this , we assumed a 95% confidence interval , 80% power of test , and parameters of diabetes and quality of life from drumond - santana et al . .
the sample calculation by comparison of proportions presented a higher number than those found by comparison of means .
this procedure assured , for a sample size of 300 individuals , a type i error = 0.05 and a power of test = 80% for all dimensions of ohip-14 , except for psychological disability , social disability , and handicap .
we sampled 300 men and women over 30 years of age with a diagnosis of either type ( i and ii ) of diabetes , and who were registered and assisted by the belo horizonte health care system .
the city of belo horizonte has nine administrative districts with differences in the number of diabetic patients registered .
thus , the number of randomly selected patients was proportional to the number of diabetic individuals registered in each district .
the number of individuals examined was determined by a sample calculation using a formula recommended by the world health organization for the anticipated population proportion with the occurrence of disease .
the inclusion criteria were individuals who had diabetes diagnose ( either type i or ii ) , up to 30 years old , were registered in the belo horizonte public health care system , were dentate , and had signed the informed consent .
additionally , teeth with gingival morphology damage , ill - adapted dental restorations , large cavitation , or third molars were excluded .
the outcome was the impact of oral health on the quality of life of adults with diabetes measured by the modified oral health impact profile ( ohip-14 ) , which has been validated for use in brazil .
as the aim was to investigate the impact of periodontal disease , the word gums was added to 13 questions on the ohip-14 .
responses were dichotomized as no impact , which only considered the option never , and impact , which considered the options
the administration of the ohip-14 was carried out at basic health units of the public healthcare system .
periodontal disease was assessed by the following clinical criteria : gingival bleeding upon probing , probing depth , and clinical insertion loss . for the determination of gingival bleeding ,
a probe was introduced into the gingival pocket until the limit of its base , waiting 30 to 60 seconds for the determination of bleeding .
bleeding was recorded ( presence or absence ) using the modified ainamo and bay index .
probing depth was determined from the measurement of the distance from the gingival margin to the bottom of the gingival sulcus or periodontal pocket . the values for each of the surfaces ( mesial , distal , vestibular , and lingual ) of all the teeth present were recorded .
clinical attachment loss was determined from the distance between the enamel - cementum junction and the bottom of the gingival sulcus or periodontal pocket .
attachment loss was measured using the enamel - cementum junction as the base , when visible .
seven sets of clinical criteria were used to diagnose periodontal disease and are described in table 1 .
all exams were performed by one researcher ( a.m.s . ) , who was submitted to the kappa test ( ) for the determination of reliability ( intraexaminer agreement ) . for the kappa test
, all clinical exams were performed at the college of dentistry , federal university of minas gerais .
a total of 588 dental surfaces were clinically examined for the kappa test ( ) calculation , with a one - week interval between each participant 's first and second examinations .
results were satisfactory for all the clinical criteria : bleeding upon probing ( 0.79 ) , probing depth ( 0.81 ) , and clinical insertion loss ( 0.91 ) . during the main study ,
clinical exams were performed in the dental offices of brazilian national health care units under artificial light , using williams periodontal probes ( hu - friedy ) , mouth mirrors , and gauze .
the data were organized in a database using the statistical package for social sciences ( spss ) version 15.0 for windows .
univariate analysis was carried out to determine the association of each set of clinical criteria used to estimate periodontal disease and the ohip-14 , using the mann - whitney , kruskall wallis and pearson chi - square tests , with the significance level of p < 0.05 .
of all study participants , the majority had diabetes type ii ( 71.3% ) , were female ( 60% ) , age 55 or older ( 50.7% ) , living without a partner ( 57.3% ) , and had low educational level ( 71.3% ) ( table 2 ) .
table 3 presents the association between ohip-14 overall results and periodontal disease , according to each one of the clinical diagnostic criteria .
only iii - machtei ( p = 0.043 ) and iv - lopez ( p <
the prevalence of periodontal disease varied depending on the clinical criteria employed : 35.3% ( i - aap ) , 30.7% ( ii - beck ) , 35.0% ( iii - machtei ) , 9.7% ( iv - lopez ) ;
92.3% ( v - albandar ) , 25.3% ( vi - tonetti ) , and 75.3% ( vii - cpi ) ( figure 1 ) . regarding ohip-14 subscales ,
functional limitation was associated with iv - lopez ( 14.9% of diabetics with periodontal disease suffered an impact on quality of life ; p = 0.006 ) and v - albandar ( 28.4% , 8.2% , and 53.7% of diabetics with mild , moderate , severe periodontal disease , resp .
suffered an impact on quality of life ; p = 0.018 ) ; pain was only associated with the v - albandar criteria ( 28.1% , 14.5% , 49.2% of diabetics with mild , moderate , and severe periodontal disease , resp . suffered an impact on quality of life
. psychological discomfort was associated with iv - lopez ( 12.3% of diabetics with periodontal disease suffered an impact on quality of life ; p = 0.018 ) .
physical disability was associated with iv - lopez ( 13.2% of diabetics with periodontal disease suffered an impact on quality of life ; p = 0.047 ) and v - tonetti ( 30.6% of diabetics with periodontal disease suffered an impact on quality of life ; p = 0.046 ) criteria .
social disability was only associated with iv - lopez ( 13.0% of diabetics with periodontal disease suffered an impact on quality of life ; p = 0.056 ) .
a handicap was associated with i - aap ( 47.0% of diabetics with periodontal disease suffered an impact on quality of life ; p = 0.025 ) and ii - beck ( 40.9% of diabetics with periodontal disease suffered an impact on quality of life ; p = 0.041 ) criteria ( tables 4 and 5 ) .
the choice of one of the different sets of clinical criteria for the diagnosis of periodontal disease is not a simple task , since there is no consensual recommendation regarding the use of these measures [ 35 ] .
the measurement of health or disease does not go beyond the tooth or site and controversial measures impede the determination of an individual as either healthy or sick .
for instance , in our study the prevalence of periodontal disease ranged from 9.7% ( iv - lopez ) to 92.3% ( v - albandar ) , depending on which set of clinical diagnostic criteria was used .
some of the clinical diagnostic criteria used in the present study consider the result of the exam by the number of affected sites , whereas others consider the number of affected teeth or a combination of teeth and sites .
moreover , depending on the manner in which the measurement is determined , a single tooth may have four or six sites .
investigated whether the application of different definition criteria of periodontal disease had an influence on the significance of the association between periodontal disease and prematurity or low birth weight .
they used fourteen definitions of periodontal disease and results showed that significance of the association between periodontal disease and pregnancy outcomes may be determined by the periodontal disease definition or measurement used .
a similar study was carried out involving cases and controls in order to compare four definitions of periodontal disease and respective associations with premature birth / low birth weight ; the authors concluded that the magnitude of the association varied depending on the definition of periodontal disease employed .
two clinical criteria of periodontal disease ( machtei and lopez ) were statistically associated with the ohip-14 overall score .
the machtei criteria may be considered the broadest for determining the prevalence of periodontal disease , as it considers insertion loss 6 mm in two or more sites and probing depth 5 mm in one or more sites for the diagnosis of the disease .
the lopez criteria appear to be more cautious regarding the extension of periodontal disease , as they consider four or more teeth with at least one site with probing depth 4 mm and insertion loss 3 mm .
it is appropriate to highlight that community periodontal index ( cpi ) was statistically associated with neither the overall ohip-14 nor with any of its subscales .
cpi is recommended as an epidemiological tool by the world health organization ( who ) , and it has been widely used internationally . in our study , we were able to classify the worst cpi criteria code 3 or 4 ( with pockets ) .
if the patient had only one site probing 4 mm , he / she was diagnosed as having periodontal disease .
this may explain why there was no association between impact on quality of life and periodontal disease .
the test of the association of each of the seven sets of criteria and the ohip-14 dimensions showed that the lopez criteria were statistically associated with functional limitation , psychological discomfort , physical disability , and social disability ( limitrophe p value for this last one ) .
this evidence may be explained considering that the more strict a clinical criterion is , the higher the chances are of it having an association with negative impact on quality of life .
psychological disability was not associated with any of sets of clinical criteria for the diagnosis of periodontal disease .
depending on the clinical criteria used , there were differences in the impact of periodontal disease on quality of life , even when measured by the same questionnaire ( ohip-14 ) .
different cut - off points for probing depth and insertion loss imply differences in prevalence rates and the results of tests on associated factors .
criteria for the definition of the presence and absence of periodontal disease are controversial , and there is a clear need to establish a cut - off point between health and disease for periodontics , as well as establish the meaning of the disease for individuals . towards this end ,
some studies have adopted more rigorous criteria to minimize overestimated rates of periodontal disease and its association with quality of life .
severe periodontal disease , measured by beck criterion , will negatively impact on the diabetic 's life , as measured by ohip-14 .
it is worth noting that the majority of studies published so far comprise the issue of periodontal disease and quality of life in individuals without diabetes .
bernab and marcenes ( 2010 ) investigated the association between quality of life and periodontal disease among 3,122 british adults and found that they were inversely associated in spite of socioeconomic conditions , demographic factors , and clinical conditions . among the few studies which focused on the relation between diabetes and quality of life , sandberg and wikblad ( 2003 ) pointed out that having diabetes played an important role in the domains physical functioning , role functioning physical , general health and social functioning , when compared to patients without a history of diabetes .
although our study included a representative sample of diabetic patients in the public healthcare system of belo horizonte , brazil , it is important to point out some limitations of this research . first , our study is cross - sectional , which does not allow any inference with regard to causality .
second , our sample size does not give us reasonable values for type i and ii errors for the dimensions psychological disability , social disability and handicap . the main issue raised by the results of the present study is that the definition of a disease established by a given set of clinical diagnostic criteria may either overestimate or underestimate the chances of an individual being considered sick or healthy .
likewise , the impact periodontal disease has on quality of life may vary depending on the definition of the disease established by one of the seven sets of diagnostic criteria analyzed here .
additionally , we did not register the mean duration of diabetes , and therefore were not able to consider development of comorbidities that negatively impact quality of life .
as health and sickness are terms that correspond to unique experiences laden with subjectivity , it seems unlikely that these terms are recognized in the totality of their meanings .
the concrete phenomenon of becoming ill , self - perception of the individual , and professional diagnosis most often fail to coexist in harmony .
this means that the same disease may be seen from different standpoints , the consequences of which are the differences identified in the impact that periodontal disease has on the daily life of affected individuals .
the concept of health or disease as determined by clinical diagnostic criteria may influence the results of the assessment of the impact of periodontal disease on quality of life of diabetics , while the perception of affected individuals is unique .
studies directed at the establishment of standardized criteria for the diagnosis of periodontal disease are needed .
such studies should be focused on the health - disease process , including its social determination .
a reference standard for epidemiological studies addressing periodontal disease could contribute to the organization of health actions in the field of collective health . | the aim of this study was to determine the impact of periodontal disease on the quality of life of individuals with diabetes according to different clinical criteria ( i - aap , ii - beck , iii - machtei , iv - lopez , v - albandar , vi - tonetti , and vii - cpi ) .
this cross - sectional study sampled 300 individuals in belo horizonte , brazil .
the oral health impact profile was used to measure the impact of periodontal disease on quality of life .
prevalence of periodontal disease was 35.3% , 30.7% , 35.0% , 9.7% , 92.3% , 25.3% , and 75.3% using criteria i , ii , iii , iv , v , vi , and vii , respectively .
the iii - machtei ( p = 0.043 ) and iv - lopez ( p < 0.001 ) criteria were associated with ohip-14 ; functional limitation was associated with iv - lopez ( p = 0.006 ) and v - albandar ( p = 0.018 ) criteria .
pain was only associated with v - albandar criteria ( p < 0.001 ) .
psychological discomfort was associated with the iv - lopez ( p = 0.018 ) criteria .
physical disability was associated with the iv - lopez ( p = 0.047 ) and v - tonetti ( p = 0.046 ) criteria .
being handicapped was associated with the i - aap ( p = 0.025 ) and ii - beck ( p = 0.041 ) criteria .
concepts of health and disease determined by clinical diagnostic criteria may influence the assessment of the impact of periodontal disease on diabetics ' quality of life . |
intraductal papillary neoplasms of the bile duct ( ipnb ) have been associated with papillary tumors of malignant potential in the extrahepatic and intrahepatic bile ducts.123 ipnb is classified as a distinct clinical and pathological entity in the 2010 world health organization classification.4 its malignant potential reportedly ranges from 19.5% to 83%.5 these tumors show papillary proliferation in the bile duct with or without mucin secretion and are considered as ipnb , the biliary counterpart of intraductal papillary mucinous neoplasm ( ipmn ) of the pancreas.467 some mucin - producing ipnbs can cause obstructive jaundice due to production of abundant high - viscosity mucin.4 herein , we presented two cases of mucin - producing ipnbs with obstructive jaundice who underwent resection of the intrahepatic primary lesions and bypass hepaticojejunostomy .
a 69 year - old male was admitted to the emergency room due to obstructive jaundice .
five years ago , he underwent lung resection for adenosquamous carcinoma of the lung , without recurrence to date . at the time of lung surgery
, he was diagnosed with gallbladder stones and hepatolithiasis of the segment iii , vi and viii ducts ( b3 , b5 and b8 ) with corresponding ductal dilatation , suggestive of associated inflammatory stricture . yearly imaging study follow - up for gallstone lesions until the recent
last visit to emergency room showed no interval changes of gallstone disease or evidence of malignant changes .
imaging study findings of dynamic liver computed tomography ( ct ) and magnetic resonance ( mr ) imaging were as follows : mucin - secreting ipnb mainly in b3 with slight interval aggravation of biliary ductal dilatation ; and multiple intrahepatic duct stones , interval increased extent and gallbladder ( fig .
cancer antigen 19 - 9 ( ca 19 - 9 ) was mildly elevated ( 38 u / ml ) .
endoscopic retrograde cholangiography ( erc ) showed multiple filling defects in the biliary system , which was compatible with mucin - producing ipnb ( fig .
two endoscopic nasobiliary tubes were inserted and retained for 10 days in order to mediate biliary decompression .
2a and 2b ) . after removing the very sticky mucin , the b3 cystic lesion identified as the source of the mucin was excised and a 2 cm - sized b3 opening was exposed ( fig . 2c and 2d ) .
frozen - section biopsy revealed that the b3 cystic wall was involved with cancer , thus the whole b3 duct wall except its connecting part to the left intrahepatic duct was resected .
intrahepatic duct stones at b5 and b8 were removed with curved stone forceps . due to some risk of residual tumor that could re - induce mucin production , we performed two separate biliary bypass drainages to the common bile duct and b3 orifice ( fig .
the operation included segment iii hepatic resection , intrahepatic stone removal , end - to - side choledochojejunostomy and b3 hepaticojejunostomy .
pathologic findings were as follows : intrahepatic cholangiocarcinoma with intraductal growth type ; confinement to bile duct wall ; no lymphovascular invasion ; no perineural invasion : no involvement of b3 margin and hepatic parenchymal resection margins ; biliary intraepithelial neoplasia 2 at the common bile duct wall ; and chronic cholangitis with bile duct dilatation , extensive erosion , multifocal rupture and abscess and xanthogranulomatous inflammation .
3 ) . since this resection was regarded as r0 resection , no additional adjuvant treatment was scheduled .
a 74 year - old female was referred for surgery due to suspected malignant mucin - producing ipnb and obstructive jaundice .
she had undergone repeated episodes of erc and percutaneous transhepatic cholangiography ( ptc ) for recurrent intrahepatic and common bile duct stones for the last 11 years .
findings of dynamic liver ct and mr imaging were as follows : mucin - producing ipnb with asymmetric disproportional dilatation in the segment iv duct ( b4 ) and mural nodule in b4 ; increased fluid collection due to bile duct rupture at the right subphrenic space adjacent to the segment iv ( fig . 4a and 4b ) .
chest ct and positron emission tomography showed multiple mild hypermetabolic lymph nodes in right internal mammary chain , suggesting metastatic lymph nodes .
ptc was performed for biliary decompression and tissue confirmation ; intra - operatively , the intraductal mass was diagnosed as ipnb of intermediate grade dysplasia ( fig . 4c and 4d ) .
5a ) . after removing the very sticky mucin , the b4 cystic lesion identified as the source of mucin
was excised and two 3 cm - sized right and left hepatic duct openings were exposed ( fig .
we decided to perform non - curative resection since residual scattered bile duct tumors at the hepatic confluence portion did not permit simple resection and left hepatectomy was contra - indicated due to patient safety .
two large openings to the right and left hepatic ducts were unified with running sutures and then reconstructed with single large hepaticojejunostomy ( fig . 5c and 5d ) .
the operation included segment iv hepatic resection , partial resection of the diaphragm and central hepaticojejunostomy .
pathologic findings were as follows : well differentiated mucinous adenocarcinoma of nodular type arising from ipnb ; extension beyond bile duct ; serosal penetration and involvement of the diaphragm ; no lymphovascular invasion ; no perineural invasion ; involvement of the radial resection margin ; and no involvement of common and hepatic bile duct resection margins .
this resection was regarded as r1/r2 resection with possibility of distant lymph node metastasis , but no additional adjuvant treatment was planned due to poor general condition of the patient .
she has been doing well for 4 months post - surgery without any evidence of tumor recurrence or distant metastasis .
a 69 year - old male was admitted to the emergency room due to obstructive jaundice .
five years ago , he underwent lung resection for adenosquamous carcinoma of the lung , without recurrence to date . at the time of lung surgery
, he was diagnosed with gallbladder stones and hepatolithiasis of the segment iii , vi and viii ducts ( b3 , b5 and b8 ) with corresponding ductal dilatation , suggestive of associated inflammatory stricture . yearly imaging study follow - up for gallstone lesions until the recent
last visit to emergency room showed no interval changes of gallstone disease or evidence of malignant changes .
imaging study findings of dynamic liver computed tomography ( ct ) and magnetic resonance ( mr ) imaging were as follows : mucin - secreting ipnb mainly in b3 with slight interval aggravation of biliary ductal dilatation ; and multiple intrahepatic duct stones , interval increased extent and gallbladder ( fig .
cancer antigen 19 - 9 ( ca 19 - 9 ) was mildly elevated ( 38 u / ml ) .
endoscopic retrograde cholangiography ( erc ) showed multiple filling defects in the biliary system , which was compatible with mucin - producing ipnb ( fig .
two endoscopic nasobiliary tubes were inserted and retained for 10 days in order to mediate biliary decompression .
2a and 2b ) . after removing the very sticky mucin , the b3 cystic lesion identified as the source of the mucin was excised and a 2 cm - sized b3 opening was exposed ( fig . 2c and 2d ) .
frozen - section biopsy revealed that the b3 cystic wall was involved with cancer , thus the whole b3 duct wall except its connecting part to the left intrahepatic duct was resected .
intrahepatic duct stones at b5 and b8 were removed with curved stone forceps . due to some risk of residual tumor that could re - induce mucin production , we performed two separate biliary bypass drainages to the common bile duct and b3 orifice ( fig .
the operation included segment iii hepatic resection , intrahepatic stone removal , end - to - side choledochojejunostomy and b3 hepaticojejunostomy .
pathologic findings were as follows : intrahepatic cholangiocarcinoma with intraductal growth type ; confinement to bile duct wall ; no lymphovascular invasion ; no perineural invasion : no involvement of b3 margin and hepatic parenchymal resection margins ; biliary intraepithelial neoplasia 2 at the common bile duct wall ; and chronic cholangitis with bile duct dilatation , extensive erosion , multifocal rupture and abscess and xanthogranulomatous inflammation .
3 ) . since this resection was regarded as r0 resection , no additional adjuvant treatment was scheduled .
a 74 year - old female was referred for surgery due to suspected malignant mucin - producing ipnb and obstructive jaundice .
she had undergone repeated episodes of erc and percutaneous transhepatic cholangiography ( ptc ) for recurrent intrahepatic and common bile duct stones for the last 11 years .
findings of dynamic liver ct and mr imaging were as follows : mucin - producing ipnb with asymmetric disproportional dilatation in the segment iv duct ( b4 ) and mural nodule in b4 ; increased fluid collection due to bile duct rupture at the right subphrenic space adjacent to the segment iv ( fig . 4a and 4b ) .
chest ct and positron emission tomography showed multiple mild hypermetabolic lymph nodes in right internal mammary chain , suggesting metastatic lymph nodes .
ptc was performed for biliary decompression and tissue confirmation ; intra - operatively , the intraductal mass was diagnosed as ipnb of intermediate grade dysplasia ( fig . 4c and 4d ) .
after removing the very sticky mucin , the b4 cystic lesion identified as the source of mucin was excised and two 3 cm - sized right and left hepatic duct openings were exposed ( fig .
we decided to perform non - curative resection since residual scattered bile duct tumors at the hepatic confluence portion did not permit simple resection and left hepatectomy was contra - indicated due to patient safety .
due to high risk of residual tumors that could re - induce mucin production , these lesions were destroyed with deep electrocautery and argon beam coagulator .
two large openings to the right and left hepatic ducts were unified with running sutures and then reconstructed with single large hepaticojejunostomy ( fig . 5c and 5d ) .
the operation included segment iv hepatic resection , partial resection of the diaphragm and central hepaticojejunostomy .
pathologic findings were as follows : well differentiated mucinous adenocarcinoma of nodular type arising from ipnb ; extension beyond bile duct ; serosal penetration and involvement of the diaphragm ; no lymphovascular invasion ; no perineural invasion ; involvement of the radial resection margin ; and no involvement of common and hepatic bile duct resection margins .
this resection was regarded as r1/r2 resection with possibility of distant lymph node metastasis , but no additional adjuvant treatment was planned due to poor general condition of the patient .
she has been doing well for 4 months post - surgery without any evidence of tumor recurrence or distant metastasis .
ipnb is considered a precursor lesion of cholangiocarcinoma.12 ipnb was proposed as a new disease entity because of striking similarities to ipmn of the pancreas , wherein the disease entity and clinicopathological features are well established.8 however , preoperative diagnosis of intrahepatic ipbn is usually difficult in practice .
the common clinical manifestations of patients with intrahepatic ipnb are recurrent abdominal pain , repeated episodes of acute cholangitis and obstructive jaundice , as presented in our previous study.1 intrahepatic duct dilatation and intraductal mass are common abnormal findings in patients with intrahepatic ipnb .
cholangiography , including indirect cholangiography ( mr cholangiography ) and direct cholangiography ( erc and ptc ) , is useful to show the entire bile duct to define the extent of the ipnb.910 mucin can not be detected on usual imaging studies .
direct cholangiography is useful for the detection of mucobilia.8 however , erc and ptc are invasive procedures , which not only risk introducing bacteria into the bile duct , but more importantly may result in dissemination of tumor cells .
mucinous obstruction of bile duct prevents visualization of the whole biliary tree as well as tumor detection by cholangiography .
ptc and erc are usually performed in patients with obstructive jaundice , but it is often not possible to relieve the jaundice by draining the inspissated mucobilia.10 cholangioscopy including percutaneous transhepatic cholangioscopy ( ptcs ) and peroral cholangioscopy can approach the bile duct directly , confirm the histology , and assess the extent of the tumor including superficial spreading along the biliary epithelium , which facilitates appropriate treatment choice.58 we prefer ptcs to peroral cholangioscopy for reliable evaluation of the intrahepatic duct in patients with mucin - producing lesions ; in addition , percutaneous transhepatic biliary drainage enables biliary decompression in the environment of sticky mucin formation .
ptcs examination is therefore an indispensable preoperative procedure for determining treatment modality and the appropriate extent of resection in intrahepatic ipnb.111 intrahepatic ipnb should not be regarded as a benign disease with low malignant potential but as a premalignant lesion with high malignant potential . in our previous study , low - grade intrahepatic ipnb was rather rare and the majority of intrahepatic ipnb cases were high - grade ipnb , and invasive ipnb with minimal and considerable invasion.1 ipnb with different malignant potentials can be ultimately diagnosed as adenoma , borderline tumor , non - invasive carcinoma or invasive carcinoma,12 a continuum of intraductal neoplastic progression .
these neoplasms arise within the ductal system and show a predominantly intraductai growth pattern macroscopically and papillary proliferation with delicate fibrovascular cores.1314 their pathological similarities are summarized as follows : macroscopic growth pattern of intraductal papillary proliferation ; occasional association with mucin hypersecretion ; microscopic feature of papillary proliferation with fibrovascular cores ; occurrence of three types of tumor cells ; occasional association with multiple lesions ; possible progression to tubular adenocarcinoma and mucinous carcinoma ; and influence of histological types of invasive lesion on survival rate .
their pathological differences are summarized as follows : high frequency of ck20 expression in ipnb than in pancreatic ipmn ; lower percentage of gastric type tumors in ipnb than in pancreatic ipmn ; and lower frequency of mucin hypersecretion in ipnb than in pancreatic ipmn.14 mucin is macroscopically identifiable in most cases of pancreatic ipmn but in only less than one - third of ipnb cases.61516 we previously reported that mucin pool formation was observed in only 7 of 43 cases ( 16.3%).1 mucin - producing ipnb is capable of secreting abundant quantities of mucus .
the liquid is composed of mucin and albumin and is rich in electrolytes , but contains neither bile salts nor pigments .
its external drainage is often contraindicated because it is followed by discharge of extraordinary quantities of mucobilia and leads to marked loss of proteins and electrolytes.17 because patients with intrahepatic ipnb often show favorable prognosis , curative surgical resection is regarded as the first - choice treatment for patients with intrahepatic ipnb without distant metastasis .
we previously showed that surgical curability was the only reliable prognostic factor for tumor recurrence and patient survival.1 palliative treatments are recommended if curative resection is not feasible.17 especially in patients with mucin - producing tumors , hepaticojejunostomy can palliate the liquid loss and is also efficient , at least temporarily , in previously reported cases.18 in this study , the 2 patients were diagnosed with intrahepatic stone disease 5 and 11 years prior to surgery , respectively .
it is likely that the pre - existing dysplastic lesions transformed to malignancy at a certain time , resulting in acceleration of mucin production .
they were referred for surgical treatment only after obstructive jaundice was developed due to massive mucin production .
in retrospect , closer follow - up may have led to earlier surgical treatment and better outcome , but poor general condition of these patients did not lead to surgery before manifestation of overt symptoms .
the long - term clinical courses of our patients highlight that abundant production of highly viscous mucin might be associated with malignant transformation . in conclusion , we presented two rare cases of mucin - producing ipnb of malignant transformation that were treated with primary resection and biliary drainage .
the case studies indicated that abundant production of highly viscous mucin inducing obstructive jaundice may be associated with malignant transformation in patients with ipnb . | intraductal papillary neoplasms of the bile duct ( ipnb ) leads to malignant transformation and mucin production . herein , we presented two cases of mucin - producing ipnb with obstructive jaundice who underwent resection of the intrahepatic lesions and bypass hepaticojejunostomy .
the first case was a 69 year - old male patient with 5-year follow up for gallstone disease .
imaging studies showed mucin - secreting ipnb mainly in the hepatic segment iii bile duct ( b3 ) and multiple intrahepatic duct stones for which , segment iii resection , intrahepatic stone removal , end - to - side choledochojejunostomy and b3 hepaticojejunostomy were conducted .
the second case was a 74 year - old female patient with 11-year follow up for gallstone disease .
imaging studies showed mucin - producing ipnb with dilatation of the segment iv duct ( b4 ) and mural nodules for which , segment iv resection , partial resection of the diaphragm and central hepaticojejunostomy were conducted .
both patients recovered uneventfully from surgery .
these cases highlight that in patients with ipnb , abundant production of highly viscous mucin inducing obstructive jaundice may be associated with malignant transformation . |
during the course of the study , harp consisted of 11 hospital study sites across the united states .
the first five patients who sought treatment each month for esophago - gastro duodenal endoscopy ( egd ) as part of treatment for h. pylori infection , confirmed by gastric biopsy , were enrolled at each site .
when fewer than five eligible study participants were seen at a study center , those eligible were enrolled .
culture of biopsied material for h. pylori was performed at the study site or at the atlanta veterans affairs hospital .
if primary isolation was performed at the harp study site , isolates were placed in trypticase soy broth ( tsb ) supplemented with glycerol and stored at 70c .
isolates were sent to the centers for disease control and prevention for antimicrobial resistance testing .
samples forwarded to the atlanta veterans affairs hospital for isolation were packed in 6 pounds of dry ice and maintained at 70c to 80c until cultured ( up to 1 month ) .
each specimen was accompanied by a standard case report form containing 145 variables of demographic , clinical , and epidemiologic information .
isolates were cultured on heart infusion agar ( hia ) with 5% rabbit blood at 37c in a microaerobic atmosphere ( 10% co2 , 85% n2 , 5% o2 ) for a minimum of 72 hours or until appearance of h. pylori colonies .
gastric biopsy specimens were homogenized by using a micropestle in a microcentrifuge tube , and the homogenates were added to hia with 5% rabbit s blood and skirrow media and incubated at 37c microaerobically for up to 2 weeks .
isolates were confirmed as h. pylori if they demonstrated typical morphologic features by dark - field microscopy and if urease , oxidase , and catalase activities were detected .
also , a polymerase chain reaction amplification assay that used primers targeted at the urea gene provided confirmation ( 7 ) .
antimicrobial susceptibility testing was performed by the agar dilution method in accordance with national committee for clinical laboratory standards ( nccls ) protocols , except that defibrinated rabbit blood was used instead of aged sheep blood ( 8) . for antimicrobial agents without an nccls - recommended breakpoint ,
case report forms and laboratory data were entered into a ms access 2000 database and analyzed by using sas , version 8.2 ( sas institute , cary , nc ) .
we defined a resistant case to be an harp isolate that demonstrated any detectable resistance to metronidazole , clarithromycin , tetracycline , or amoxicillin .
harp study sites were assigned to the following four regions : northeast ( washington , dc ; new york , ny ; and philadelphia , pa ) , south ( atlanta , ga ; nashville , tn ; and galveston , tx ) , midwest ( detroit , mi ; cleveland , oh ; indianapolis , in ; and milwaukee , wi ) , and west ( palo alto , ca ) .
differences between patients infected with resistant and susceptible strains of h. pylori were assessed by using the fisher exact test , mantel - haenszel chi - square , or student t test , as appropriate . a one - way analysis of variance ( anova ) was used to determine antimicrobial resistance rate trends for clarithromycin and metronidazole . when the difference in the number of study participants that harbored a resistant h. pylori infection and those who did not for a given risk factor was < 2 , we chose to exclude the risk factor from further analysis because of the possibility of arriving at a spurious association caused by small cell - size variations .
this method enhanced the statistical reliability of all possible exposure and resistance associations considered for multivariate modeling .
risk factors that met these criteria were subjected to a chi - square score model selection procedure to obtain the most significant and stable multivariate model .
multivariate analyses were performed by using logistic regression to assess the independent association of identified risk factors with any detectable antimicrobial resistance using two different models .
a second logistic regression model was created that we conditioned on geographic location of harp site , previous antimicrobial treatment for h. pylori infection , and antacid use to resolve colinearity issues with race .
isolates were cultured on heart infusion agar ( hia ) with 5% rabbit blood at 37c in a microaerobic atmosphere ( 10% co2 , 85% n2 , 5% o2 ) for a minimum of 72 hours or until appearance of h. pylori colonies .
gastric biopsy specimens were homogenized by using a micropestle in a microcentrifuge tube , and the homogenates were added to hia with 5% rabbit s blood and skirrow media and incubated at 37c microaerobically for up to 2 weeks .
isolates were confirmed as h. pylori if they demonstrated typical morphologic features by dark - field microscopy and if urease , oxidase , and catalase activities were detected .
also , a polymerase chain reaction amplification assay that used primers targeted at the urea gene provided confirmation ( 7 ) .
antimicrobial susceptibility testing was performed by the agar dilution method in accordance with national committee for clinical laboratory standards ( nccls ) protocols , except that defibrinated rabbit blood was used instead of aged sheep blood ( 8) . for antimicrobial agents without an nccls - recommended breakpoint , breakpoints were selected after a review of the literature .
case report forms and laboratory data were entered into a ms access 2000 database and analyzed by using sas , version 8.2 ( sas institute , cary , nc ) .
we defined a resistant case to be an harp isolate that demonstrated any detectable resistance to metronidazole , clarithromycin , tetracycline , or amoxicillin .
harp study sites were assigned to the following four regions : northeast ( washington , dc ; new york , ny ; and philadelphia , pa ) , south ( atlanta , ga ; nashville , tn ; and galveston , tx ) , midwest ( detroit , mi ; cleveland , oh ; indianapolis , in ; and milwaukee , wi ) , and west ( palo alto , ca ) .
differences between patients infected with resistant and susceptible strains of h. pylori were assessed by using the fisher exact test , mantel - haenszel chi - square , or student t test , as appropriate . a one - way analysis of variance ( anova ) was used to determine antimicrobial resistance rate trends for clarithromycin and metronidazole . when the difference in the number of study participants that harbored a resistant h. pylori infection and those who did not for a given risk factor was < 2 , we chose to exclude the risk factor from further analysis because of the possibility of arriving at a spurious association caused by small cell - size variations .
this method enhanced the statistical reliability of all possible exposure and resistance associations considered for multivariate modeling .
risk factors that met these criteria were subjected to a chi - square score model selection procedure to obtain the most significant and stable multivariate model .
multivariate analyses were performed by using logistic regression to assess the independent association of identified risk factors with any detectable antimicrobial resistance using two different models .
a second logistic regression model was created that we conditioned on geographic location of harp site , previous antimicrobial treatment for h. pylori infection , and antacid use to resolve colinearity issues with race .
of 317 enrolled harp study participants for whom complete demographic information was available , 205 ( 65% ) were male , 116 ( 37% ) were white , 172 ( 54% ) were black , 14 ( 4% ) were asian , 3 ( 1% ) were native american , and 12 ( 4% ) were of other ethnic backgrounds .
the median age was 57 years ( range 394 years ) . among males , 97 ( 31% ) were white , 90 ( 28% ) were black , and 9 ( 3% ) were asian , 1 ( 0.3% ) was native american , and 8 ( 3% ) were of other ethnic backgrounds . among females , 19 ( 6% ) were white , 82 ( 26% ) were black , 5 ( 2% ) were asian , 2 ( 0.6% ) were native american , and 4 ( 1% ) were of other ethnic backgrounds .
the most common endoscopic diagnoses for conditions of harp participants were gastric erosions , gastritis , duodenal and gastric ulcers , and esophagitis ( table 1 ) .
347 h. pylori isolates submitted to harp from 1998 to 2002 , 118 ( 34% ) were resistant to > 1 antimicrobial agent , 101 ( 29% ) h. pylori isolates were resistant to one agent only , and 17 ( 5% ) h. pylori isolates were resistant to more than one antimicrobial agent .
three isolates were resistant to amoxicillin ( 1% ) , 45 were resistant to clarithromycin ( 13% ) , 87 were resistant to metronidazole ( 25% ) , and no isolate was found to be resistant to tetracycline .
multiple - agent resistance was observed for clarithromycin and amoxicillin ( 1 isolate , 0.3% ) and clarithromycin and metronidazole ( 16 isolates , 5% ) .
a test of trend showed no significant trend for resistance to metronidazole , but a significant trend ( r = 0.76 ) was noted for a decline in resistance to clarithromycin during the study period ( figure ) .
resistance of helicobacter pylori isolates submitted to the helicobacter pylori antimicrobial resistance monitoring project , 19982002 ( n = 347 ) .
1998 , n = 7 isolates ; 1999 , n = 137 isolates ; 2000 , n = 117 isolates ; 2001 , n = 47 isolates ; 2002 , n = 39 isolates . resistance to tetracycline was found in 0% of isolates during each year of the monitoring project .
most isolates were submitted by centers in the northeast region , followed by the south , midwest , and the west . submitted h. pylori isolates from the northeast region had the highest frequency of single- and dual - agent resistance ( table 2 ) .
the midwest region submitted h. pylori isolates with the second - highest single agent resistance rate , while the southern region had the second - highest dual agent resistance rate .
the highest proportion of h. pylori isolates resistant to clarithromycin , metronidazole , and amoxicillin was in the northeast ( table 2 ) .
the four sites reporting the highest proportion of isolates resistant to one agent were indianapolis , indiana ; galveston , texas ; philadelphia , pennsylvania ; and washington , dc ( table 3 ) .
indianapolis had the highest overall resistance : 50% of all isolates submitted were resistant to at least one agent .
the atlanta site reported the highest proportion of clarithromycin - resistant isolates ( table 3 ) .
isolates resistant to amoxicillin were found in nashville , tennessee , philadelphia , and washington , dc .
demographic , clinical , and epidemiologic data were analyzed for association with infection by a resistant h. pylori strain .
results of univariate analysis of risk factors for antimicrobial resistance are summarized in table 4 .
on univariate analysis , significant risk factors for h. pylori resistance included past treatment of h. pylori infection with clarithromycin , treatment of past h. pylori infection for a second time in the past 5 years , treatment of past h. pylori infection with clarithromycin more than once , and treatment of past h. pylori infection with a proton pump inhibitor more than once . no protective factors for resistant h. pylori resistance approached significance .
n = 347 ; harp , helicobacter pylori antimicrobial resistance monitoring project ; egd , esophagogastro - duodenal endoscopy ; ppi , proton pump inhibitor ; ci , confidence interval .
these risk factors were not considered for multivariate analysis because of small cell - size differences between subjects with a resistant h. pylori infection and those without .
on multivariate analysis , the most significant risk factor for h. pylori resistance was previous treatment of h. pylori infection within the past 5 years ( table 5 ) .
protective factors for h. pylori infection were treatment with an antacid ( mylanta ) and submission of specimens from new york , palo alto , california , atlanta , or nashville .
however , a high degree of colinearity was found between geographic location and race in this model .
for example , 84% of washington , dc , participants were black , compared to 20% of participants from nashville .
accordingly , a second multivariate model was constructed in which geographic location was controlled by conditioning . in this model ,
black race was the only risk factor significantly associated with infection with a resistant h. pylori strain ( table 6 ) .
n = 347 ; harp , helicobacter pylori antimicrobial resistance monitoring project ; ci , confidence interval .
n = 235 ; harp , helicobacter pylori antimicrobial resistance project ; ci , confidence interval .
the emergence of antimicrobial resistance in h. pylori represents a serious public health challenge because of the prevalence of infection and incidence of severe sequelae .
a 12-fold increase in resistance to antimicrobial agents such as clarithromycin has been documented in cases in which antimicrobial therapy regimens do not eliminate h. pylori ( 9 ) .
once resistance to clarithromycin appears , cross - resistance to all other macrolides also occurs , which eliminates the potential for their use in salvage therapies . in the past 5 years , amoxicillin and tetracycline resistance has also been described ( 6,10 ) .
however , most published studies have been based on cross - sectional designs , not prospective population - based surveillance of resistance . a meta - analysis of 20 nationwide clinical trials estimated h. pylori resistance to clarithromycin , metronidazole , and amoxicillin to be 10.1% , 36.9% , and 1.4% , respectively ( 6 ) .
resistance rates may be higher in pediatric populations ; for example , a study in michigan demonstrated that clarithromycin resistance in pediatric h. pylori strains was 2.5 times higher than that reported in adults in the same region and elsewhere in the united states ( 12 ) .
single - site and multicenter surveillance network studies in britain , croatia , the czech republic , turkey , greece , poland , russia , slovenia , turkey , estonia , italy , bulgaria , germany , and belgium have observed primary metronidazole and clarithromycin resistance with rates as high as 40.3% and 10.6% , respectively ( 1317 ) .
a large prospective surveillance project in northeastern germany showed primary metronidazole and clarithromycin resistance rates of 26.2% and 2.2% , respectively , in 1,644 clinical h. pylori isolates collected from 1995 to 2000 ( 17 ) .
high resistance rates to metronidazole and clarithromycin have been reported in portugal , brazil , hong kong , saudia arabia , and lebanon ( 1822 ) .
overall , h. pylori resistance to metronidazole among single and multicenter surveillance networks is greater than all other resistant antimicrobial agents combined .
harp is the only multicenter network providing ongoing prospective antimicrobial resistance and associated risk factor data for h. pylori in north america .
the first 4 years of data show that resistance to antimicrobial drugs commonly used to treat h. pylori infections is widespread , though rates varied from year to year ; this finding was true for both clarithromycin and metronidazole .
we speculate that the precipitous decrease in metronidazole resistance in 2002 could be attributed to the reduction in isolates received by harp , which limited the power and representiveness of our sample .
we performed a two - phased multivariate analysis on risk factors significantly associated with resistance on univariate analysis . in the first - phase , multivariate model , antimicrobial resistance
was significantly associated with previous antimicrobial treatment for h. pylori , and antacid use was protective . however , in this model , geographic location was highly collinear with race , which necessitated the second - phase multivariate model , in which the contribution of race was assessed by conditioning on geographic location , previous antimicrobial treatment for h. pylori infection , and antacid use .
this model clearly demonstrated an association between black race and infection with an antimicrobial - resistant h. pylori .
this association may indicate persistent transmission of resistant strains or in vivo induction of resistance .
the association between h. pylori infection and blacks in the united states has been amply documented ( 2,3 ) .
however , the association between incidence of h. pylori resistance and black race is new .
the biologically plausible association between previous antimicrobial use and resistance approached significance in the second - phase model ; however , many study participants could not recall whether they were previously treated .
enrollment of additional participants in harp may sufficiently enhance the statistical power of the study to show such an association , while enrollment in sites serving a more racially diverse population may help clarify the contributions of geographic location as a risk factor for resistant infection .
while drawn from various regions in the continental united states , the 11 harp sites represent a convenience sample of academic medical centers that is not truly representative of the u.s . population . some participating sites enrolled more patients per month than others , so that cumulative data are biased towards individual study sites .
most of the participating hospital sites serve adult populations , and thus the results largely reflect resistance incidence towards adult patients .
patients undergoing endoscopy for h. pylori - related illness constitute a minority of infected persons , whose clinical condition and h. pylori - resistance status may not be representative of the total population with underlying h. pylori infection . although this trend has not been documented , many public health analysts suspect that the proportion of h. pylori infections diagnosed and treated in academic medical centers is decreasing compared with those in community practices .
we suspect that this trend may have accounted for the reduced number of isolates submitted to harp over time .
although these limitations primarily affected our sample size and how the sample represented the population , they did not invalidate the epidemiologic significance of the results generated from this study . given the limited population - based surveillance data on h. pylori resistance , harp offers the best available data on h. pylori resistance in the united states .
the accuracy and usefulness of this prospective , multicenter network can be enhanced in a number of ways . increasing
given the fact that only 12 study participants < 12 years of age were included , enrollment of pediatric - care sites will improve the age representation of the harp cohort .
this feature is particularly important considering the increasing number of reports that describe higher resistance rates in pediatric strains compared to those isolated from adults , particularly resistance to macrolides ( 12,23 ) .
enrolling study participants in community practices will provide data on resistance rates in h. pylori isolates from infected persons not treated at academic medical centers , a population in which resistance rates in h. pylori are unknown and may differ significantly from the current harp cohort .
future harp findings may serve as the basis for specific therapeutic recommendations and pretreatment antimicrobial susceptibility testing in high - risk populations . in summary
, we have shown that antimicrobial resistance in clinical h. pylori isolates is extensive , that it varies from year to year , and that resistant isolates are more common among blacks .
ongoing , prospective surveillance of h. pylori resistance is essential to assure [ or ensure ? ] that appropriate data are available to guide the choice of therapy , particularly in high - risk populations . | helicobacter pylori is the primary cause of peptic ulcer disease and an etiologic agent in the development of gastric cancer .
h. pylori infection is curable with regimens of multiple antimicrobial agents , and antimicrobial resistance is a leading cause of treatment failure .
the helicobacter pylori antimicrobial resistance monitoring program ( harp ) is a prospective , multicenter u.s .
network that tracks national incidence rates of h. pylori antimicrobial resistance . of 347 clinical h. pylori
isolates collected from december 1998 through 2002 , 101 ( 29.1% ) were resistant to one antimicrobial agent , and 17 ( 5% ) were resistant to two or more antimicrobial agents .
eighty - seven ( 25.1% ) isolates were resistant to metronidazole , 45 ( 12.9% ) to clarithromycin , and 3 ( 0.9% ) to amoxicillin . on multivariate analysis ,
black race was the only significant risk factor ( p < 0.01 , hazard ratio 2.04 ) for infection with a resistant h. pylori strain . formulating pretreatment screening strategies or providing alternative therapeutic regimens for high - risk populations may be important for future clinical practice . |
the existence of dark matter ( dm ) in the universe is highly motivated by many astrophysical and cosmological observations [ 14 ] .
one of the best motivated candidates for a dm particle is a weakly interacting massive particle ( wimp ) . at the large hadron collider ( lhc )
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\begin{document}$$m_{\chi } \le $ $ \end{document}m 10 gev ) , and therefore provide information complementary to direct dm searches , which are most sensitive to larger dm masses [ 69 ] .
if the particles that mediate the interactions between dm and standard model ( sm ) particles are too heavy to be produced directly in the experiment , their interactions can be described by contact operators in the framework of an effective field theory [ 1012 ] . for each operator
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\begin{document}$$t$$\end{document}t ) was proposed in ref . .
to search for these processes , dedicated selections are defined to reconstruct the various production and decay modes of these heavy - quark final states . for final states containing a semileptonic decay of a top quark
, the results of the search for a supersymmetric partner of the top quark are used .fig .
1dominant feynman diagrams for dm production in conjunction with a a single \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \mathcal{o}_\text { scalar}= \sum _ q \frac{m_q}{m_*^n}\bar{q}q\bar{\chi } \chi , \end{aligned}$$\end{document}oscalar=qmqmnqq,where \documentclass[12pt]{minimal }
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\begin{document}$$m_q$$\end{document}mq , which mitigates contributions to flavour - changing processes , strongly constrained by flavour physics observables [ 25 , 26 ] , through the framework of minimal flavour violation ( mfv ) .
the dependence on the quark mass makes final states with bottom and top quarks the most sensitive to these operators .
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the results are also interpreted in light of a bottom - flavoured dark matter model ( \documentclass[12pt]{minimal }
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\begin{document}$$\phi $ $ \end{document} , mediates the interactions between dm and quarks as shown in fig . 2 .
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1234
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angular selections
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\begin{document}$$\delta \phi ( b_{1},e_\mathrm { t}^{\mathrm { miss}})\ge 1.6$$\end{document}(b1,etmiss)1.6
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\begin{document}$$\delta \phi ( \ell , e_\mathrm { t}^{\mathrm { miss}})>0.6$$\end{document}(,etmiss)>0.6
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\begin{document}$$\delta r(\ell , j_{1})<2.75$$\end{document}r(,j1)<2.75
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\begin{document}$$\delta r(\ell , b)<3.0$$\end{document}r(,b)<3.0
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\begin{document}$$r>$$\end{document}r>0.75
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\begin{document}$$>{190}$$\end{document}>190 gev
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\begin{document}$$>{130}$$\end{document}>130 gev
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}/ \sqrt{h_\text { t}^{4j } } $ $ \end{document}etmiss / ht4j
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\begin{document}$$\sqrt{\text { gev } } $ $ \end{document}gev
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\begin{document}$$r$$\end{document}r are used to reject the abundant top quark background
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\begin{document}$$\phi $ $ \end{document}. it consists of an inner tracking detector surrounded by a superconducting solenoid , electromagnetic and hadronic calorimeters , and an external muon spectrometer incorporating large superconducting toroidal magnets .
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\begin{document}$$\sqrt{s } = 8$$\end{document}s=8 tev with stable beam conditions during the 2012 lhc run .
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\begin{document}$$k_t$$\end{document}kt clustering algorithm with a radius parameter of 0.4 . the inputs to this algorithm are three - dimensional topological clusters .
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\begin{document}$$\eta $ $ \end{document}-dependent calibration factors based on simulation and validated with extensive test - beam and collision - data studies . in the simulation , this procedure calibrates the jet energies to those of the corresponding jets constructed from stable simulated particles .
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\begin{document}$$b$$\end{document}b - tagging algorithm combines the measurement of several quantities distinguishing heavy quarks from light quarks based on their longer lifetime and heavier mass .
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0.090.290.240.18allowed non sm events
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\begin{document}$$m_\phi $ $ \end{document}m is 600 gev .
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\begin{document}$$p$$\end{document}p values , are also given .
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\begin{document}$$t\overline{t}$$\end{document}tt pair , where either both top quarks decay hadronically ( sr3 ) or one top quark decays hadronically and the other semileptonically ( sr4 ) .
sr4 was developed for a top squark search by the atlas collaboration and coincides with the
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\begin{document}$$>$$\end{document } > 100 gev .
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\begin{document}$$z$$\end{document}z bosons .
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}$$\end{document}etmissand for an example signal with the operator d1 .
other backgrounds are composed of diboson and multijet production . the expected signal for \documentclass[12pt]{minimal }
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\begin{document}$$m_*= 100/40/600$$\end{document}m=100/40/600 gev for the d1/c1/d9 operators , respectively .
events with values exceeding the range presented are included in the highest bin comparison between data and expected sm background .
a , b
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\begin{document}$$r$$\end{document}r and for an example signal with the operator d1 .
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}$$\end{document}etmissand for an example signal with the operator d1 .
other backgrounds are composed of diboson and multijet production . the expected signal for \documentclass[12pt]{minimal }
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\begin{document}$$m_*= 100/40/600$$\end{document}m=100/40/600 gev for the d1/c1/d9 operators , respectively .
events with values exceeding the range presented are included in the highest bin to enrich the sample in \documentclass[12pt]{minimal }
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}$$\end{document}etmiss triggers , and require exactly one isolated lepton ( electron or muon ) with \documentclass[12pt]{minimal }
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\begin{document}$$p_\mathrm { t}$$\end{document}pt .
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\begin{document}$$\tau $ $ \end{document} leptons in the final state .
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\begin{document}$$t\bar{t}$$\end{document}tt background .
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\begin{document}$$\epsilon $ $ \end{document} for the selections described above varies between 0.1 and 8 % depending on the signal region , operator , and specific channel considered .
sr1 and sr2 have the highest efficiencies ( \documentclass[12pt]{minimal }
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\begin{document}$${a } \times \epsilon > 2\,\%$$\end{document}a>2% ) for the d9 operator , while sr3 and sr4 are most efficient for the d1 and c1 operators ( \documentclass[12pt]{minimal }
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\begin{document}$${a } \times \epsilon > 1\,\%$$\end{document}a>1% ) .
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\begin{document}$$z\rightarrow \nu \overline{\nu } $ $ \end{document}z events produced in conjunction with one or more jets .
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\begin{document}$$\gamma + $ $ \end{document}+jets control region substantially increases the number of events at large missing transverse momentum .
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\begin{document}$$z\rightarrow \nu \bar{\nu } $ $ \end{document}z background .
corrections to compensate for the differences in efficiency and acceptance between the \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document}b - tagged jets before applying any requirements on the missing transverse momentum .
a muon control region is chosen because the energy loss of muons in the detector is comparatively small .
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\begin{document}$$10$$\end{document}10 % , mainly from the flavour composition of background processes , kinematic differences between the control and signal regions and relative normalizations of backgrounds .
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\begin{document}$$z$$\end{document}z is also a substantial source of background for sr1 and sr2 when the resulting charged leptons fail to be identified or if the \documentclass[12pt]{minimal }
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\begin{document}$$w(\ell \nu ) + $ $ \end{document}w()+jets mc samples generated using alpgen2.3 with the cteq6l1 parton distribution function ( pdf ) set .
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\begin{document}$$b$$\end{document}b ) per event .
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\begin{document}$$w+b$$\end{document}w+b production is highly suppressed and therefore negligible .
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\begin{document}$$w(\ell \nu ) + $ $ \end{document}w()+jets events is selected by adding a lepton requirement to the selection and is used to validate the estimate of this background . the purity of \documentclass[12pt]{minimal }
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\begin{document}$$w(\ell \nu ) + $ $ \end{document}w()+jets in the control region for sr1 ( sr2 ) is 67 % ( 47 % ) .
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\begin{document}$$w(\ell \nu ) $ $ \end{document}w()+jets background is approximately 39 % ( 38 % ) for sr1 and 52 % ( 37 % ) for sr2 . the systematic uncertainty on
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\begin{document}$$t\bar{t}$$\end{document}tt is estimated using mc samples and validated in data control regions before applying signal selection requirements .
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\begin{document}$$t\bar{t}$$\end{document}tt process is selected with very high purity by requiring events with one lepton and large jet multiplicities.fig .
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\begin{document}$$m_*$$\end{document}m at 90 % cl for the sr1 ( red ) , sr2 ( black ) , sr3 ( green ) , and sr4 ( blue ) as a function of \documentclass[12pt]{minimal }
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\begin{document}$$m_{\chi } $ $ \end{document}m for the operators a d1 , b c1 , and c d9 .
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\begin{document}$$m_{\chi } $ $ \end{document}m for the operators a d1 , b c1 , and c d9 .
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\begin{document}$$g_q g_\chi < 4\pi $ $ \end{document}gqg<4 , the dashed lines and hollow makers represent the full collider constraints the dominant source of background for sr3 and sr4 is \documentclass[12pt]{minimal }
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\begin{document}$$t\bar{t}$$\end{document}tt events .
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\begin{document}$$t\bar{t}$$\end{document}tt events with one of the two top quarks decaying semileptonically .
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}+m_\mathrm { t}>25~(30)$$\end{document}etmiss+mt>25(30 ) gev for the electron ( muon ) channel .
the uncertainties are small because the sr3 data control region uses a kinematic region similar to the signal region with the lepton veto and jet multiplicity being the main difference .
corrections to compensate for the differences in efficiency and acceptance between hadronic and semi - leptonic top decays are derived from mc samples generated using the powheg box generator interfaced with jimmy4.31 with the next - to - leading - order ( nlo ) pdf set ct10 . the systematic uncertainty on the \documentclass[12pt]{minimal }
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\begin{document}$$7\,\%$$\end{document}7%
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}/ \sqrt{h_\text { t}^{4j}}$$\end{document}etmiss / ht4j . a similar selection , but applying an inverted \documentclass[12pt]{minimal }
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\begin{document}$$w(\ell \nu ) + $ $ \end{document}w()+jets background . the uncertainty on the \documentclass[12pt]{minimal }
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\begin{document}$$t\bar{t}$$\end{document}tt background is estimated to be approximately 20 % , which is larger than the uncertainty in sr3 due to the limited statistics .
these uncertainties are evaluated by varying the renormalisation and factorisation scale of the simulations , comparing alternative pdf sets , and studying the effects of different shower generators and of isr and final - state radiation .
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\begin{document}$$t\bar{t}+z / w$$\end{document}tt+z / w , and diboson production , are estimated in all signal regions using simulations and nlo cross sections [ 54 , 55 ] .
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\begin{document}$$wt$$\end{document}wt background is generated using the powheg generator .
the single - top t - channel is generated with acermc3.8 interfaced with pythia6 . associated production of \documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}z ) are generated with madgraph5 with up to two additional partons interfaced with pythia6 .
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\begin{document}$$z$$\end{document}z ) boson are determined using the mstw2008 nlo ( cteq6.6 m ) pdf sets .
the diboson samples are generated using herwig6.520 [ 58 , 59 ] and jimmy4.31 with the cteq6l1 pdf set .
the multijet background is estimated using data - driven methods and is found to be negligible in all signal regions after full selection .
object reconstruction efficiencies in simulated events are corrected to reproduce the performance measured in data .
the systematic uncertainty of the background estimates derived from simulation combines the uncertainties on the efficiency of the \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document}b - tagging algorithm , the uncertainties on the determination of the energy scale and resolution of the jet energy and \documentclass[12pt]{minimal }
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}$$\end{document}etmiss , the theoretical uncertainty on the various cross - sections , changes in the shapes of distributions used to extrapolate event counts from control regions to the signal region , data driven corrections and the pdf uncertainties .
overall , the systematic uncertainty on the background estimated from simulation is calculated to be between 12 and 18 % , depending on the signal region .
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\begin{document}$$b$$\end{document}b - fdm employs the madgraph5 generator interfaced with pythia6 using the cteq6l1 pdf .
samples are generated for operators d1 , c1 , and d9 , assuming \documentclass[12pt]{minimal }
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\begin{document}$$m_{\chi } $ $ \end{document}m between 10 and 1300 gev .
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\begin{document}$$m_{\phi } $ $ \end{document}m , between 5 and 3000 gev .
the instrumental uncertainties on the simulated signal yields for d1 , c1 , and d9 operators are between 11 and 15 % , depending on the signal region .
the equivalent uncertainties for the \documentclass[12pt]{minimal }
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\begin{document}$$m_{\chi } $ $ \end{document}m and the mediator mass .
the uncertainties from the pdf are computed by comparing the rates obtained with the default pdf set ( cteq6l1 ) with those obtained with two alternative sets ( mstw2008lo and nnpdf21lo [ 61 , 62 ] ) .
the uncertainties on the signal acceptance from pdf and scale variations are estimated to be approximately 10 % for the d1 , c1 , and d9 operators for \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document}b - fdm models .
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\begin{document}$$m$$\end{document}m of the heavy mediator .
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\begin{document}$$g_q g_\chi < 4\pi $ $ \end{document}gqg<4 this leads to the following validity requirements on mc truth level : \documentclass[12pt]{minimal }
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\begin{document}$$q_{\mathrm tr}<(4 \pi ) ^2 m_*^2 / m_q$$\end{document}qtr<(4)2m2/mq ( c1 ) .
table 2 shows the expected background from various sources in the four signal regions as well as the observed yields in data . the expected signal yields for the operators d1 , c1 , and d9 , as well as for the \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}p values , for the signal regions sr1 , sr2 , sr3 , and sr4 are 0.09 , 0.29 , 0.24 , and 0.18 , respectively . as no significant excess
is observed , limits on the signal yield are set using a profile likelihood ratio test following the \documentclass[12pt]{minimal }
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\begin{document}$$cl_s$$\end{document}cls prescription .
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\begin{document}$$m_\phi = 600$$\end{document}m=600 gev .
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\begin{document}$$m _ * = 1$$\end{document}m=1 tev until it is equal to the observed upper limit on beyond - the - sm events .
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\begin{document}$$m_\chi = 10$$\end{document}m=10 gev is 38 ( 221 ) fb .
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\begin{document}$$e_\mathrm { t}^{\mathrm { miss}}$$\end{document}etmiss distributions for ( a ) sr1 , ( b ) sr2 , and ( d ) sr4 and ( c ) the \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document}r variable for sr3.fig .
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\begin{document}$$\sigma _ { \chi -\hbox { n}}^\mathrm{si}$$\end{document}-nsi ) for the scalar operator d1 ( red ) as a function of \documentclass[12pt]{minimal }
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\begin{document}$$m_{\chi } $ $ \end{document}m. the yellow and green curves represent the exclusion limits recently set by the lux and super - cdms collaborations [ 6 , 7 , 65 ] . the coupling is assumed to be \documentclass[12pt]{minimal }
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\begin{document}$$m_{\chi } $ $ \end{document}m. the yellow and green curves represent the exclusion limits recently set by the coupp and picasso collaborations [ 8 , 9 , 65 ] .
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\begin{document}$$b$$\end{document}b - fdm model from combined results of sr1 and sr2 .
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\begin{document}$$\pm 1~\sigma $ $ \end{document}1 uncertainty .
the observed limit , largely dominated by sr1 , is given by the solid red line .
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\begin{document}$$m_{\chi } $ $ \end{document}m. the yellow and green curves represent the exclusion limits recently set by the lux and super - cdms collaborations [ 6 , 7 , 65 ] .
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\begin{document}$$m_{\chi } $ $ \end{document}m. the yellow and green curves represent the exclusion limits recently set by the coupp and picasso collaborations [ 8 , 9 , 65 ] .
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\begin{document}$$\pm 1~\sigma $ $ \end{document}1 uncertainty .
the observed limit , largely dominated by sr1 , is given by the solid red line .
the region beneath the curve indicating the observed limit is excluded figure 4 shows the 90 % cl exclusion curves for the effective mass scale \documentclass[12pt]{minimal }
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\begin{document}$$m_{\chi } $ $ \end{document}m. the results for the operators d1 , c1 , and d9 are presented individually for all four signal regions .
the best limits on the d1 and c1 operators are obtained using sr4 , while sr1 provides the best limits on the d9 operator , as shown in fig . 4 . these limits are then converted into limits on the \documentclass[12pt]{minimal }
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figures 5 and 6 show the corresponding 90 % cl exclusion curves for the spin - independent and spin - dependent \documentclass[12pt]{minimal }
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\begin{document}$$m_{\chi } $ $ \end{document}m for the strongest results obtained in any signal region .
the most stringent limits set by direct detection experiments [ 69 ] are also shown . only \documentclass[12pt]{minimal }
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\begin{document}$$90\,\%$$\end{document}90% of the events fulfill the effective field theory validity constraints are shown in figs . 5 and 6 . the limits shown are especially strong in the low - mass region where several collaborations [ 28 , 6668 ] have recently claimed possible observations of dm .
the results reported in this article represent the first atlas limits on the scalar operator c1 and they significantly improve the sensitivity to \documentclass[12pt]{minimal }
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\begin{document}$$\chi $ $ \end{document}nucleon interactions mediated by the scalar operator d1 compared to previous atlas results [ 14 , 16 , 18 , 19 ] .
figure 7 shows the exclusion curves observed and expected for the \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document}b - fdm model as a function of the mediator and dm masses .
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\begin{document}$$m_\phi $ $ \end{document}m ) , the signal region with the best expected sensitivity is used , with sr1 dominating over the other signal regions . for a dm particle of approximately 35 gev , as suggested by the interpretation of data recorded by the fermi - lat collaboration , mediator masses between approximately 300 and 500 gev are excluded at 95 % cl .
in summary , this article reports a search for dark - matter pair production in association with bottom or top quarks .
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we recently reported that concentrations of circulating tumor necrosis factor receptors 1 and 2 ( tnfr1 and tnfr2 ) are strong predictors of future progression to chronic kidney disease ( ckd ) 3 in patients with type 1 diabetes and microalbuminuria ( 1 ) .
these biomarkers also predict the onset of end - stage renal disease ( esrd ) in patients with type 2 diabetes ( 2 ) .
their effects are equivalent and are independent of traditional clinical characteristics measured at the beginning of follow - up such as estimated glomerular filtration rate ( egfr ) , urinary albumin / creatinine ratio ( acr ) , and glycated hemoglobin a1c ( hba1c ) . here
we seek to examine the association of the concentration of circulating tnfr2 with risk of esrd in a different study group , namely patients with type 1 diabetes and persistent proteinuria , part of a previously described joslin proteinuria cohort ( 3 ) .
in contrast to the previous studies where threshold - based outcomes such as ckd 3 or esrd were used , in this study we used rate of renal decline as the quantitative measure of intensity of disease process leading to esrd ( 4 ) . steeper rate of renal decline results in shorter time to onset of esrd .
this quantitative approach helps us to overcome problems associated with variable renal function at entry and variable duration of follow - up in our cohort .
esrd develops in 40% of type 1 diabetic patients with proteinuria after 15 years of follow - up ( 3 )
. as we recently demonstrated , the process of renal function loss leading to esrd is approximately linear and can be expressed as a constant rate of renal decline , or egfr slope ( 4 ) .
thus , if the slope is known , we can estimate the time to esrd , conditionally on the level of renal function at the beginning of the follow - up ( 4 ) . in this study of the association between circulating tnfr2 and the rate of renal decline , we used serial creatinine based egfr obtained during follow - up together with information about time of onset of esrd .
these two types of information ( longitudinal egfr data and time to esrd ) were combined in a joint longitudinal - survival model to estimate the rate of renal decline ( egfr loss ) and time to esrd , taking into account variable egfr at entry , variable duration of follow - up , and variable number of egfr estimates during follow - up ( 58 ) .
the study group has been described previously ( 3 ) . in brief , the joslin proteinuria cohort ( n = 423 ) was ascertained between 1991 and 2004 in the population of adult type 1 diabetic patients receiving long - term care at the joslin clinic , boston , ma ( 3,500 adult patients ) ( 3 ) .
all patients were caucasian with urinary albumin excretion within the proteinuria range in at least two out of the three consecutive determinations of the acr performed at the joslin clinic laboratory during a 2-year interval preceding enrollment into the study ( 3,4 ) .
informed consent procedures and protocols for examinations were approved by the joslin institutional review board , as were methods for ascertaining dates of onset of esrd and death .
descriptions of baseline and follow - up examinations , definitions of esrd , time of its onset , and natural history of esrd in this cohort have been reported previously ( 3,4 ) .
out of 423 patients enrolled in the joslin proteinuria cohort , 349 patients were in ckd stage 13 at enrollment .
in addition to collecting and storing baseline and follow - up serum samples , we retrieved 4,097 serum creatinine measurements performed in the joslin clinic laboratory for routine visits during follow - up . throughout the period of study , they were assayed by jaffe s modified picrate method on a ciba corning express plus chemistry analyzer ( medfield , ma ) . for 1,113 of these measurements ( 27% ) , serum obtained from the same blood draw as the sample submitted for assay in the joslin clinic laboratory was also submitted to the advanced research and diagnostic laboratory at the university of minnesota , where creatinine was measured with the roche enzymatic assay ( product 11775685 ) q8 on a roche / hitache mod p analyzer ( indianapolis , in ) , calibrated to be traceable to an isotope dilution mass spectrometry reference assay .
these duplicate measurements were used to develop a conversion formula to calibrate the other joslin clinic laboratory results to the isotope dilution mass spectrometry traceable method , as previously described ( 4 ) .
egfr was estimated using calibrated serum creatinine measurements and the ckd - epi formula ( 9 ) .
biomarkers were measured in serum samples collected at the enrollment into the study and stored at 80c .
tnfr2 was measured with an elisa assay ( catalog no . drt200 ; r&d systems , minneapolis , mn ) as described earlier ( 2 ) . in our previous study
because serum concentrations of tnfr1 and tnfr2 are highly correlated and provide redundant information regarding prediction of ckd 3 or esrd , only the latter was measured in the current study .
urinary albumin concentration was measured at the joslin clinic laboratory by immunonephelometry on a bn100 instrument with n albumin kits ( behring , somerville , nj ) and urinary creatinine concentration with jaffe 's modified picrate method on a ciba corning express plus chemistry analyzer , as previously described ( 4 ) .
hba1c was measured at the joslin clinic laboratory in the 1990s with bio - rad hplc analyzer ( bio - rad laboratories , hercules , ca ) and in 20012004 with tosoh 2 + 2 hplc analyzer ( tosoh bioscience , south san francisco , ca ) .
both methods were standardized to diabetes control and complications trial ( dcct ) and were consistent throughout the study period .
first , we studied the association of serum tnfr2 with traditional clinical risk factors and egfr at study entry .
we also estimated cumulative incidence of esrd during follow - up in quartiles of tnfr2 and tested differences between quartiles with a log - rank test .
for descriptive purpose , we estimated renal function change as regression slope fitted to each patient s longitudinal egfr data and expressed it in ml / min/1.73 m / year .
second , for measuring the strength of a covariate s association with renal function changes during follow - up , we applied a joint longitudinal - survival model ( described in detail in the supplementary data ) . in brief , this model overcomes the bias that would be present in overall estimates of the slopes in the cohort due to the truncated follow - up observations from patients with rapid egfr loss ( 11 ) .
it uses both the longitudinal egfr data and observed times to esrd for the whole study group to obtain unbiased estimates of individual rates of renal decline ( egfr loss ) ( 58,11 ) .
this is a novel approach to estimate the impact of a covariate ( such as tnfr2 ) on the rate of egfr loss , the most important manifestation of the underlying disease process leading to esrd .
the approach also imputes a value of egfr at zero follow - up time ( intercept ) and a time to esrd ( whether observed or not ) .
the method is an extension of mixed - effects models and involves two levels of data .
first , longitudinal egfr data and observed times of onset of esrd serve to estimate patient - specific regression parameters . on the second level ,
statistical inferences are drawn about the magnitudes of the covariate associations with the rate of egfr loss . to minimize confusion between the least squares and joint model parameters , we refer to the least squares regression estimate as a slope of egfr and joint model estimate as a rate of renal decline . similarly , we refer to the observed egfr at entry to the study as
entry egfr and refer to the joint model intercept as imputed baseline egfr .
to assess performance of tnfr2 and other covariates in predicting time to esrd , we calculated c - index ( 12 ) using a score derived from the log times to event imputed by the joint model ( x ) with a sas macro developed in the mayo clinic ( 13 ) . to test interactions between tnfr2 and another risk factor , we used the joint model to compare the association of tnfr2 with renal outcomes across strata formed by the quartiles of the interacting variable using a likelihood ratio test .
analyses were performed in sas for windows , version 9.3 ( sas institute , cary , nc ) .
the study group has been described previously ( 3 ) . in brief , the joslin proteinuria cohort ( n = 423 ) was ascertained between 1991 and 2004 in the population of adult type 1 diabetic patients receiving long - term care at the joslin clinic , boston , ma ( 3,500 adult patients ) ( 3 ) .
all patients were caucasian with urinary albumin excretion within the proteinuria range in at least two out of the three consecutive determinations of the acr performed at the joslin clinic laboratory during a 2-year interval preceding enrollment into the study ( 3,4 ) .
informed consent procedures and protocols for examinations were approved by the joslin institutional review board , as were methods for ascertaining dates of onset of esrd and death .
descriptions of baseline and follow - up examinations , definitions of esrd , time of its onset , and natural history of esrd in this cohort have been reported previously ( 3,4 ) .
out of 423 patients enrolled in the joslin proteinuria cohort , 349 patients were in ckd stage 13 at enrollment .
in addition to collecting and storing baseline and follow - up serum samples , we retrieved 4,097 serum creatinine measurements performed in the joslin clinic laboratory for routine visits during follow - up . throughout the period of study , they were assayed by jaffe s modified picrate method on a ciba corning express plus chemistry analyzer ( medfield , ma ) . for 1,113 of these measurements ( 27% ) , serum obtained from the same blood draw as the sample submitted for assay in the joslin clinic laboratory was also submitted to the advanced research and diagnostic laboratory at the university of minnesota , where creatinine was measured with the roche enzymatic assay ( product 11775685 ) q8 on a roche / hitache mod p analyzer ( indianapolis , in ) , calibrated to be traceable to an isotope dilution mass spectrometry reference assay .
these duplicate measurements were used to develop a conversion formula to calibrate the other joslin clinic laboratory results to the isotope dilution mass spectrometry traceable method , as previously described ( 4 ) .
egfr was estimated using calibrated serum creatinine measurements and the ckd - epi formula ( 9 ) .
biomarkers were measured in serum samples collected at the enrollment into the study and stored at 80c .
tnfr2 was measured with an elisa assay ( catalog no . drt200 ; r&d systems , minneapolis , mn ) as described earlier ( 2 ) . in our previous study , we found that storage time did not affect tnfr2 concentration ( 10 ) .
because serum concentrations of tnfr1 and tnfr2 are highly correlated and provide redundant information regarding prediction of ckd 3 or esrd , only the latter was measured in the current study .
urinary albumin concentration was measured at the joslin clinic laboratory by immunonephelometry on a bn100 instrument with n albumin kits ( behring , somerville , nj ) and urinary creatinine concentration with jaffe 's modified picrate method on a ciba corning express plus chemistry analyzer , as previously described ( 4 ) .
hba1c was measured at the joslin clinic laboratory in the 1990s with bio - rad hplc analyzer ( bio - rad laboratories , hercules , ca ) and in 20012004 with tosoh 2 + 2 hplc analyzer ( tosoh bioscience , south san francisco , ca ) .
both methods were standardized to diabetes control and complications trial ( dcct ) and were consistent throughout the study period .
first , we studied the association of serum tnfr2 with traditional clinical risk factors and egfr at study entry .
we also estimated cumulative incidence of esrd during follow - up in quartiles of tnfr2 and tested differences between quartiles with a log - rank test .
for descriptive purpose , we estimated renal function change as regression slope fitted to each patient s longitudinal egfr data and expressed it in ml / min/1.73 m / year .
second , for measuring the strength of a covariate s association with renal function changes during follow - up , we applied a joint longitudinal - survival model ( described in detail in the supplementary data ) . in brief , this model overcomes the bias that would be present in overall estimates of the slopes in the cohort due to the truncated follow - up observations from patients with rapid egfr loss ( 11 ) .
it uses both the longitudinal egfr data and observed times to esrd for the whole study group to obtain unbiased estimates of individual rates of renal decline ( egfr loss ) ( 58,11 ) .
this is a novel approach to estimate the impact of a covariate ( such as tnfr2 ) on the rate of egfr loss , the most important manifestation of the underlying disease process leading to esrd .
the approach also imputes a value of egfr at zero follow - up time ( intercept ) and a time to esrd ( whether observed or not ) .
the method is an extension of mixed - effects models and involves two levels of data .
first , longitudinal egfr data and observed times of onset of esrd serve to estimate patient - specific regression parameters . on the second level ,
statistical inferences are drawn about the magnitudes of the covariate associations with the rate of egfr loss . to minimize confusion between the least squares and joint model parameters
, we refer to the least squares regression estimate as a slope of egfr and joint model estimate as a rate of renal decline .
entry egfr and refer to the joint model intercept as imputed baseline egfr .
to assess performance of tnfr2 and other covariates in predicting time to esrd , we calculated c - index ( 12 ) using a score derived from the log times to event imputed by the joint model ( x ) with a sas macro developed in the mayo clinic ( 13 ) . to test interactions between tnfr2 and another risk factor , we used the joint model to compare the association of tnfr2 with renal outcomes across strata formed by the quartiles of the interacting variable using a likelihood ratio test .
analyses were performed in sas for windows , version 9.3 ( sas institute , cary , nc ) .
this study includes 349 patients with proteinuria and ckd stage 13 who were enrolled into the joslin proteinuria cohort ( 3 ) .
median age was 38 years , diabetes duration 24 years , and bmi 25 kg / m . by design ,
their urinary albumin excretion was within the proteinuric range , and median entry egfr was 81 ml / min/1.73 m ( 55 , 104 ) .
systolic and diastolic blood pressures were close to their therapeutic target ( medians 130 and 78 mmhg , respectively ) .
ace inhibitors ( ace - i ) or angiotensin ii receptor blockers ( arb ) were already prescribed for 69% of them .
this proportion is somewhat lower than is currently the case but reflects the fact that use of these medications was not widespread in the early 1990s when a large proportion of the study cohort entered follow - up .
glycemic control was predominantly poor ; median hba1c was 8.9% ( 74 mmol / mol ) .
the median of serum concentration of tnfr2 ( 25th and 75th percentiles ) was 4,415 pg / ml ( 3,497 , 5,777 ) at entry .
clinical characteristics of the 349 patients in the joslin proteinuria cohort in ckd stages 13 at the study entry data are median ( 25th , 75th percentile ) or percent ( n ) unless otherwise indicated .
* data are incidence rate per 100 person - years ( number of events ) .
renal function changes during 518 years of follow - up were evaluated with the 4,097 creatinine determinations ( median seven per patient ) and assigned egfr = 10 ml / min/1.73 m at onset of esrd in 111 patients .
using least square regression mean ( sd ) , egfr slope was 5.9 ( 8.2 ) ml / min/1.73 m / year . using the joint modeling ,
mean rate of renal decline was 5.2 ( 4.9 ) ml / min/1.73 m / year .
the incidence rate of esrd was 3.9 per 100 person - years , and mortality rate unrelated to esrd was 0.9 per 100 person - years .
neither hba1c nor blood pressure varied with increasing concentration of tnfr2 ; spearman correlation coefficients were 0.01 ( p = 0.87 ) and 0.10 ( p = 0.06 ) , respectively .
the tnfr2 concentration was significantly ( p = 0.006 ) lower in the patients without renoprotective treatment ( medians : 3,883 vs. 4,574 pg / ml ) for reasons that are obscure because their physicians had no knowledge of their low tnfr2 .
acr at entry increased ( r = 0.36 , p < 0.001 ) whereas entry egfr decreased ( r = 0.70 , p < 0.001 ) with increasing concentration of tnfr2 .
1 . after 12 years of follow - up , the risk was 14 , 30 , 35 , and 88% in the first through fourth quartiles , respectively ( p < 0.001 ) .
subsequently , we used a joint longitudinal - survival model to examine the associations of clinical covariates at entry with the rate of egfr loss and with the imputed baseline egfr and time to esrd ( table 2 ) . in this analysis ,
serum tnfr2 was transformed in percentile ranks and scaled to one quartile units , as in our previous publications ( 1,2 ) , and acr was log transformed , such that 1 unit change corresponds to doubling of its value .
estimates from univariate ( top ) and multivariate ( bottom ) joint models of the effects of tnfr2 and clinical characteristics on three components of renal decline : rate of egfr loss ( expressed in ml / min/1.73 m / year ) , egfr at baseline ( ml / min/1.73 m ) , and time to esrd ( percent change of time to esrd ) data are point estimates ( 95% cis ) .
the primary outcome in the study , the rate of egfr loss , is in boldface . in univariate analysis , serum concentration of tnfr2 had a strong impact on the rate of renal decline . a one quartile increase in tnfr2
was associated with a 1.3 ml / min/1.73 m / year steeper rate of egfr loss and a 17.4 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) .
together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 38.4% ( p < 0.001 ) .
doubling of acr was associated with a 1.7 ml / min/1.73 m / year steeper rate of egfr loss and a 6.9 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) .
together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 29.0% ( p < 0.001 ) . a 1% ( 11 mmol / mol ) increase in hba1c
was associated with a 1.0 ml / min/1.73 m / year steeper rate of egfr loss ( p < 0.001 ) but was not associated with imputed baseline egfr .
the result was a 10.4% shorter imputed time to esrd ( p < 0.001 ) .
a counterintuitive effect of diabetes duration was observed on egfr loss . a 10-year increase in duration of diabetes
was associated with a 0.8 ml / min/1.73 m / year ( p = 0.024 ) reduction in the rate of egfr loss , an 8.6 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 ) , and no reduction in imputed time to esrd . a 1 mmol / l increase in total serum cholesterol level
was associated with a 1.0 ml / min/1.73 m faster rate of egfr loss ( p < 0.001 ) , no change in imputed baseline egfr , and 8.1% reduction in imputed time to esrd ( p = 0.026 ) .
diastolic ( but not systolic ) blood pressure was also associated with a slightly steeper rate of egfr loss ( p = 0.014 ) but not with the imputed baseline egfr or time to esrd .
renoprotective treatment was associated with diminished rate of egfr loss ( p = 0.021 ) but with significantly lower imputed baseline egfr ( p < 0.001 ) .
other covariates measured at enrollment such as sex , age , bmi , and smoking did not have a significant impact on the rate of renal decline during follow - up ( data not shown ) .
to help understand the results of the analyses described above , we use a simplified example with tnfr2 dichotomized by its median value .
2 . it illustrates relationships between imputed baseline egfr , rate of renal decline , and imputed time to esrd .
the average rate of egfr loss in patients below median is 4.1 ml / min/1.73 m / year , and imputed baseline egfr and time to esrd are 96.4 ml / min/1.73 m and 20.2 years , respectively . in patients with tnfr2 above median , the rate of egfr loss is steeper ( 6.2 ml / min/1.73 m / year ) , their imputed baseline egfr lower ( 62.3 ml / min/1.73 m ) , and their imputed time to esrd substantially shorter ( 8.2 years ) .
the differences in the joint model trajectories for patients above versus below the median tnfr2 are indicated in the figure with thin black lines .
they are as follows : rate of egfr loss steeper by 2.1 ml / min/1.73 m / year , imputed baseline egfr lower by 34.1 ml / min/1.73 m
( p < 0.001 ) , and imputed time to esrd shortened by 59.3% , i.e. , 0.9 in log(time ) ( p
in analyses from table 2 , the predictors are modeled as continuous variables ( except renoprotection at baseline ) .
thick lines depict imputed egfr trajectories , and thin solid lines indicate a covariate s ( tnfr2 ) associations with imputed baseline egfr , rate of renal decline , and imputed time to esrd . thin interrupted line indicates ckd stage 5 .
e , imputed time of esrd . in multivariate analysis , only three covariates were significantly associated with the rate of renal decline ( see lower part of table 2 ) .
the changes of the rate of egfr loss associated with tnfr2 , acr , and hba1c were 0.8 , 1.3 , and 1.0 ml / min/1.73 m / year , respectively , and all were significant at p < 0.001 .
tnfr2 was the only one of the three that remained significantly associated with imputed baseline egfr .
the coefficient for its association remained unchanged in comparison with the univariate model ( 17.2 ml / min/1.73 m , p < 0.001 ) .
the strong association of tnfr2 with the rate of egfr loss and imputed baseline egfr produced the largest change in imputed time to esrd , a reduction by 34.6% ( p < 0.001 ) .
this was more than twice the magnitude of the associations of acr and hba1c with imputed time to esrd : reductions of 15.0 and 9.6% , respectively ( p < 0.001 for each ) .
in addition to the analyses on the determinants of the rate of renal decline described above , we compared the predictive performances of tnfr2 , acr , and hba1c for discrimination between patients who developed and did not develop esrd during 518 years of follow - up . in this analysis , we used the c - index derived from the time to esrd component of the joint model .
the highest discrimination in predicting time to esrd was provided by the serum concentration of tnfr2 ; the c - index from a single - variable model was 0.79 ( 95% ci 0.75 , 0.83 ) .
the index was 0.72 ( 0.66 , 0.77 ) for acr and 0.62 ( 0.57 , 0.68 ) for hba1c .
in the multivariate joint model presented in the lower portion of table 2 , tnfr2 , acr , and hba1c together had a c - index 0.86 ( 0.84 , 0.89 ) .
the results of this analysis were comparable with our previous findings regarding determinants of progression to esrd in type 2 diabetes ( 2 ) . to evaluate whether the association of serum tnfr2 with the rate of egfr loss depended upon the value of egfr at entry , we tested the association for heterogeneity across strata of the study group defined by ckd stage .
similarly , we tested for its heterogeneity across quartiles of other covariates ( hba1c and acr ) . the only statistically significant interaction ( p = 0.030 ) was that between tnfr2 and hba1c .
3 , where the study group was subdivided according to quartiles of tnfr2 and quartiles of hba1c . in the first quartile of hba1c , the relationship between tnfr2 and the rate egfr loss was weak ; the difference in mean rates of egfr loss between first and fourth quartiles of tnfr2 was only 1.9 ml / min/1.73 m / year .
this difference increased in three successive quartiles to 3.0 , 5.4 , and 5.4 ml / min/1.73 m / year , respectively .
expressed as linear trend across tnfr2 quartiles , the coefficients were 0.8 ( p = 0.157 ) , 1.1 ( p = 0.051 ) , 1.7 ( p = 0.001 ) , and 1.9 ( p < 0.001 ) in the first , second , third , and fourth quartiles of hba1c , respectively .
equivalent results were obtained when we examined the effect of hba1c within quartiles of tnfr2 ( reading down the columns in fig .
2 rather than across the rows ) . mean and standard error of the rate of egfr loss ( joint model ) according to quartiles of tnfr2 within quartiles of hba1c .
it is important to note that baseline tnfr2 and hba1c are independent ( spearman correlation coefficient 0.01 , p = 0.87 ) .
the first , second , and third quartiles of the distribution of tnfr2 in patients with hba1c < 8.9% or < 74 mmol / mol ( median ) were 3,542 , 4,439 , and 5,725
pg / ml , nearly the same as in patients with hba1c 8.9% or 74 mmol / mol ( 3,481 , 4,409 , and 5,887
this study includes 349 patients with proteinuria and ckd stage 13 who were enrolled into the joslin proteinuria cohort ( 3 ) .
median age was 38 years , diabetes duration 24 years , and bmi 25 kg / m . by design ,
their urinary albumin excretion was within the proteinuric range , and median entry egfr was 81 ml / min/1.73 m ( 55 , 104 ) .
systolic and diastolic blood pressures were close to their therapeutic target ( medians 130 and 78 mmhg , respectively ) .
ace inhibitors ( ace - i ) or angiotensin ii receptor blockers ( arb ) were already prescribed for 69% of them .
this proportion is somewhat lower than is currently the case but reflects the fact that use of these medications was not widespread in the early 1990s when a large proportion of the study cohort entered follow - up .
glycemic control was predominantly poor ; median hba1c was 8.9% ( 74 mmol / mol ) .
the median of serum concentration of tnfr2 ( 25th and 75th percentiles ) was 4,415 pg / ml ( 3,497 , 5,777 ) at entry .
clinical characteristics of the 349 patients in the joslin proteinuria cohort in ckd stages 13 at the study entry data are median ( 25th , 75th percentile ) or percent ( n ) unless otherwise indicated .
* data are incidence rate per 100 person - years ( number of events ) .
renal function changes during 518 years of follow - up were evaluated with the 4,097 creatinine determinations ( median seven per patient ) and assigned egfr = 10 ml / min/1.73 m at onset of esrd in 111 patients .
using least square regression mean ( sd ) , egfr slope was 5.9 ( 8.2 ) ml / min/1.73 m / year . using the joint modeling ,
mean rate of renal decline was 5.2 ( 4.9 ) ml / min/1.73 m / year .
the incidence rate of esrd was 3.9 per 100 person - years , and mortality rate unrelated to esrd was 0.9 per 100 person - years .
neither hba1c nor blood pressure varied with increasing concentration of tnfr2 ; spearman correlation coefficients were 0.01 ( p = 0.87 ) and 0.10 ( p = 0.06 ) , respectively .
the tnfr2 concentration was significantly ( p = 0.006 ) lower in the patients without renoprotective treatment ( medians : 3,883 vs. 4,574 pg / ml ) for reasons that are obscure because their physicians had no knowledge of their low tnfr2 .
acr at entry increased ( r = 0.36 , p < 0.001 ) whereas entry egfr decreased ( r = 0.70 , p < 0.001 ) with increasing concentration of tnfr2 .
, the risk was 14 , 30 , 35 , and 88% in the first through fourth quartiles , respectively ( p < 0.001 ) .
subsequently , we used a joint longitudinal - survival model to examine the associations of clinical covariates at entry with the rate of egfr loss and with the imputed baseline egfr and time to esrd ( table 2 ) . in this analysis ,
serum tnfr2 was transformed in percentile ranks and scaled to one quartile units , as in our previous publications ( 1,2 ) , and acr was log transformed , such that 1 unit change corresponds to doubling of its value .
estimates from univariate ( top ) and multivariate ( bottom ) joint models of the effects of tnfr2 and clinical characteristics on three components of renal decline : rate of egfr loss ( expressed in ml / min/1.73 m / year ) , egfr at baseline ( ml / min/1.73 m ) , and time to esrd ( percent change of time to esrd ) data are point estimates ( 95% cis ) . the primary outcome in the study , the rate of egfr loss , is in boldface . in univariate analysis ,
serum concentration of tnfr2 had a strong impact on the rate of renal decline . a one quartile increase in tnfr2
was associated with a 1.3 ml / min/1.73 m / year steeper rate of egfr loss and a 17.4 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) .
together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 38.4% ( p < 0.001 ) .
doubling of acr was associated with a 1.7 ml / min/1.73 m / year steeper rate of egfr loss and a 6.9 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 for each ) .
together , the lower baseline and steeper rate of egfr loss shortened the imputed time to esrd by 29.0% ( p
a 1% ( 11 mmol / mol ) increase in hba1c was associated with a 1.0 ml / min/1.73 m / year steeper rate of egfr loss ( p < 0.001 ) but was not associated with imputed baseline egfr .
the result was a 10.4% shorter imputed time to esrd ( p < 0.001 ) .
a counterintuitive effect of diabetes duration was observed on egfr loss . a 10-year increase in duration of diabetes
was associated with a 0.8 ml / min/1.73 m / year ( p = 0.024 ) reduction in the rate of egfr loss , an 8.6 ml / min/1.73 m lower imputed baseline egfr ( p < 0.001 ) , and no reduction in imputed time to esrd . a 1 mmol / l increase in total serum cholesterol level was associated with a 1.0 ml / min/1.73 m faster rate of egfr loss ( p < 0.001 ) , no change in imputed baseline egfr , and 8.1% reduction in imputed time to esrd ( p = 0.026 ) .
diastolic ( but not systolic ) blood pressure was also associated with a slightly steeper rate of egfr loss ( p = 0.014 ) but not with the imputed baseline egfr or time to esrd .
renoprotective treatment was associated with diminished rate of egfr loss ( p = 0.021 ) but with significantly lower imputed baseline egfr ( p < 0.001 ) .
other covariates measured at enrollment such as sex , age , bmi , and smoking did not have a significant impact on the rate of renal decline during follow - up ( data not shown ) .
to help understand the results of the analyses described above , we use a simplified example with tnfr2 dichotomized by its median value .
2 . it illustrates relationships between imputed baseline egfr , rate of renal decline , and imputed time to esrd
. the average rate of egfr loss in patients below median is 4.1 ml / min/1.73 m / year , and imputed baseline egfr and time to esrd are 96.4 ml / min/1.73 m and 20.2 years , respectively . in patients with tnfr2 above median , the rate of egfr loss is steeper ( 6.2 ml / min/1.73 m / year ) , their imputed baseline egfr lower ( 62.3 ml / min/1.73 m ) , and their imputed time to esrd substantially shorter ( 8.2 years ) .
the differences in the joint model trajectories for patients above versus below the median tnfr2 are indicated in the figure with thin black lines .
they are as follows : rate of egfr loss steeper by 2.1 ml / min/1.73 m / year , imputed baseline egfr lower by 34.1 ml / min/1.73 m
( p < 0.001 ) , and imputed time to esrd shortened by 59.3% , i.e. , 0.9 in log(time ) ( p < 0.001 ) . in analyses from table 2 ,
thick lines depict imputed egfr trajectories , and thin solid lines indicate a covariate s ( tnfr2 ) associations with imputed baseline egfr , rate of renal decline , and imputed time to esrd .
in multivariate analysis , only three covariates were significantly associated with the rate of renal decline ( see lower part of table 2 ) .
the changes of the rate of egfr loss associated with tnfr2 , acr , and hba1c were 0.8 , 1.3 , and 1.0 ml / min/1.73 m / year , respectively , and all were significant at p < 0.001 .
tnfr2 was the only one of the three that remained significantly associated with imputed baseline egfr .
the coefficient for its association remained unchanged in comparison with the univariate model ( 17.2 ml / min/1.73 m , p < 0.001 ) .
the strong association of tnfr2 with the rate of egfr loss and imputed baseline egfr produced the largest change in imputed time to esrd , a reduction by 34.6% ( p < 0.001 ) .
this was more than twice the magnitude of the associations of acr and hba1c with imputed time to esrd : reductions of 15.0 and 9.6% , respectively ( p < 0.001 for each ) .
in addition to the analyses on the determinants of the rate of renal decline described above , we compared the predictive performances of tnfr2 , acr , and hba1c for discrimination between patients who developed and did not develop esrd during 518 years of follow - up . in this analysis , we used the c - index derived from the time to esrd component of the joint model .
the highest discrimination in predicting time to esrd was provided by the serum concentration of tnfr2 ; the c - index from a single - variable model was 0.79 ( 95% ci 0.75 , 0.83 ) .
the index was 0.72 ( 0.66 , 0.77 ) for acr and 0.62 ( 0.57 , 0.68 ) for hba1c . in the multivariate joint model presented in the lower portion of table 2 , tnfr2 , acr , and hba1c together had a c - index 0.86 ( 0.84 , 0.89 ) .
the results of this analysis were comparable with our previous findings regarding determinants of progression to esrd in type 2 diabetes ( 2 ) .
to evaluate whether the association of serum tnfr2 with the rate of egfr loss depended upon the value of egfr at entry , we tested the association for heterogeneity across strata of the study group defined by ckd stage .
similarly , we tested for its heterogeneity across quartiles of other covariates ( hba1c and acr ) . the only statistically significant interaction ( p = 0.030 ) was that between tnfr2 and hba1c .
3 , where the study group was subdivided according to quartiles of tnfr2 and quartiles of hba1c . in the first quartile of hba1c , the relationship between tnfr2 and the rate egfr loss was weak ; the difference in mean rates of egfr loss between first and fourth quartiles of tnfr2 was only 1.9 ml / min/1.73 m / year .
this difference increased in three successive quartiles to 3.0 , 5.4 , and 5.4 ml / min/1.73 m / year , respectively .
expressed as linear trend across tnfr2 quartiles , the coefficients were 0.8 ( p = 0.157 ) , 1.1 ( p = 0.051 ) , 1.7 ( p = 0.001 ) , and 1.9 ( p < 0.001 ) in the first , second , third , and fourth quartiles of hba1c , respectively .
equivalent results were obtained when we examined the effect of hba1c within quartiles of tnfr2 ( reading down the columns in fig .
2 rather than across the rows ) . mean and standard error of the rate of egfr loss ( joint model ) according to quartiles of tnfr2 within quartiles of hba1c .
it is important to note that baseline tnfr2 and hba1c are independent ( spearman correlation coefficient 0.01 , p = 0.87 ) .
the first , second , and third quartiles of the distribution of tnfr2 in patients with hba1c < 8.9% or < 74 mmol / mol ( median ) were 3,542 , 4,439 , and 5,725 pg / ml , nearly the same as in patients with hba1c 8.9% or 74 mmol / mol ( 3,481 , 4,409 , and 5,887
the current study showed a strong association between a single baseline measurement of serum concentration of tnfr2 and the future rate of renal function decline in type 1 diabetic patients with proteinuria .
it replicates and expands our previous findings of a strong association of circulating tnfrs with the risk of ckd stage 3 in patients with type 1 diabetes and microalbuminuria , and risk of esrd in patients with type 2 diabetes ( 1,2 ) .
in contrast to the previous studies , this study focused on serum tnfr2 as a determinant of the rate of egfr loss , the most direct measure available of the intensity of the underlying disease process that leads to esrd . our analytical method , joint longitudinal - survival analysis , estimates the rate of egfr loss together with two other characteristics of the trajectory of renal decline during follow - up : the intercept ( imputed baseline egfr ) and the imputed time to onset of esrd . in both univariate and multivariate analyses , the serum concentration of tnfr2 is strongly associated with the rate of egfr loss , secondarily with the imputed baseline egfr ( a consequence of what damage has already accumulated ) and ultimately the imputed time to esrd .
the strength of this association is unchanged by inclusion of acr and other clinical risk factors in the multivariate model , indicating that its role is independent of them .
the mechanisms through which circulating levels of tnfrs might impact renal decline have been discussed in our previous publications ( 1,2 ) .
urinary albumin excretion ( measured as acr ) is also associated with the rate of egfr loss , and although that association is attenuated in multivariate analysis with tnfr2 , it is not fully accounted for . regarding the respective roles of serum tnfrs and urinary albumin excretion , several aspects are known . in type 1 diabetic patients , circulating tnfr2 predicts the development of proteinuria , as shown in the dcct / epidemiology of diabetes interventions and complications ( edic ) study ( 14 ) .
although that study has not yet examined its effect on renal decline , our recent study has shown that tnfr 1 and 2 are major determinants of early renal decline in type 1 diabetic patients with normoalbuminuria , as well as in those with microalbuminuria ( 15 ) and those with proteinuria ( current study ) .
thus , one may conclude that a high serum concentration of tnfrs increases both urinary albumin excretion and risk of renal decline .
regarding precedence , early renal decline is demonstrable before the onset of microalbuminuria and its progression to proteinuria ( 15 ) .
therefore , the level of urinary albumin excretion can be considered more of an intermediate or accompanying phenotype rather than an independent determinant of renal decline .
the effect of hba1c on the rate of egfr loss is strong and independent of other clinical risk factors .
the fact that , unlike tnfr2 , it has no impact on the imputed baseline egfr suggests that these two determinants play different roles in the disease process leading to esrd . one plausible hypothesis accounting for this difference is that hba1c is an environmental exposure that comes into play only at specific stages of diabetic nephropathy .
the decline itself is perhaps a consequence of a genetic susceptibility that is reflected in the serum concentration of tnfr2 .
an exposure , such as hba1c , may vary over time , and the single baseline measurement may not reflect well its prior level or that level s influence on baseline egfr .
in contrast , a constitutive susceptibility , such as tnfr2 , may be more constant and the baseline egfr will reflect its prior effect on the disease process .
for example , patients with a high serum tnfr2 will have a lower baseline egfr resulting from the renal decline that took place before the study entry . despite different associations with the imputed baseline egfr , hba1c and tnfr2 had strong and similar associations with the rate of egfr loss , and these effects were synergistic .
in patients with good and moderate glycemic control , the association of tnfr2 with egfr loss was much less than in patients with poor glycemic control . by the same token ,
the association of poor glycemic control with the rate of egfr loss was blunted in patients with low serum tnfr2 . in the remaining patients ,
i.e. , in those with poor glycemic control and high serum levels of tnfr2 , egfr loss was rapid and might lead to esrd within a short follow - up time . despite this strong finding , the biological interpretation of this synergism is not clear .
changes in clinical covariates ( such as blood pressure , treatment with renoprotective drugs , and other characteristics ) that went unrecognized during follow - up may have contributed to egfr trajectories and limit the biological interpretation of our findings .
nevertheless , this study provides an assessment that does not need qualification of the predictive ability of a one - time measurement of tnfr2 and hba1c together for identifying patients at high risk of rapid renal decline and rapid progression to esrd .
our study design resembles the typical patient visit during which endocrinologists or nephrologists try to predict the future from laboratory results without knowing what pathogenetic mechanisms might be involved or what changes might occur before the outcome ( in this case esrd ) is reached many years in the future .
tnfr2 is a strong determinant of renal decline in patients with type 1 diabetes and proteinuria . by combining this measurement with levels of hba1c
patients at highest risk should be enrolled in therapeutic programs to retard the rapid rate of renal function loss .
although such programs need to be developed , there is some evidence that in patients with proteinuria and high hba1c , significantly improved glycemic control maintained for 4 or more years can slow the rate of egfr loss and postpone the onset of esrd after a several - year lag time ( 16 ) .
an important strength of our study and its novelty is the focus on the annual rate of egfr loss as the renal outcome measure .
the joint longitudinal - survival model simultaneously uses information about temporal changes in egfr and the observed times to esrd to estimate this quantitative outcome . traditionally , observational studies and clinical trials have used threshold - based outcomes such as time to ckd stage 3 , time to a doubling of serum creatinine , or time to onset of esrd ( 17,18 ) . within the typical time horizon of a study ( several years ) , only a fraction , the highest - risk patients or those closest to the threshold , develop the outcome .
they depend on measurements of single serum creatinine values , whose assay has considerable random variation .
in addition , the precision of measurements of event times is a function of the frequency of study visits and missed appointments .
the rate of egfr loss , on the other hand , is estimated from multiple egfr observations and is less sensitive to biologic and assay variation in serum creatinine , missed appointments , and frequency of study visits .
first , we used serum creatinine based estimates of gfr , which are less accurate than direct measurements or estimates based on serum cystatin c. direct gfr measurements might increase the strength of the associations .
second , we assessed exposure to hyperglycemia by the value of hba1c at entry to the study only . similarly , as for other risk markers , we considered only their value at entry .
accounting for possible changes in these covariates before or after the start of follow - up , most likely , might have improved their strength as predictors of the rate of renal decline .
| objectivewe studied the serum concentration of tumor necrosis factor receptor 2 ( tnfr2 ) and the rate of renal decline , a measure of the intensity of the disease process leading to end - stage renal disease ( esrd).research design and methodsa cohort of 349 type 1 diabetic patients with proteinuria was followed for 518 years .
serum tnfr2 , glycated hemoglobin a1c ( hba1c ) , and other characteristics were measured at enrollment .
we used a novel analytic approach , a joint longitudinal - survival model , fitted to serial estimates of glomerular filtration rate ( egfr ) based on serum creatinine ( median seven per patient ) and time to onset of esrd ( 112 patients ) to estimate the rate of renal decline ( egfr loss).resultsat enrollment , all patients had chronic kidney disease stage 13 .
the mean ( sd ) rate of egfr loss during 518 years of follow - up was 5.2 ( 4.9 ) ml / min/1.73 m2/year .
serum tnfr2 was the strongest determinant of renal decline and esrd risk ( c - index 0.79 ) .
the rate of egfr loss became steeper with rising concentration of tnfr2 , and elevated hba1c augmented the strength of this association ( p = 0.030 for interaction ) . in patients with hba1c 10.1% ( 87 mmol / mol ) ,
the difference in the rate of egfr loss between the first and fourth quartiles of tnfr2 was 5.4 ml / min/1.73 m2/year , whereas it was only 1.9 in those with hba1c
< 7.9% ( 63 mmol / mol).conclusionscirculating tnfr2 is a major determinant of renal decline in patients with type 1 diabetes and proteinuria .
elevated hba1c magnifies its effect .
although the mechanisms of this synergism are unknown , our findings allow us to stratify patients according to risk of esrd . |
hypopharyngeal cancer comprises one - third of these cases and is usually diagnosed at an advanced stage , leading to extensive surgical resection , post - surgical loss of function of swallowing and/or speaking , and poor prognosis.1 - 3 with the initiation of the national cancer screening program , routine diagnostic endoscopy is increasing in korea .
however , the oropharyx is commonly overlooked in many cases due to patient discomfort or under - appreciation of this anatomical region by endoscopists.4 current developments in high definition endoscopy and novel imaging endoscopy , such as narrow band imaging ( nbi ) , magnifying endoscopy and the i - scan , have enabled gastrointestinal ( gi ) endoscopists to detect hypopharyngeal carcinoma at an early stage.5,6 patients with such early lesions can be treated with endoscopic resection . there have been only a few reports of successful endoscopic resection for hypopharyngeal cancer , almost exclusively from japan.1 - 5
a 55-year - old asian male was referred to our department for the management of suspicious hypopharyngeal cancer diagnosed by endoscopic biopsy at a primary medical clinic .
the patient was not present with symptoms related to hypopharyngeal disease , such as dysphagia or throat discomfort .
he was a 40-pack / year current smoker and consumed alcohol on a regular basis ( > 3 times per week ) .
he had previously undergone ivor - lewis operation for esophageal squamous cell carcinoma ( pt1n0m0 ) 1 year ago .
endoscopic examination disclosed a round slightly elevated hyperemic mucosal lesion , approximately 8 mm at the longest diameter , at the right pyriform recess ( fig .
nbi showed dilated , tortuous and irregular intrapapillary ca - pillary loops ( ipcl ) , consistent with type v ipcl pattern described by inoue et al .
1b).7 contrast enhanced neck and chest computed tomography ( ct ) scans showed no evidence of focal lesions or lymph node enlargement in the head and neck region , and positron emission tomography - ct scans were unrevealing .
based on these findings we diagnosed stage i early hypopharyngeal squamous cell carcinoma ( ct1n0m0 ) .
endoscopic resection was performed under general anesthesia . a cap fitted , forward - viewing , sterilized endoscope ( eg2001i ; pentax medical systems corp . ,
lugol 's solution was applied for demarcation of the lesion , and marking around the lesion was performed with the dual knife ( kd-650q ; olympus , tokyo , japan ) . saline solution with 0.001% epinephrine and 0.004% indigo carmine dye
was injected with a 23-gauge needle ( nm-200l-0423 ; olympus ) into the submucosa under and around the lesion for mucosal elevation .
after sufficient mucosal elevation around the lesion had been achieved , a snare was introduced into the accessory channel and suction was applied .
the lesion was ensnared and resected en bloc using the cap assisted endoscopic mucosal resection ( emr - c ) technique .
the total procedure time was 35 minutes , and no complications occurred during the procedure .
the resected specimen measured 109 mm and an erythematous elevated lesion was observed in the center of the specimen ( fig .
immunohistochemical staining and pathologic analysis was consistent with squamous cell carcinoma in situ with p53 protein overexpression in the full thickness of the epithelial layer ( fig .
six months later on routine endoscopic follow - up , a slightly depressed hyperemic lesion was observed at the opposite left pyriform sinus ( fig .
final pathologic results showed low grade dysplasia with focal p53 protein overexpression confined to the lower one - third of the epithelial layer ( fig .
diagnostic screening endoscopy is performed routinely in korea , and with it the incidence of early stage gi cancers is increasing .
although the detection of early stage hypopharyngeal carcinoma is difficult , such lesions can be identified as demarcated reddish areas on careful endoscopic examination.4,5 high definition imaging and new endoscopic imaging modalities such as nbi , magnifying endoscopy , and i - scan technology can further enhance the detection of such lesions .
muto et al.4 reported that nbi was superior to conventional endoscopy for detection of early stage oropharyngeal and hypopharyngeal cancer .
endoscopy may also play an important role for the surveillance and early detection of synchronous and/or metachronous squamous cell carcinoma of the oropharynx and esophagus in previously treated patients . as in our case
, these cancers are known to have a high risk of developing second primary malignancies ( field cancerization ) .
the reported overall incidence of second primary malignancies , which is higher in smokers and alcohol drinkers than in non - smokers and nondrinkers , ranges between 5% and 30% , with an annual incidence of 1.5% to 7% per year , even in long - term survivors .
squamous cell carcinoma of the hypopharynx and oropharynx are known to be associated to secondary primary malignancies in the esophagus , and tumors at the same location.8,9 currently in korean and japan , endoscopic resection of early gastric cancer is considered a standard therapy . however
, the indication for endoscopic resection in the case of hypopharyngeal carcinoma is unclear because experience of surgical resection for early stage hypopharyngeal carcinoma is rare .
furthermore , the muscularis mucosa , which divides the gi mucosa from the submucosa , does not exist in the hypopharynx .
some authors have suggested that carcinoma in situ or slightly invaded carcinoma may be an indication for endoscopic resection.1 - 5 however , there is limited data to support this hypothesis .
endoscopic resection of early stage hypopharyngeal cancer is less invasive , and has the potential to improve patients ' quality of life with regard to swallowing disorder , aspiration pneumonia , speech , and cosmetic defects associated with extensive resection .
however , for further expansion of endoscopic resection in cases of early stage hypopharyngeal carcinoma , multicenter studies aimed at establishing indication with regard to depth of invasion , tumor size , and frequency of complications are needed .
although further evidence is needed , the authors believe that endoscopic resection , preferably en bloc resection , is a viable option for early stage cancer or carcinoma in situ of the hypopharynx . | hypopharyngeal cancers are often diagnosed at an advanced stage and have a poor prognosis .
even when they are diagnosed at an operable stage , surgery often results in substantial morbidity and decreased patients ' quality of life .
although the endoscopic diagnosis of early hypopharyngeal cancer is difficult , recent developments in advanced imaging endoscopy have enabled easier diagnosis of these lesions .
endoscopic resection of early hypopharyngeal cancer is a potential minimally invasive treatment that can preserve the function and quality of life of patients .
reports of this procedure are limited , however .
we report a case of hypopharygeal cancer treated with endoscopic resection . |
as mentioned earlier , during the early phases of throwing the medial elbow structures are subjected to medial tensile forces .
this leads to the common overuse syndromes presenting with medial elbow pain including medial epicondylar aphophysitis , medial epicondyle avulsions , ulnar collateral ligament sprains / tears , and common wrist flexor / pronator strains .
the spectrum of these disorders have been given the general term of little leaguer s elbow .
the medial epicondyle is the last physis of the elbow to close , and both the common wrist flexors / pronators and proximal ulnar collateral ligament originate at this epiphysis .
little leaguer s elbow will present with progressive medial elbow pain , diminished throwing effectiveness , and loss of throwing velocity .
physical exam findings usually will show tenderness to palpation over the medial epicondyle , with increasing pain noted with resisted wrist flexion .
conventional radiographs are the initial imaging test of choice , which may show irregularity and fragmentation of the medial epicondyle .
comparison views of the contralateral elbow should be obtained which may show a widening of the physeal line on the affected side .
treatment is almost always conservative , with a period of rest with no throwing recommended for 46 weeks .
elbow and shoulder strengthening exercises and physical therapy are also recommended , followed by a slow interval return to throwing program .
along the same continuum of medial elbow overuse injury , frank avulsions of the medical epicondyle may occur .
the physis is the weak link in the immature skeleton explaining why this injury is much more common than ulnar collateral ligament tears in the younger patients . for
non - displaced fractures , a period of rest from throwing is always required , usually 3 months , along with protection from valgus forces and resisted wrist flexion . a short period of immobilization ( 13 weeks )
other strengthening exercises should be started , followed by a slow interval throwing program at the end of the rest period .
treatment of displaced medial epicondylar fractures tends to be more controversial , but most feel that displacement > 5 mm requires surgical fixation . minimally displaced fragments may heal with solid bone union , but sometimes healing only occurs by fibrous union formation . this may be associated with chronic pain and dysfunction in high level throwers . in our practice at andrews sports medicine and orthopaedic center
, any pitcher or overhead athlete who has even minimal displacement usually gets surgical fixation secondary to the fear that these athletes will not be able to return to full activities with conservative management .
a retrospective study is currently being conducted to better answer the question of the need to fix all minimally displaced medial epicondylar physeal fractures .
it is important to be aware of two major causes of lateral elbow pain seen in pediatric and adolescent patients .
osteochondritis dessicans of the capitellum and panner s disease both can present with lateral pain , and are differentiated by the age of presentation , etiology , and radiographic findings .
the cause of ocd of the capitellum has largely been unknown and controversial , but many feel it is related to the combination of repetitive microtrauma across the radio - capitellar joint and the tenuous blood supply of the capitellum . baseball pitchers and gymnasts seem to be most prone to these high stresses across the radio - capitellar joint . in baseball pitching ,
compression and shear forces occur at the radio - capitellar joint during the late cocking phase . in gymnasts , 60% of the force across the elbow joint when in full extension occurs at the radio - capitellar joint .
in addition to these increased forces seen in active athletes , the radial head cartilage seems to be stiffer than the cartilage of the lateral capitellum , thus creating a mechanical mismatch , further leading to capitellar injury .
the tenuous blood supply of the capitellar epiphysis is a result of the lack of humeral metaphyseal collateral circulation meaning that the capitellar epiphysis receives its entire blood supply from one or two isolated posterior vessels that must transverse the entire cartilaginous capitellum . because of this , the ability for the capitellar epiphysis to heal itself after episodes of microtrauma is compromised , possibly leading to osteonecrosis and eventually an ocd lesion .
ocd of the capitellum usually presents in the 11- to 15-year - old adolescent , and baseball pitchers and gymnasts are the most commonly affected persons .
a total of 90% of patients will complain of pain localized on the lateral aspect of the elbow , but some may have diffuse pain , or no pain at all .
other symptoms may include loss of motion ( usually extension ) , locking , or catching .
if the latter two features are present , one must consider the possibility that articular cartilage fragmentation and resulting loose body formation has occurred .
physical exam findings may reveal tenderness to palpation over the radial capitellar joint and occasionally loss of extension .
they may also have a positive active radio - capitellar compression test which is considered positive if the patient s pain is reproduced as they actively pronate and supinate their forearm with the elbow locked in full extension while applying a valgus force .
conventional radiographs are the preferred initial diagnostic test of choice when evaluating for capitellar ocd lesions .
ap views of the elbow in 45 of flexion may be the most helpful in identifying the abnormalities associated with this lesion .
mri may show earlier changes that may not yet be visible on plain radiographs , and the earliest change may be seen as low signal intensity at the capitellar surface on t1-weighted images only .
non - operative treatment is usually reserved for early lesions without mechanical symptoms or obvious loose bodies .
if the overlying cartilage is still intact , these tend to be managed best conservatively .
patients should be removed from the inciting trauma ( pitching , gymnastics ) for at least 6 months , and usually pitchers should not return to this position in the future .
surgical intervention usually is reserved for patients who fail to respond to 6 months of conservative treatment , or those who have mechanical symptoms , unstable fragments , or obvious loose bodies .
surgical options may include arthroscopic loose body removal , debridement of lesion with abrasion chondroplasty , osteochondral autologous transplantation , or rarely fragment reimplantation . despite treatment , about half of patients
panner s disease affects younger patients than those typically with ocd lesions , usually in the age group of 712 years .
it represents an avascular necrosis of the capitellum , similar to the other pediatric osteochondroses including legg
in contrast to osteochondritis dessicans , it is not associated with repetitive trauma and the capitellum typically reconstitutes with time without any associated long term sequelae .
treatment is conservative , with restriction from aggravating activities , including those involving axial loading such as throwing and gymnastics .
common causes of posterior elbow pain in the young patients include olecranon apophysitis or avulsion , olecranon bursitis , posterior olecranon impingement , or olecranon osteochondrosis .
repetitive forced extension as well as locking the elbow in full extension , which are commonly seen in the follow - through / deceleration phases of pitching , can lead to traction across the olecranon apophyis where the triceps tendon inserts , resulting in olecranon apophysitis .
frank avulsions can also occur , but they usually preceded the chronic posterior elbow pain indicative of ongoing olecranon apophysitis .
treatment for olecranon apophysitis and non - displaced physeal fractures involves rest and suspension of those overhead activities such as throwing that cause repetitive extension and forced hyperextension at the elbow .
throwing athletes typically may need 23 months of rest to decrease risk of reoccurrence . for displaced fractures , surgical fixation
olecranon bursitis can also occur in young athletes secondary to acute or chronic trauma occurring over the dorsal aspect of the elbow .
the presence of extensive erythema , tenderness , or systemic symptoms should alert the clinician to the possibility of a co - existing cellulitis .
olecranon bursitis can usually be managed conservatively with rest , ice , nsaids , compression wrappings , and even elbow pads to allow return to play .
if these methods are unsuccessful , aspiration and subsequent corticosteroid into the bursa can be considered . in rare cases
, bursectomy may need to be performed . if a throwing athlete presents with posterior elbow pain , and no pain over the olecranon apophysis or significant soft tissue swelling
is evident , one should consider the possibility of posterior olecranon impingement ( i.e. , valgus extension overload syndrome ) .
physical exam findings include a positive valgus extension overload test , in which pain is reproduced when a valgus force is applied at the elbow joint while the elbow is passively forced into maximal extension .
the impingement may be caused by medial olecranon osteophytes or intra - articular loose bodies .
symptoms of locking may indicate a foreign body , which may also be seen on radiographs .
treatment initially may consist of physical therapy aimed at improving extension range of motion , but if a loose body is present , arthroscopic removal is usually necessary .
the presence of posterior olecranon impingement is usually secondary to valgus extension overload , which should alert the clinician to the possibility of an underlying ulnar collateral ligament injury or insufficiency and thus needs thorough evaluation .
elbow fractures are relatively common in children , accounting for at least 10% of all pediatric fractures .
elbow fractures are often more likely to require reduction than some of the other common pediatric fractures , and can be associated with a higher complication rate .
however , conventional radiographs of the elbow in the skeletally immature individual can be sometimes difficult to evaluate owing to the secondary ossification centers that may be mistaken for fractures .
it is important for providers who care for these injuries be aware of the normal order of appearance of the six secondary ossification centers .
this is best accomplished by knowing the pneumonic crmtol , which is illustrated in the chart below ( table 1).table 1 ossification centerage at appearance ( years)ccapitellum1rradius3mmedial epicondyle5ttrochlea7oolecranon9llateral epicondyle11 of the elbow fractures , supracondylar fractures are the most common and important to be aware of owing to a high rate of malunion and complications such as neurovascular injury or compartment syndrome .
these are usually seen in children during the first decade of life ( most common ages 57 years ) .
the classic mechanism of injury is usually a child who falls on an outstretched upper extremity and suffers a hyperextension type injury at the elbow .
the distal fragment usually lies posterior , and this is best seen on lateral radiographs by drawing an imaginary line along the cortex of the anterior humerus and visualizing the capitellum lying posterior to this line ( in the normal elbow , the anterior humeral line should bisect the capitellum ) .
the posterior fat pad may also be present ( always an abnormal finding ) as well as an anterior displaced anterior fat pad ( sail sign ) , both of which are indicative of a joint effusion .
prompt diagnosis is critical owing to the potential complications of malunion and injury to the neurovascular structures that lie in close proximity , namely the brachial artery , median , radial , and ulnar nerves .
the quality of the brachial and radial pulses should be assessed , as well as for the presence of cool , pale distal extremities and decreased capillary refill .
sign ( radial nerve ) , tight fist covering thumb ( median nerve ) , and the ok sign by having them approximate the thumb and index finger ( anterior interosseous nerve ) .
it is also important to assess for signs of compartment syndrome , including increasing pain on passive extension of the fingers , parasthesias , pallor , pulselessness , or paralysis .
supracondylar fractures are typically designated as type i , ii , or iii depending on the amount of displacement .
type i fractures are non - displaced and may be treated conservatively in a long arm cast with elbow held in at least 90 of flexion for 34 weeks .
type ii fractures are partially displaced but have the posterior periosteal hinge intact , while type iii fractures are completely displaced with no contact between the two fragments .
both type ii and type iii fractures usually require closed reduction with percutaneous k wire insertion to hold normal position .
operative reduction is usually required if rotational deformity is noted , significant medial comminution exists , or if the reduction can not be held in 100120 of flexion .
volkmann s ischemia is a dreaded complication , which can occur if a displaced fracture is treated with a cast in hyperflexion .
the restriction that the cast provides coupled with extensive swelling can cause permanent neurovascular damage .
nursemaid s elbow , more accurately termed annular ligament displacement ( ald ) , is the most common upper extremity injury presenting to emergency departments in children less than 6 years of age .
patients usually will complain of elbow pain and parents may notice the sudden decreased movement of that arm .
ald occurs when the annular ligament is displaced in between the humeral capitellum and the radial head .
this is usually caused by an abrupt axial traction placed on the forearm while the elbow is fully extended and forearm pronated .
these injuries can usually be easily corrected by simple reduction techniques , involving either a supination / flexion sequence or a hyperpronation technique .
classically , the supination / flexion technique has been utilized by most practitioners , but studies seem to indicate that the hyperpronation technique may be more effective than supination / flexion on first attempt and may be associated with less discomfort . one study involving several pediatric emergency departments randomly assigned patients presenting with findings suggestive of ald to either the flexion / supination or hyperpronation technique .
a total of 85 patients were included in the study and 95% of those randomized to the hyperpronation group had successful reduction on first attempt compared to 77% successful reduction in the supination / flexion group .
the hyperpronation group also required fewer attempts at successful reduction than did the flexion / supination group , with overall success rate of the hyperpronation group being 97.5% compared to 86% in the supination / flexion group .
another prospective study indicated that the forced pronation technique was perceived as being less painful than the supination
when evaluating a pediatric or adolescent with elbow pain it is important to consistently take a thorough history and accurate physical exam .
if acute , serious fractures , including supracondylar fractures should be ruled out , as these usually need prompt orthopaedic referral .
mechanism of injury , or the types of activities that exacerbate the pain should also be elucidated . for the chronic , overuse types of injuries that cause elbow pain , determining what part of the throwing interval can be very helpful .
third , the location of the pain can also be of use , especially for the chronic types of elbow pain . the physical exam can further delineate the pathology , and close attention should be paid to range of motion , or lack thereof and the location of maximal tenderness .
conventional radiographs can then be used , and sometimes further imaging modalities may be necessary .
it is vitally important to know the sequence of appearance of the secondary ossification centers when evaluating these images . by following these steps in comprehensive and systemic manner , | the pediatric and adolescent elbow is subject to both acute and chronic overuse injuries .
the practitioner should develop a classification system to evaluate all such injuries , with first focusing on whether the injury represents an acute episode or rather it represents a more chronic problem .
in addition , localizing the area of pain as being either medial , lateral , or posterior can better help differentiate the diagnosis .
youth baseball pitchers and throwers are particularly at risk for overuse injuries of the elbow , most of which are related to an injury mechanism termed
valgus extension overload . the most common entity related to this
is termed little leaguer s elbow .
treatment is usually conservative , but for some injuries surgery may be required , especially for displaced medial epicondylar avulsion fractures .
other acute injuries also should be easily recognizable by the general clinician including annular ligament displacement ( nursemaid s elbow ) which represents one of the most common upper extremity injuries presenting to emergency rooms in youngsters under the age of 6 .
most studies seem to indicate a hyperpronation reduction technique may be more successful then the flexion / supination technique .
it is also important to have an awareness of some of the common elbow fractures seen in the younger patient , in particularly supracondylar fractures owing to their high propensity for complications .
when evaluating the elbow for fractures , it is necessary to have an understanding of the appearance of the ossification centers seen on the pediatric elbow . |
injury to the spleen is a recognized complication during percutaneous renal access due to the close anatomical relationship of the spleen and the left kidney .
however , transsplenic renal access is a rare complication of percutaneous nephrolithotomy and can also result in considerable morbidity , often requiring emergent splenectomy .
we present our experience with splenic injury during percutaneous nephrolithotomy managed conservatively with the use of a collagen - thrombin hemostatic sealant ( d - stat ; vascular solutions , inc . , minneapolis , mn ) after delayed removal of the nephrostomy tubes .
in select hemodynamically stable patients , nonoperative management with the adjunctive use of hemostatic sealants may be considered .
percutaneous nephrolithotomy ( pcnl ) has become the standard of care for large and complex renal calculi , demonstrating excellent stone clearance rates with acceptable morbidity in most patients .
although some reviews note overall high complication rates with pcnl , the majority of morbidity is related to percutaneous renal access and can be managed conservatively without significant sequelae .
major complications requiring intervention during pcnl occur in 0.9% to 4.7% of cases , and include bleeding , sepsis , and injury to adjacent viscera .
injuries to the lung , pleura , liver , colon , duodenum , and spleen have been reported during percutaneous access to the kidney . due to the close anatomic relationship of the left kidney and the spleen ,
iatrogenic splenic injuries are associated with renal surgery and percutaneous access ; however , splenic injuries during pcnl are uncommon .
the early diagnosis and treatment of splenic injuries can prevent associated morbidity and mortality , as missed splenic injuries are potentially fatal .
some authors advocate the management of splenic injuries during pcnl with emergent exploratory laparotomy and splenectomy .
we describe a case of iatrogenic splenic injury during pcnl , resulting from transsplenic renal access and tract dilation , managed conservatively with the use of a collagen - thrombin hemostatic sealant .
a 33-year - old woman was discovered to have a large left staghorn calculus on investigation of lower back pain with recurrent proteus urinary tract infections .
a noncontrast ct of her abdomen revealed a left staghorn calculus occupying the entire left renal pelvis and calices .
the patient elected to undergo a percutaneous nephrolithotomy and was admitted one day prior to scheduled surgery to receive intravenous antibiotics .
the lower , middle , and upper pole calices were accessed by using a 21-gauge needle , and 6-french pyeloureteral catheters were advanced to the distal ureter over glide wires .
the patient then underwent pcnl , and all 3 nephrostomy tracts were sequentially enlarged by using a 30-french by 15-cm balloon dilating catheter ( nephromax , boston scientific corporation , boston , ma ) .
the most inferior nephrostomy tract was dilated first , followed by the mid and upper pole tracts . bleeding occurred after dilating the upper nephrostomy tract , which was placed at the tenth posterior intercostal space .
after the majority of the patient 's stone burden was removed , three 22-french nephrostomy catheters and a double j ureteral stent were placed under fluoroscopic guidance .
the estimated blood loss from the procedure was 750 ml . during the first 2 postoperative days ,
the patient had continued hematuria and was anemic with a hemoglobin of 6.0 g / dl .
she was transfused a total of 6 units of packed red blood cells and otherwise remained hemodynamically stable . a noncontrast ct scan to assess any remaining stone burden revealed that the 2 upper nephrostomy tubes traversed through the spleen ( figure 1a ) . following consultation with
the general surgery service , the patient underwent continued observation as she remained hemodynamically stable and did not require any further blood products .
the patient continued on bed rest in a monitored unit , with all 3 nephrostomy tubes in situ , and her urine gradually cleared .
the 3 nephrostomy tubes were removed by interventional radiology on postoperative day 4 , at which time a collagen - thrombin hemostatic sealant , d - stat ( vascular solutions , inc . ,
a repeat ct shortly after removal of the nephrostomy tubes confirmed that no active bleeding or hematoma was present .
the patient otherwise made an uneventful recovery and was discharged home on postoperative day 6 .
she returned one month later for management of her residual stone disease within the left kidney and underwent ureterorenoscopy and laser lithotripsy .
delayed imaging at 6 months revealed successful stone clearance without perisplenic fluid collections ( figure 1b ) .
( a ) axial computed tomographic images after percutaneous nephrolithotomy to assess remaining stone burden , which demonstrated transsplenic renal access with a nephrostomy catheter after percutaneous nephrolithotomy and ( b ) at 6-month follow - up demonstrating resolution of injury without significant sequelae .
pcnl remains the treatment of choice for staghorn and complex renal calculi and is generally well tolerated with minimal morbidity . although vascular complications are reported with pcnl , injury to the spleen is rare .
due to the close anatomical relationship of the spleen to the left kidney , the risk of injury to the spleen during upper pole renal access is increased .
while splenic injuries may present acutely with hemodynamic instability , clinical signs and symptoms vary widely .
a heightened suspicion for vascular injuries or splenic trauma in the perioperative period should be considered in patients undergoing pcnl with excessive blood loss , hemodynamic instability , or severe abdominal pain . in our patient ,
splenic injury resulted from initial percutaneous access and was exacerbated after dilation of the nephrostomy tract to 30 french .
occult splenic injuries may be underreported and unrecognized , as ct imaging is not routinely used postoperatively .
we recommend ct imaging in patients with excessive perioperative bleeding , because ct has a high specificity and sensitivity to facilitate identification of splenic trauma or injury to surrounding structures .
a careful review of preoperative imaging could identify anatomical variants , such as splenomegaly or a retrorenal colon or spleen . in these complicated cases ,
the use of additional imaging modalities such as ultrasound or ct guidance during initial percutaneous renal access should be considered instead of fluoroscopy to avoid potential injury to surrounding organs .
historically , the nonoperative management of splenic injuries was associated with a high mortality rate .
however , advances in imaging and new grading systems of splenic injuries have allowed select patients to be successfully managed conservatively , thus avoiding laparotomy and splenectomy . although the incidence of splenic injury with percutaneous renal access is more common , splenic injuries during pcnl are rare and have potential for significant bleeding due to a larger dilation of the nephrostomy tract .
the morbidity of such an injury would depend on the number of attempts used to gain renal access as well as the diameter and location of the nephrostomy tract within the spleen .
we reviewed reported cases of splenic injury during pcnl and noted that a significant number of cases were managed operatively ( table 1 ) .
some authors have recommended management with emergent laparotomy and splenectomy for splenic injuries ; however , a nonoperative approach may be pursued in select hemodynamically stable patients .
these patients can be managed with bed rest , close monitoring , and the placement of large bore catheters to tamponade bleeding within the nephrostomy tracts .
most importantly , early recognition of such splenic injuries is vital to ensure adequate patient resuscitation and to prepare for possible emergent splenectomy if conservative management fails . in the present case ,
the patient was closely observed after delayed removal of the nephrostomy tubes with injection of a collagen - thrombin hemostatic sealant ( d - stat ; vascular solutions , inc . ,
these hemostatic agents have also been successfully utilized as adjuncts to reduce bleeding from splenic injuries during renal surgery , and have been shown to be safe and effective in controlling bleeding after the removal of nephrostomy catheters for tubeless pcnl .
alternatively , splenic artery angioembolization may be used as a nonoperative management of splenic bleeding in select cases .
repeat ct imaging can be performed to detect early signs of active bleeding ; however , emergent splenectomy is warranted if conservative management fails or for patients with evidence of hemodynamic instability
. literature review of splenic injuries during percutaneous nephrolithotomy exploratory laparotomy / splenectomy exploratory laparotomy and hemostasis with fibrin glue transfused 4 units prbcs .
uneventful recovery direct pressure over nephrostomy tract followed by serial monitoring for 3 days discharge home with delayed nephrostomy tube removal on postoperative day 15 delayed nephrostomy tube removal on postoperative day 12 perisplenic hematoma without further active bleeding .
the early recognition and management of splenic injuries during pcnl is paramount to prevent potential morbidity and mortality .
although patients may require emergent laparotomy with splenectomy , hemodynamically stable patients may be managed conservatively with close monitoring , delayed removal of the nephrostomy catheters , and with the adjunctive use of hemostatic sealants . | background : injury to the spleen is a recognized complication during percutaneous renal access due to the close anatomical relationship of the spleen and the left kidney .
however , transsplenic renal access is a rare complication of percutaneous nephrolithotomy and can also result in considerable morbidity , often requiring emergent splenectomy.methods:we present our experience with splenic injury during percutaneous nephrolithotomy managed conservatively with the use of a collagen - thrombin hemostatic sealant ( d - stat ; vascular solutions , inc . ,
minneapolis , mn ) after delayed removal of the nephrostomy tubes.results:the patient had an uneventful recovery and was discharged home on postoperative day 6.conclusion:in select hemodynamically stable patients , nonoperative management with the adjunctive use of hemostatic sealants may be considered . |
the most commonly used tool for diagnosis of non - functional thyroid nodules is fine - needle aspiration biopsy ( fnab ) [ 13 ] . however
, fnab still do not provide a diagnosis in 3040% of cases [ 411 ] ; consequently , non - diagnosed patients must be subjected to diagnostic surgery , which could impact in recurrent laryngeal nerve and parathyroid gland [ 1214]morbidity .
these methods include ultrasound [ 1518 ] , ultrasonographic elastography [ 1921 ] , repeated fnab , determination of mutations of braf and rearrangements of ret / ptc [ 2327 ] , and assessing genetic panels . despite these efforts ,
when it is decided not to operate on a patient based on these alternate diagnostic tools , the risk exists of not diagnosing a cancer and leaving it to evolve freely in at least 2530% of the cases . in an attempt to resolve this problem , metabolic assessment of the thyroid nodule by gammagraphy with technetium-99 m (
it was found that all thyroid cancer lesions accumulate mibi ( understood as accumulation of the net result of uptake and outflow of the radio - molecule ) , obtaining a 100% sensitivity and a negative predictive value of 100% .
this negative predictive value has been of great clinical use in ruling - out cancer .
this characteristic has been found since use of the first in vitro tests of tumor tissue , as well as in clinical studies performed on thyroid nodules around the world [ 3241 ] .
when this tool is applied to cases with a non - diagnostic fnab , 39% of the cases can be reclassified to the benign group . when there is no mibi concentration in the nodule , cancer is ruled - out in 100% of cases . however , in 61% of the cases , no definitive diagnosis can be reached .
mibi s concentration within the lesion ( due to metabolic activity ) may be found to be high , which occurs in all patients with thyroid cancer , but it may also be found to be high in some benign thyroid nodules . the same diagnostic value ( negative predictive value of 97100% [ 4246 ] ) can be achieved in thyroid nodules assessed through non - diagnostic aspiration biopsy by means of 18f - fluorodeoxyglucose positron emission tomography ( pet ) .
mibi is not retained in benign cells ; it enters into the mitochondrial matrix because it is a lipophilic cation and there is a natural negative charge in the mitochondrial matrix , but the moment at which oxidative phosphorylation occurs , mibi is washed out of the mitochondrial matrix [ 4749 ] . in cancer cells , oxidative phosphorylation and atp production
in contrast , pumping of hydrogen ions ( protons ) from the mitochondrial matrix to the intermembrane space continues due to the function of the respiratory chain , which intensifies the negative polarity in the mitochondrial matrix ( warburg effect ) ; in consequence , mibi retention in the mitochondrial matrix is higher and persistent [ 5053 ] .
it has been demonstrated that the electrical gradient difference of the mitochondrial transmembrane potential ( m ) increases from 60 mv ( normal in the mitochondrial matrix ) to between 150 and 170 mv [ 5459 ] in cancerous tissue .
this warburg effect is the reason why pet also has the above - mentioned diagnostic value , because by not producing atp the cell must consume a large amount of glucose [ 6063 ] .
accordingly , this situation could be related to differences in mitochondrial atpases between normal and neoplastic cells .
the mitochondrial atpase is characterized as having 2 main multiprotein components : f0 , immersed in the internal mitochondrial membrane and consisting of 3 types of proteins ( subunits ) called a1 , b2 and c12 ; and f1 , which is oriented towards the mitochondrial matrix and has 5 different polypeptides ( subunits ) called alpha3 , beta3 , delta , gamma , and epsilon . under normal conditions ,
the flow of protons produced by oxidative phosphorylation is pumped from the mitochondrial matrix to the mitochondrial intermembrane space .
subsequently , these protons return to the mitochondrial matrix by means of the proton channel located in subunit a1 of component f0 , inducing rotation of subunit c12 of f0 and this in turn induces rotation of subunits gamma and epsilon of component f1 .
this rotation produces changes in the binding affinity of atpase induced by adp and pi , which finally leads to production of 3 atp molecules for each 360 rotation of subunit gamma . in cancer cells
it is still not known whether any subunit of f0 or f1 presents a structural dysfunction .
however , some authors have attributed this dysfunction to component f1 ; protein beta has specifically been involved in liver cancer [ 6578 ] . based on the above
, we studied the difference in the relative expression of mrna of the atpase subunits between paired malignant lesions and healthy tissues from papillary thyroid cancer patients .
we measured alpha , beta , gamma , and epsilon of subunit f1 and that of the c12 of subunit f0 .
the objective of this work was to assess the difference in the relative mrna expression of the thyroid mitochondrial atpase subunits between benign and malignant lesions .
the protocol was approved by the investigation and ethics committee of the general hospital of mxico .
thirty - one papillary thyroid carcinoma and 35 nodular colloid goiter fresh tissues samples were managed at the thyroid clinic of the hospital general de mexico and the instituto politecnico nacional .
after a conventional thyroidectomy procedure , a 0.50.50.5 cm piece of the tumor tissue was resected , as well as a sample from distal healthy thyroid tissue . both samples were immediately snap - frozen and stored at 70c .
the remnant tissue was sent for histological analysis , which is the diagnostic criterion standard .
tissue samples ( 100 mg ) were homogenized in 1 ml of trizol ( invitrogen co. , carlsbad , ca ) using a polytron device ( kinematica inc . , bohemia , ny ) .
total rna was purified using the purelink rna mini kit ( invitrogen co. ) following the protocol provided by the manufacturer .
integrity of all rna samples was verified through 1.5% agarose gel electrophoresis and formaldehyde , according to the standard technique .
cdna synthesis was accomplished using the first strand cdna synthesis kit ( thermo - fisher scientific inc . ,
reactions were prepared in 20 l , containing 5 g of total rna and oligo-(dt)18 primers , following the manufacturer s instructions .
the obtained cdna was quantified by spectrophotometer in a nanodrop 2000 device ( thermo scientific , wilmington , de ) .
differential gene expression of the mitochondrial atpase components between tumor and healthy tissues were assessed by quantitative pcr , using real - time system stepone plus ( applied biosystems inc .
, including the following taqman assays : hs00900735_m1 ( atp5a1 ) , hs00969569_m1 ( atp5b ) , hs01101219_g1 ( atp5c1 ) , hs00829069_s1 ( atp5g1 ) , hs01086654_g1 ( atp5g2 ) , hs00266085_m1 ( atp5g3 ) , hs04194825_s1 ( atp5e ) .
all target probes were marked with fam fluorophore and covered contiguous exons ( so that the genomic dna would not be detected ) , except for the probe for gene atp5a1 . as a reference ,
reactions were prepared in 10 l containing 1 g cdna , 1x gene expression master mix , as well as the specific probes to detect the target and constitutive genes , respectively .
cycling conditions were as recommended by the manufacturer , and the relative values of expression were calculated with the delta - delta ct method .
all reactions were performed in duplicate and the reported result is the geometric mean of 2 independent assays .
relative quantification relates the pcr signal of the target transcript in the malignant nodule to that of healthy tissue from the same patient and in the colloid goiter nodule to that of healthy tissue from the same thyroid gland .
results were normalized to z - score ( z - scores are expressed in terms of standard deviations from their means ) .
the basis of our hypothesis is : atp is not generated by mitochondrial atpase of malignant cells due to a down - regulation in some of the atpase subunits studied .
therefore , odds ratio was calculated using the z - score , to quantify how strongly the down - regulation , equal or less than z=1.96 ( 95%interval confidence ) , is associated with malignant cells .
a 1-sided fisher s exact test was done , with a significance level set at p=0.05 .
thirty - one ptc patients were included in this study , with an average age of 43.7 years ( range 1878 ) ; 29 were women ( 93.5% ) , having an average age of 44.2 years .
thirty - five patients with colloid goiter were studied , with an average age of 45 years ( range 2087 ) ; 32 were women ( 91.4% ) , with an average age of 44.5 years .
thirty - one ptc patients were included in this study , with an average age of 43.7 years ( range 1878 ) ; 29 were women ( 93.5% ) , having an average age of 44.2 years .
thirty - five patients with colloid goiter were studied , with an average age of 45 years ( range 2087 ) ; 32 were women ( 91.4% ) , with an average age of 44.5 years .
eleven patients showed down - regulation of this transcript in ptc . in this group ,
twenty patients showed up - regulation in ptc ; in this group , only 1 had equal to or more than z=+1.96 .
the colloid goiter group had 12 patients with down - regulated transcript level , of which none had equal to or less than z=1.96 and 23 had up - regulation , of which none had equal to or more than z=+1.96 . in comparing down - regulation ( equal or less than z=1.96 ) ,
the 1-sided fisher s exact test result was p=0.478 . in the papillary cancer group , 7 patients had down - regulated transcript level , of which none had equal to or less than z=1.96 .
twenty - four patients had up - regulation , of which none had equal to or more than z=+1.96 . in the colloid goiter group , 3 patients had down - regulated transcript level , of which none had equal to or less than z=1.96 and 32 patients had up - regulation , of which none had equal to or more than z=+1.96 . in comparing down - regulation ( equal or less than z=1.96 ) ,
the papillary cancer group had 12 patients with down - regulated transcript level , of which none had equal or less than z=1.96 and 19 had up - regulation , of which no patients had equal to or more than z=+1.96 .
the colloid goiter group had 12 patients with down - regulated transcript level . in this group
no patients had equal to or less than z=1.96 ; 23 had up - regulation , of which no patients had equal to or more than z=+1.96 .
in comparing down - regulation ( equal to or less than z=1.96 ) , the 1-sided fisher s exact test score was p=1 .
sixteen patients with ptc showed down - regulation of this transcript , of which 6 patients had equal to or less than z=1.96 and 15 showed up - regulation , of which none had equal to or more than z=+1.96 .
the colloid goiter group had 9 patients with down - regulation of the transcript , of which none had equal to or less than z=1.96 ; 26 patients had up - regulation , of which none had equal to or more than z=+1.96 .
the or was 11.76 ( 95% confidence interval , 1.245237.98 ) in comparing down - regulation ( equal or less than z=1.96 ) and the 1-sided fisher s exact test score was p=0.045 ( figure 1 ) . in the papillary cancer group 21 patients had down - regulated transcript level , of which 5 patients had equal to or less than z=1.96 and 8 had up - regulation , of which none had equal to or more than z=+1.96 .
the colloid goiter group had 23 patients with down - regulated transcript level , of which 2 patients had equal to or less than z=1.96 and 12 up regulation , of which none had equal to or more than z=+1.96 .
or=3.28 ( 95% confidence interval , 0.5619.15 ) . in comparing down - regulation ( equal or less than z=1.96 ) , the 1-sided fisher s exact test score was p= 0.17 . in the papillary cancer group 16
patients had down - regulated transcript level , of which only 1 patient had equal to or less than z=1.96 and 15 had up - regulation , of which none had equal to or more than z=+1.96 .
the colloid goiter group had 15 patients with down - regulated transcript level , of which none had equal to or less than z=1.96 and 20 had up - regulation , of which none had equal to or more than z=+1.96 .
the or was 3.0 ( 95% confidence interval , 0.1179.49 ) . in comparing down - regulation ( equal or less than z=1.96 )
, the 1-sided fisher s exact test score was p=0.51 . in the papillary cancer group 13
patients had down - regulated transcript level , of which 4 had equal to or less than z=1.96 , and 18 patients had up - regulation , of which none had equal to or more than z=+1.96 . in the colloid goiter group 21
patients had down - regulated transcript level , of which 4 had equal to or less than z=1.96 and 14 patients had up - regulation , of which none had equal to or more than z=+1.96 .
the or was 1.88 ( 95% confidence interval , 0.379.39 ) . in comparing down - regulation ( equal or less than z=1.96 ) ,
the 1-sided fisher s exact test result was p=0.35 . according to several distance - based clustering algorithms ( figure 2 ) , at the level of genes ( y axis ) , the behavior of atp5g1 and atp5g2 was similar since they are located in the same branch .
the order of similarity was atp5e , atp5c1 , and finally atp5g2 , all of which belong to 1 major cluster .
this may represent biological coherence , as each major branch corresponds to f0 and f1 complexes , respectively .
eleven patients showed down - regulation of this transcript in ptc . in this group ,
twenty patients showed up - regulation in ptc ; in this group , only 1 had equal to or more than z=+1.96 .
the colloid goiter group had 12 patients with down - regulated transcript level , of which none had equal to or less than z=1.96 and 23 had up - regulation , of which none had equal to or more than z=+1.96 . in comparing down - regulation ( equal or less than z=1.96 ) ,
in the papillary cancer group , 7 patients had down - regulated transcript level , of which none had equal to or less than z=1.96 .
twenty - four patients had up - regulation , of which none had equal to or more than z=+1.96 . in the colloid goiter group , 3 patients had down - regulated transcript level , of which none had equal to or less than z=1.96 and 32 patients had up - regulation , of which none had equal to or more than z=+1.96 .
in comparing down - regulation ( equal or less than z=1.96 ) , the 1-sided fisher s exact test score was p=0.277 .
the papillary cancer group had 12 patients with down - regulated transcript level , of which none had equal or less than z=1.96 and 19 had up - regulation , of which no patients had equal to or more than z=+1.96 .
the colloid goiter group had 12 patients with down - regulated transcript level . in this group
no patients had equal to or less than z=1.96 ; 23 had up - regulation , of which no patients had equal to or more than z=+1.96 .
in comparing down - regulation ( equal to or less than z=1.96 ) , the 1-sided fisher s exact test score was p=1 .
sixteen patients with ptc showed down - regulation of this transcript , of which 6 patients had equal to or less than z=1.96 and 15 showed up - regulation , of which none had equal to or more than z=+1.96 .
the colloid goiter group had 9 patients with down - regulation of the transcript , of which none had equal to or less than z=1.96 ; 26 patients had up - regulation , of which none had equal to or more than z=+1.96 .
the or was 11.76 ( 95% confidence interval , 1.245237.98 ) in comparing down - regulation ( equal or less than z=1.96 ) and the 1-sided fisher s exact test score was p=0.045 ( figure 1 ) .
in the papillary cancer group 21 patients had down - regulated transcript level , of which 5 patients had equal to or less than z=1.96 and 8 had up - regulation , of which none had equal to or more than z=+1.96 .
the colloid goiter group had 23 patients with down - regulated transcript level , of which 2 patients had equal to or less than z=1.96 and 12 up regulation , of which none had equal to or more than z=+1.96 .
or=3.28 ( 95% confidence interval , 0.5619.15 ) . in comparing down - regulation ( equal or less than z=1.96 ) , the 1-sided fisher s exact test score was p= 0.17 .
in the papillary cancer group 16 patients had down - regulated transcript level , of which only 1 patient had equal to or less than z=1.96 and 15 had up - regulation , of which none had equal to or more than z=+1.96 .
the colloid goiter group had 15 patients with down - regulated transcript level , of which none had equal to or less than z=1.96 and 20 had up - regulation , of which none had equal to or more than z=+1.96 .
the or was 3.0 ( 95% confidence interval , 0.1179.49 ) . in comparing down - regulation ( equal or less than z=1.96 ) ,
in the papillary cancer group 13 patients had down - regulated transcript level , of which 4 had equal to or less than z=1.96 , and 18 patients had up - regulation , of which none had equal to or more than z=+1.96 . in the colloid goiter group 21 patients had down - regulated transcript level , of which 4 had equal to or less than z=1.96 and 14 patients had up - regulation , of which none had equal to or more than z=+1.96 .
the or was 1.88 ( 95% confidence interval , 0.379.39 ) . in comparing down - regulation ( equal or less than z=1.96 ) ,
the 1-sided fisher s exact test result was p=0.35 . according to several distance - based clustering algorithms ( figure 2 ) , at the level of genes ( y axis ) ,
the behavior of atp5g1 and atp5g2 was similar since they are located in the same branch .
the order of similarity was atp5e , atp5c1 , and finally atp5g2 , all of which belong to 1 major cluster .
this may represent biological coherence , as each major branch corresponds to f0 and f1 complexes , respectively .
it is well established that during cancer progression , alteration of the mitochondrial respiratory chain occurs . some of the changes that the mitochondria may exhibit during the development and progression of most cancer cells are the differential expression of respiratory chain subunits and of glycolytic enzymes .
it is important to remember that elevated glycolysis in cancer cells does not necessarily mean that these cells only use the glycolytic pathway for atp generation .
however , this peculiar aerobic metabolism of cancer cells , now called the seven hallmarks of the cancer phenotype , may in the future help in the diagnosis , prognosis , and treatment of the disease . under aerobic conditions ,
normal cells utilize glucose , which is converted to pyruvate and then to co2 , and water through the tricarboxylic acid cycle ( tca ) and oxidative phosphorylation in mitochondria .
when the cells have a limited supply of oxygen or have an impairment that impedes oxidation of pyruvate , this molecule then is reduced to lactate in the cytoplasm .
this is an inefficient process producing 2 moles of atp for every mole of glucose consumed .
cancer cells convert most of their glucose into lactate ( regardless of presence of oxygen ) , aiding their own natural selection . in accordance with these findings
, it has been shown that most cancer cells have mutations in the tca cycle enzymes , such as succinate dehydrogenase and fumarate hydratase , leading to metabolic changes that have already been observed at the rna level .
transcriptome changes in carcinogenesis have been widely studied and a great amount of data has been published [ 8689 ] .
however , most of these data require confirmation by means of a highly accurate method of quantitative real - time pcr .
finding a considerable differential gene expression of mitochondrial f0 and f1 atpase subunits between papillary thyroid cancer and a benign condition would be of great help because the diagnosis of malignancy in the thyroid nodule has not yet been resolved .
genes encoding alpha , beta , gamma , and epsilon of f1 , and c12 of subunit f0 , were analyzed at the rna level ( q pcr by duplicate ) to determine if abnormal expression of these genes could partially explain the warburg effect occurring in ptc .
we propose that transcriptional analysis of several atpase genes expressed by thyroid tumors should provide a means to define a signature fingerprint of molecular mechanisms underlying this type of cancer and might progressively improve conventional diagnosis parameters .
simultaneous expression levels of several genes offer better possibilities to understand and characterize the molecular mechanisms underlying ptc .
for example , in breast cancer sorlie found certain expression patterns of mrna related to tumor subclasses with clinical implications .
few alterations have been reported to be associated with ptc at the molecular level . to the best of our knowledge ,
assessment of mitochondrial rna expression of the atpase subunits from thyroid tissue has not been analyzed by qpcr .
it is very clear from the results of the present study that there is a high degree of individual variability of the atpase subunits expression for each patient ( regardless of the type of illness ) , showing a unique profile of mrna expression for each of the 6 genes measured . from all 6 genes included in the present study ,
atp5e transcript down - expression was highly associated only with ptc diagnosis or=11.76 ( 95% ci 1.245237.98 , p=0.04 ) .
we recognize the limitation of having a very wide confidence interval , which means that we must confirm this initial information with a larger sample .
this deregulation in ptc may explain why the rotation of gamma and epsilon f1 subunits stops , thus , avoiding changes in the binding affinity of the catalytic subunits for adp and pi , and finally altering the atp production .
atp5e down - regulation transcript expression was highly associated only with ptc diagnosis and not with colloid goiter . | backgroundcancer development involves an injury to the respiratory machinery ( warburg effect ) due to decreased or impaired mitochondrial function .
this circumstance results in a down regulation of some of the atpase subunits of the malignant tissue .
the objective of this work was to assess and compare the relative expression of mrna of mitochondrial atpase subunits between samples of thyroid cancer and benign nodules.material/methodssamples from 31 patients who had an operation for ptc at the general hospital of mexico were snap - frozen and stored at 70c .
thirty - five patients who had an operation for benign tumors were also included in the study .
mrna expression levels of alpha , beta , gamma , and epsilon subunits of f1 and c12 of subunit fo were determined by real - time rt - pcr ( by duplicate ) , in order to determine if abnormal expression of these genes could partially explain the warburg effect in papillary thyroid cancer ( ptc).resultsatp5e transcript alteration ( down - expression ) was highly associated to ptc diagnosis or=11.76 ( 95% confidence interval , 1.245237.98 ; p=0.04).conclusionsrelative down - expression of atp5e transcript was highly associated with ptc diagnosis .
this transcript alteration may be used as a tumoral marker in papillary thyroid cancer . |
first described in 1886 by hochstetter , left - sided gallbladder remains a rare anomaly . in a multicenter series of laparoscopic cholecystectomies completed in hungary
true left - sided gallbladder ( sinistroposition ) occurs when the gallbladder is found to the left of the falciform ligament , beneath the left lobe of the liver .
it is located between segments iii and iv or on segment iii of the liver .
the course of the cystic duct in sinistroposition has been classically described as joining the hepatic duct on the right side in a right - to - left manner , thus creating a hairpin turn anterior to the hepatic duct .
less common , the cystic duct has been noted to join the common hepatic duct or left hepatic duct from the left side .
given this variable ductal anatomy , proper identification is essential for the safe completion of cholecystectomy when this anomaly is encountered .
sinistroposition often is encountered unexpectedly in the operating room because common preoperative imaging , such as ultrasonography ( us ) , fails to recognize this variant anatomy .
initially , the safe completion of laparoscopic cholecystectomy in patients with sinistroposition was questioned and reasonably so , given its variable ductal anatomy . however , numerous case reports have documented safe laparoscopic removal of the rare left - sided gallbladder .
as expected , these reports stress the proper identification and management of sinistroposition 's ductal anatomy . if the anatomy is ever in doubt , intraoperative cholangiography is recommended .
single - incision laparoscopy is currently among the techniques at the forefront of minimally invasive surgery .
many techniques have been described , from a single skin incision with multiple ports placed through the fascia within the area under the single skin opening to use of a commercially available single port with multiple working channels .
regardless of technique , reports have shown single - incision laparoscopy to be safe and effective in cholecystectomy .
some of the proposed benefits of single - incision surgery include improved cosmesis , less postoperative pain , and the ability to convert to traditional 4-port laparoscopy . at our institution , for our single - incision laparoscopic cholecystectomies
a single incision is made at the umbilicus with insertion of two ports : one for the camera and one for working . by passing sutures through the gallbladder fundus , body , and infundibulum ,
we are able to manipulate the gallbladder in an extracorporeal manner , eliminating the need for another port site .
her diagnosis was confirmed by a right upper quadrant us , cholecystokinin - hepatobilary scan , and esophagogastroduodenoscopy .
her cholecystokinin - hepatobilary scan showed a decreased gallbladder ejection fraction of 19% and reproduction of symptoms at the time of cholecystokinin injection .
of note , none of the preoperative imaging obtained provided any indication as to a diagnosis of sinistroposition . in the operating room ,
this entails entering the abdomen by use of techniques that allow us both a single site and the traditional , multiple - port laparoscopic approach . once the laparoscope is placed in the abdomen and the abdomen is surveyed , the decision to proceed with single - site or traditional 4-port laparoscopic cholecystectomy is made .
the abdomen was insufflated to 15 mm hg at which time a laparoscope was placed through the trocar and the abdomen was inspected .
the gallbladder was normal in color , that is , the so - called robin 's egg blue .
it was encountered to the left of the falciform ligament , attached to segment iii of the liver . because the traditional port placement in the right upper quadrant would be less than optimal , the decision was made to proceed with a single - incision technique .
this was based on the idea that the flexibility obtained by the suture retraction technique would be beneficial in this situation .
a second 5-mm trocar was placed through the same skin incision , just to the left and superior to the original trocar .
a suture was then placed through the left anterior abdominal wall , into the fundus of the gallbladder , and back through the abdominal wall , essentially suspending the gallbladder from the abdominal wall .
a second suture was placed through the anterior abdominal wall ( epigastric region ) , into the body of the gallbladder in a figure - of-8 fashion , and back out the left lateral abdominal wall , thus allowing for manipulation .
a third suture was placed in the same fashion as described earlier , this time passing through the infundibulum of the gallbladder and again exiting through the left anterior abdominal wall .
this allowed us to manipulate the gallbladder in such a way as to expose the triangle of calot ( figure 1 ) .
both were dissected circumferentially , and the liver edge was exposed , allowing us to obtain a true critical view of the patient 's anatomy ( figure 2 ) .
as shown in figure 2 , we found that the ductal anatomy in our patient with sinistroposition held true because the cystic duct entered the hepatic duct from the right side .
the cystic duct and artery were then clipped and transected ( figures 3 and 4 ) .
an ultrasand dissection device scalpel was used to dissect the gallbladder off of the fossa ( figure 5 ) .
the suture through the infundibulum was securely grasped , allowing us to remove the gallbladder through the umbilical fascial defect .
one should note that the falciform ligament lies to the right of the gallbladder and the gallbladder bed is within segment iii of the liver .
one should note that the cystic duct enters the common bile duct ( cbd)/common hepatic duct from the right side .
first pioneered in the late 1980s , laparoscopic cholecystectomy has since revolutionized the surgeon 's approach to gallbladder disease . with the advancement of laparoscopic skills and
the continued high volume of gallbladder disease , laparoscopic cholecystectomy is one of the most common surgical operations in the united states .
given this fact , it is not surprising that despite its rarity , left - sided gallbladder ( sinistroposition ) is encountered at times during laparoscopic cholecystectomy .
as previously mentioned , multiple case reports have documented the safe removal of the gallbladder by means of conventional laparoscopic cholecystectomy when sinistroposition is encountered .
these reports stress the proper identification of unanticipated ductal anatomy as the key to safe completion of the operation .
in addition to the unexpected ductal anatomy , port placement and ergonomics also provide a challenge when sinistroposition is encountered .
this is often more challenging for the right - handed surgeon , as has been documented in the literature regarding cholecystectomy in situs inversus totalis .
dissection can be carried out in one of two ways . in the first manner ,
thus the right hand would provide retraction and manipulation of the gallbladder , whereas the left would be used for dissection and clipping .
alternatively , some surgeons have advocated a crossover technique in which the dominant hand ( assuming right ) is used for dissection and clipping whereas the nondominant hand ( left ) provides retraction .
although a single - incision technique does not entirely solve the difficulty of the right - handed surgeon operating on a left - sided gallbladder , it does simplify port placement and ergonomics .
we believe that the use of single - incision laparoscopic surgery ( sils ) is a safe alternative among the options for laparoscopic cholecystectomy in patients with sinistroposition .
although there are numerous methods for performing sils , the overall concept bodes well for the unexpected anatomy encountered with sinistroposition . with a single port site ,
no further ports need be placed and the troublesome ergonomics faced by the right - handed surgeon when presented with a left - sided gallbladder is significantly reduced .
however , once one is facile with this technique , the same skill set is needed for both right- and left - sided gallbladders alike .
the multichannel - port single - site technique is based on the surgeon 's ability to work in tight quarters and have significant crossover of instruments .
likewise , the suture retraction ( marionette ) technique is easily adapted to the left - sided gallbladder . in our case and with the chosen single - site technique specifically , we believe that the ability to remove and replace the sutures to optimize exposure without creating additional incisions was of true benefit . in addition , the single working port allows the surgeon to operate with his or her dominant hand .
we believe that this eliminates the difficulties of relying heavily on the nondominant hand when sinistroposition is encountered and allows for safe removal of the gallbladder .
we believe that single - incision laparoscopic cholecystectomy represents a valid and safe option for cholecystectomy when sinistroposition is encountered , granted that the operating surgeon is facile with the single - incision technique and aware of the aforementioned anatomic ductal variations . | true left - sided gallbladder ( sinistroposition ) is a rare anatomic anomaly in which the gallbladder is found to the left of the falciform ligament , under the left lobe of the liver .
though uncommon , it is important for the surgeon to recognize this finding because the ductal anatomy is unique and the mechanics of the operation provide a technical challenge .
multiple case reports have documented safe management of sinistroposition encountered during conventional laparoscopic cholecystectomy .
we present a case of sinistroposition encountered during a single - incision laparoscopic cholecystectomy .
we believe that the single - site laparoscopic technique is not only a safe option but may actually provide certain benefits in approaching this difficult anatomy in the properly selected patient and the experienced single - site surgeon 's hands . |
the temporomandibular joint ( tmj ) is a joint present above the spine . from a kinematic
standpoint ,
the alignment of the tmj directly affects the stability and alignment of the
spine , and the tmj is the only bilateral joint among all joints in the human body1 . when the position of the lower jaw is
misplaced , it is generally pushed toward the rear of the head . in this case ,
due to the
homeostasis of the human body that tends to maintain balance in any event , the center of
gravity of the head leans toward the rear of the head to make the entire head lean backward .
when the position of the tmj is changed , the axis of the center of gravity of the head is
changed , leading to misalignment of the entire spine .
the position of the tmj is closely
related with masticatory functions , and this factor is important for control of the entire
body s postures such as the functions of the cerebellum , vestibula functions , oculomotor
functions , and proprioceptive senses2 .
mouth guards are used in a wide range of sport activities in order to increase the
stability of the tmj , the importance of which has been emphasized , to protect the tmj from
external resistance , and to protect teeth and periodontal tissues .
they are also used in
patients with any stomatognathic disability that may affect exercise habits or postures or
any abnormality in occlusion in order to induce normal occlusion .
thanks to the development of science , these mouth guards have been developed into many
shapes fit for their purposes depending of their manufacturing methods and materials .
mouth
guards can be largely divided into three types based on their manufacturing methods .
stock - type mouth guards ( stm ) are ready - made mouth guard that can not be adjusted , and
boil - and bite - type mouth guards ( btm ) are those that are to be molded by the user .
finally ,
custom - made mouth guards ( ctm ) are those made from models after obtaining impressions of the
teeth of the athletes who will use them . with regard to materials , those made of soft
materials have been developed for the prevention of sport injuries , and those made of hard
materials have been developed for oral functions and alignment3 .
all oral appliances including mouth guards developed with a wide
range of methods and materials are called mandibular orthopedic repositioning appliances
( moras ) .
moras are known to affect the relief of neck stress and postures , improve respiratory
functions4 , and improve body
balance4 .
thus , moras affect the entire
body by causing changes in the stomatognathic system . however , since studies of the
relationship between muscle activation and a mora that directly affects body balance are
insufficient , basic data on the effects of a mora on the entire body are insufficient .
therefore , the purpose of this study was to measure the muscle activities of the trunk
muscles and upper limb muscles during maximum isometric contraction when tmj alignment was
achieved with a mora in order provide basic data on the effects of a mora on the entire
body .
the present study was conducted with healthy adults in their 20s ( males=10 , females=10 ) who
had not been diagnosed with any medical conditions within the last six months .
all the
subjects voluntarily participated in this study and signed an agreement for this experiment
that stated that they were able to reject participation anytime if they wanted .
an 8 ch
surface electromyography ( myosystem dts , noraxon u.s.a . inc . ,
scottsdale , ca , usa ) system
was used to measure the muscle activities of the upper limb muscles and neck muscles of the
subjects during maximum isometric contraction when a mora was used and when no mora was
used .
the myoresearch xp master edition 1.06 program was used to store and process
electromyographic signals with a sampling rate of 1,000 hz , a 20500 hz band - pass filter , a
60 hz notch filter , and iwc - dts electrodes ( 9113a - dts ) .
the sites for electrodes were shaved
with a razor , and the horny substance was removed with sandpaper .
the electrodes were
attached after cleaning the sites with an alcohol swab to ensure collection of accurate
electromyogram data .
agcl emg surface electrodes were applied with a
3 cm center - to - center spacing over the lumbar and cervical erector spinae , upper trapezius ,
biceps , triceps , rectus abdominis , internal abdominal obliques , external abdominal obliques ,
and sternocleidomastoid muscle .
the maximum isometric contraction of each muscle was
achieved in the manual muscle testing position5 . regardless of whether a mora was used or not , all the measurements
were conducted when the tmj was closed at the maximum force .
the root mean square data of each muscle was measured for five seconds in maximal voluntary
isometric contraction .
the level of muscle activation during maximum isometric contraction
was expressed as % mvic by calculating the relative muscle contraction of the 100% average
muscle contraction in the middle one second of a 3-second measurement , i.e. , by ignoring the
first and last second of the measurement .
the mora used in the experiment was made of hard - type splint 020 ( easy - vac gasket , 3a
medes , goyang , south korea ) , and ortho - jet ( fast curing orthodontic acrylic resin , lang
dental , wheeling , il , usa ) was used to set the occlusion of the hard - type mora . to obtain elaborate maxillary and mandibular casts of the study subjects ,
the subjects
impressions were obtained using alginate impression materials and plaster casts were
made .
when mandibular centric bites were obtained , each subject was instructed to bite down on
cotton rolls on both sides of his / her oral cavity and maintain the state for approximately
five minutes to eliminate the effects of teeth on the muscles and stabilize the muscles . to
eliminate the effects of gravity on the mandibular bites ,
the maxillary cast was attached to a semi - adjustable articulator using a facebow , and the
mandibular cast was attached to the semi - adjustable articulator using the obtained
mandibular centric waxbite .
a random point was marked on the attached gingiva between the right canine tooth and the
first premolar of the attached maxillary and mandibular casts on the working model , the
distance between the maxillary and mandibular casts was measured based on the marked point ,
and the incisal guide pins of the articulator were lifted so that each of them was lifted by
3 mm from their position .
the prepared primary mora was mounted on the maxillary working cast , the occlusal vertical
dimension was increased by 3 mm at the centric occlusion , and the maxillary overbite - type
primary mora was attached to the maxillary front teeth and set at the lifted occlusal
vertical dimension by covering it with paraffin wax .
then , the mora was adjusted using a
self - curing resin so that all teeth would evenly come into contact with the centric
occlusion , canine guidance would be given during lateral movements to the left and right ,
and the front teeth group would be evenly guided anteriorly during anterior movements .
although the deep fossae formed by the occlusal tables of antagonist teeth are generally not
included in moras that are widely used to treat tmj disorders , 1 mm deep fossae were
artificially formed so that all cusp tips of antagonist teeth would come into contact with
the mora evenly and no locational change would occur .
the fabricated moras were placed into the oral cavities of the study subjects and adjusted
so that all teeth would evenly come into contact with them so that the centric relation and
centric occlusion would match and the mutually protected occlusion would be precise .
the
occlusions were adjusted so that anterior movements would be guided by the front teeth group
and lateral movement would be guided by the canines .
then , the set occlusal vertical
dimension was rechecked , and the experimental moras was completed .
the subjects general characteristics were checked through percentage and frequency
analyses , and the differences in the muscle activities of individual muscles between using
mora and not - using mora , were analyzed using independent t - tests .
the maximum isometric contractions of the trunk and upper limb muscles when a mora was used
were compared with those when no mora was used .
the results showed that the
sternocleidomastoid muscle , cervical and lumbar erector spinae , upper trapezius , biceps ,
triceps , rectus abdominis , and internal oblique and external oblique muscles all showed
significant increases in maximum isometric contractions when using a mora ( table 1table 1 .
comparison of upper limb and trunk muscle activation during maximum isometric
contraction between each condition ( unit : % mvic)musclewithout a morawith a morascm*111.6 6.0125.4 15.6c - es*115.0
8.2138.1 18.0ut*114.5 7.7123.7 9.2biceps*114.7 5.1125.2 7.6triceps*111.2 4.9120.9 8.6ra*116.0 6.3128.8 6.7iao*116.3 4.2128.1 5.0eao*117.7 5.1128.1 6.0l - es*118.7 4.7130.8 6.2mean sd , p<0.05 .
scm , sternocleidomastoid muscle ; c - es , cervical erector spinae ;
iao , internal abdominal oblique ; eao , external abdominal oblique ; l - es , lumbar erector
spinae ; ra , rectus abdominis ) ( p<0.05 ) .
scm , sternocleidomastoid muscle ; c - es , cervical erector spinae ;
iao , internal abdominal oblique ; eao , external abdominal oblique ; l - es , lumbar erector
spinae ; ra , rectus abdominis
human postures are determined and maintained by the coordinated movements of muscles ,
proprioceptive senses , the sense of equilibrium , and the positions and functions of
joints6 . among joint positions ,
the
position of the tmj , which is characterized as a bilateral joint , can control the position
of the head , which is the heaviest part of the body , and thus it affects the stability and
functions of the entire body2 .
therefore , the present study was performed to examine the effects of the use of a mora that
can provide positional changes and stability to the lower joint , which in turn moves the
tmj , on the body .
the maximum isometric contractile power of the trunk and upper limb muscles when a mora was
used was compared with that when no mora was used , and the results showed significant
increases in power in all the muscles measured . given these results
, it seems like the
increase in stability of the tmj resulting from use of a mora affected the stability and
balance of the entire body so that the maximal isometric contraction of the trunk and upper
limb muscles increased7 .
muscle activation
is closely related to the stability of the spine and the entire body8 , and proper body alignment increases the muscle activation
of distal part of the body9 , 10 . according to the results of a study conducted by bates
and atkinson11 , a mora affects the tmj
and moves it into its normal position .
therefore , moras can be widely used in treating
craniomandibular disorders , and treatment with them can improve motor abilities , muscle
strength , endurance , and concentration12 , 13 .
the results of these studies indicate
that use of a mora is one method of increasing muscle activation in the entire body in
stable positions .
therefore , the use of a mora is considered to be a method for normal adults to improve the
stability of the entire body through improvement of the stability of the tmj ; that is , the
proximal improvement in stability improves not only muscle strength with increased muscle
activation but also stability during exercises . | [ purpose ] the purpose of this study was to measure the muscle activities of the trunk
muscles and upper limb muscles during maximum isometric contraction when temporomandibular
joint alignment was achieved with a mandibular orthopedic repositioning appliance in order
provide basic data on the effects of mandibular orthopedic repositioning appliance on the
entire body . [ subjects ] the present study was conducted with healthy korean adults in
their 20s ( males=10 , females=10 ) .
[ methods ] an 8 channel surface electromyography system
was used to measure the muscle activities of the upper limb muscles and neck muscles of
the subjects during maximum isometric contraction with and without use of a mandibular
orthopedic repositioning appliance .
[ results ] the maximum isometric contractions of the
trunk and upper limb muscles when mandibular orthopedic repositioning appliance were used
were compared with those when no mandibular orthopedic repositioning appliance was used .
the results showed that the sternocleidomastoid muscle , cervical and lumbar erector
spinae , upper trapezius , biceps , triceps , rectus abdominis and internal oblique and
external oblique muscles all showed significant increases in maximum isometric
contractions with a mandibular orthopedic repositioning appliance .
[ conclusion ] the use of
a mandibular orthopedic repositioning appliance is considered to be a method for normal
adults to improve the stability of the entire body with the improvement of the stability
of the tmj .
the proximal improvement in stability improves of the proximal thereby
improving not only muscle strength with increased muscle activation but also stability
during exercises . |
copd is a serious , debilitating condition that has become a major public health concern and by 2020 is projected to rank fifth in global burden of disease and third in global mortality.1 for a proportion of patients , copd is a progressive disease characterized by periodic acute exacerbations of symptoms .
these exacerbations pose an immediate threat to patients and also hasten progression of the disease .
exacerbations accelerate decline of lung function , so that patients often fail to return to baseline levels .
although symptoms may last a few days , recovery of lung function can take weeks to months , resulting in prolonged periods of functional limitation and general worsening of quality of life , often with some degree of permanent functional decline .
exacerbations are also associated with substantial risk of hospitalization and death , as well as considerable economic costs that increase with exacerbation frequency.1,2 preventing exacerbations is therefore a key goal of copd management1,35 and hence the need to predict who are likely to experience exacerbations .
the recent global initiative for chronic obstructive lung disease ( gold ) categories a d were developed to aid in assessing future risk of exacerbations and performed well in one study;6,7 however , patient assignment to categories may vary depending on the choice of symptom measure , limiting their applicability.8 indices of copd severity such as the body mass index [ bmi ] , obstruction , dyspnea , and exercise ( bode ) index and the age , dyspnea , and obstruction ( ado ) index have been used to try to predict future exacerbations among patients with copd but with only moderate ( 60%70% ) prediction success ; moreover , they require specific data ( eg , 6-minute walk test ) that may not be routinely available.9,10 the dyspnea , airflow obstruction , smoking status , and exacerbation frequency ( dose ) index has been shown to predict future exacerbations in a large primary care data set , and the index was stronger than previous exacerbation frequency or the ado or bode index.9,11,12 other researchers have developed de novo statistical models to identify independent clinical predictors . however , many of these studies included relatively small sample sizes,10,1316 patients with severe copd,14,17,18 and/or severe outcomes such as hospitalization or death.13,15,17,1922 the goal of the current study was to develop a robust , clinically based predictive model that would encompass all levels of copd severity as well as moderate or severe exacerbation severity .
such a model could help in earlier targeting of patients for review to optimize drug therapy and other interventions , with the aim of reducing hospital admissions , decline in lung function , and the morbidity and mortality associated with copd .
a secondary objective was to compare the model s predictive value in relation to existing predictive tools .
this was a historical follow - up study of patients with copd identified from the optimum patient care research database ( opcrd).23 the opcrd is a quality - controlled , longitudinal , respiratory - focused database containing anonymous data from general practices in the uk and has been approved by the trent multicentre research ethics committee for clinical research use ( approval reference 10/h0405/3 ) , and this study was approved by the anonymised data ethics protocols and transparency committee , the independent scientific advisory committee for the opcrd .
informed consent was not required or possible as we worked with anonymous data , and this was not an interventional study .
however , patients could opt out of having their data used in research . at the time of the study , the opcrd contained records of > 50,000 patients with copd from > 300 uk general practices .
the database combines routine data from electronic patient records with linked patient - reported data collected using disease - specific questionnaires .
routine clinical data , including patient demographic characteristics , comorbidities , exacerbation history , modified medical research council ( mmrc ) score,24 and current therapy , were extracted from primary care practice management systems . in addition , a proportion of patients with relevant disease codes were invited to complete validated disease assessment questionnaires , sent via a secure mailing house .
the questionnaires enabled calculation of the mmrc scores and the copd assessment test ( cat ) scores.25 the current study was divided into model - building and model - validation components .
patients were eligible for inclusion in the model - building phase if , on or before march 12 , 2013 , they had at least one recorded eosinophil count , were at least 40 years of age , had been diagnostically coded for copd , and had a forced expiratory volume in 1 second / forced vital capacity ( fev1/fvc ) ratio < 0.7 recorded within 5 years of their last eosinophil count ( defined as the index date ) . all eligible patients needed to have at least 1 year of observation before ( baseline year ) and 1 year after ( outcome year ) the index date . included patients also needed to have complete data on the candidate predictors analyzed .
those with chronic respiratory diseases other than asthma , such as bronchiectasis , were excluded .
the validation cohort consisted of patients with similar eligibility criteria identified between march 2013 and february 2014 .
potentially important variables within the opcrd were identified from a search of the literature and from expert opinion of the authors :
sociodemographic factors : sex , age , height , weight , bmi , smoking statussymptom severity : mmrc dyspnea score , cat score , number of exacerbations in the previous yearcomorbidities : asthma , eczema , allergic or nonallergic rhinitis , nasal polyps , diabetes mellitus , gastroesophageal reflux disease ( gerd ) , ischemic heart disease , heart failure , anxiety / depression , charlson comorbidity indexspirometry : fev1 ( % predicted ) , fev1/fvc ratioperipheral blood eosinophilia ( defined as 500 cells/l ) .
sociodemographic factors : sex , age , height , weight , bmi , smoking status symptom severity : mmrc dyspnea score , cat score , number of exacerbations in the previous year comorbidities : asthma , eczema , allergic or nonallergic rhinitis , nasal polyps , diabetes mellitus , gastroesophageal reflux disease ( gerd ) , ischemic heart disease , heart failure , anxiety / depression , charlson comorbidity index spirometry : fev1 ( % predicted ) , fev1/fvc ratio peripheral blood eosinophilia ( defined as 500 cells/l ) . to enhance diagnostic specificity and to be consistent with earlier research , as well as with the gold cut - point for risk of future events,7 we defined the outcome of interest as frequent ( 2 ) exacerbations .
exacerbations were defined as either 1 ) unscheduled hospital admission or accident / emergency attendance for copd or lower respiratory events , 2 ) an acute course of oral corticosteroids prescribed with evidence of respiratory review , or 3 ) antibiotics prescribed with evidence of respiratory review .
where one or more oral corticosteroid course , hospitalization , or antibiotic prescription occurred within a 2-week window , these events were considered to be the result of the same exacerbation .
prior to analysis , continuous variables were evaluated via likelihood ratio test to see if quadratic or cubic transformation improved model fit .
akaike information criteria ( aic ) were compared to test whether model fit was improved by categorical or continuous classification .
univariable logistic regressions were performed to gauge the importance of individual variables and to help define the best form ( eg , continuous , categorical ) of each variable .
however , all potentially important variables were fed into a multiple logistic regression with backward selection of the model having the lowest aic .
the questionnaire variables dealing with symptoms and pack - years were excluded from this analysis owing to small sample size . by way of sensitivity analysis
sub - population 1 consisted of all patients without an overlapping diagnosis of asthma , defining asthma using the sensitive definition of any asthma - related read code at any time in the data set .
the second , smaller subpopulation consisted of those with questionnaire information on symptoms ( cat and mmrc score ) and pack - years of smoking .
calibration plots were performed by comparing observed with predicted risk among 150 groups of ~110 patients each .
goodness of fit was judged using both the c statistic ( area under the curve ) and the hosmer
external validity was judged by the c statistic when the full multivariable model was applied to the validation cohort .
in addition , we assessed the predictive accuracy of the model to predict two or more exacerbations compared with the dose index and gold categories a d using the mmrc , together with exacerbations and fev1 , to assign categories.7
to enhance diagnostic specificity and to be consistent with earlier research , as well as with the gold cut - point for risk of future events,7 we defined the outcome of interest as frequent ( 2 ) exacerbations .
exacerbations were defined as either 1 ) unscheduled hospital admission or accident / emergency attendance for copd or lower respiratory events , 2 ) an acute course of oral corticosteroids prescribed with evidence of respiratory review , or 3 ) antibiotics prescribed with evidence of respiratory review .
where one or more oral corticosteroid course , hospitalization , or antibiotic prescription occurred within a 2-week window , these events were considered to be the result of the same exacerbation .
prior to analysis , continuous variables were evaluated via likelihood ratio test to see if quadratic or cubic transformation improved model fit .
akaike information criteria ( aic ) were compared to test whether model fit was improved by categorical or continuous classification .
univariable logistic regressions were performed to gauge the importance of individual variables and to help define the best form ( eg , continuous , categorical ) of each variable .
however , all potentially important variables were fed into a multiple logistic regression with backward selection of the model having the lowest aic .
the questionnaire variables dealing with symptoms and pack - years were excluded from this analysis owing to small sample size . by way of sensitivity analysis , the model - building process was repeated for two different subpopulations .
sub - population 1 consisted of all patients without an overlapping diagnosis of asthma , defining asthma using the sensitive definition of any asthma - related read code at any time in the data set .
the second , smaller subpopulation consisted of those with questionnaire information on symptoms ( cat and mmrc score ) and pack - years of smoking .
calibration plots were performed by comparing observed with predicted risk among 150 groups of ~110 patients each .
goodness of fit was judged using both the c statistic ( area under the curve ) and the hosmer
external validity was judged by the c statistic when the full multivariable model was applied to the validation cohort .
in addition , we assessed the predictive accuracy of the model to predict two or more exacerbations compared with the dose index and gold categories a d using the mmrc , together with exacerbations and fev1 , to assign categories.7
approximately 51,000 patients with copd were identified from the opcrd ; 16,565 met all inclusion criteria .
the study index dates ( ie , dates of last eosinophil count ) ranged from 1993 to 2012 ( median year , 2009 ; interquartile range , 20072010 ) . of the 16,565 patients included in the full population ,
9,393 did not have any recorded asthma read code at any time ( subpopulation 1 ) and 3,713 had questionnaire data for determining cat score ( subpopulation 2 ) .
the characteristics of these three populations ( table 1 ) and the frequencies of copd exacerbations ( table 2 ) were similar , with only minor differences .
most patients had moderate copd , and 92% of lung function measurements were taken within 2 years of the index date ( 80% within 1 year ) .
patient numbers for the categorized mmrc and dose index scores and for gold category are in table s1 .
approximately 20% of the total population had two or more exacerbations in the outcome year ( table 2 ) .
the final multivariable model contained eleven variables , of which the number of exacerbations in the preceding year had the strongest association .
most other variables were associated with relatively modest odds ratios ( ors ) ( table 4 ) .
the overall c statistic for the model was 0.751 ( 95% ci 0.7420.761 ) ( figure 2 ) .
the hosmer lemeshow test had a p - value of 0.30 , suggesting no significant departures from goodness of fit .
the model developed using the asthma - free subset ( subpopulation 1 ) was very similar to that for the full population ( with the obvious exception of asthma ) and had similar ors ( table 5 ) .
the smaller subset of patients for whom cat questionnaire data were available ( subpopulation 2 ) also produced a similar model , although several variables important to the full data set were no longer retained in the subpopulation 2 model .
this latter model included two additional variables ( cat score and female sex ) not in the full model , but including these variables did not appreciably alter the c statistic for the full model .
age was a much stronger risk factor in subpopulation 2 than in the other models ( table 5 ) .
summary measures of internal validity were unremarkable and suggested adequate fit and predictive ability for the full - population model . applying the full - population model to the validation data set ( n=2,713 ; baseline characteristics in table s2 ) resulted in a c statistic of 0.735 ( 95% ci 0.7130.757 ) , suggesting good external validity within the validation cohort ( figure 3 ) .
the dose index score ( table 1 ) and gold group categorization were determined using the mmrc score for 3,558 patients with available data ( table s1 ) .
the c statistic ( 95% ci ) for a model using the dose index was 0.641 ( 0.6170.664 ) and that using the gold groups was 0.644 ( 0.6220.666 ) as compared with 0.751 ( 0.7420.761 ) for our full - population model .
summary measures of internal validity were unremarkable and suggested adequate fit and predictive ability for the full - population model . applying the full - population model to the validation data set ( n=2,713 ; baseline characteristics in table s2 ) resulted in a c statistic of 0.735 ( 95% ci 0.7130.757 ) , suggesting good external validity within the validation cohort ( figure 3 ) .
the dose index score ( table 1 ) and gold group categorization were determined using the mmrc score for 3,558 patients with available data ( table s1 ) . the c statistic ( 95% ci ) for a model using the dose index was 0.641 ( 0.6170.664 ) and that using the gold groups was 0.644 ( 0.6220.666 ) as compared with 0.751 ( 0.7420.761 ) for our full - population model .
using a large database of routinely collected electronic health records from patients with copd in the uk , we developed and validated a model incorporating eleven variables that performed well in predicting two or more copd exacerbations in the subsequent year ( c statistic of 0.751 ) .
sensitivity analyses in the subpopulations with no asthma ever recorded ( c statistic 0.742 ) and with patient - recorded questionnaire data ( c statistic 0.745 ) supported the main results .
the frequency of exacerbations in the previous year was the major predictor of future exacerbation risk .
our findings provide evidence that routinely collected health care data can be used to predict frequent copd exacerbations . moreover ,
our model performed better for predicting copd exacerbations when applied to our heterogeneous study population than models using the dose index or gold groups calculated using the mmrc score .
many other predictive studies have focused on risk of death or hospitalization , often among patients with severe copd.13,15,17,1921 a strength of the current study is the inclusion of all individuals with copd in a general population and all subsequent exacerbations , regardless of whether the exacerbation required hospitalization .
exacerbation rates were relatively low in the study , with > 50% of patients having no acute exacerbation in either the baseline or the outcome years , possibly a result of the broadly inclusive eligibility criteria that produced a distribution of copd severities , from mild to very severe , within the study population;27 primary care copd populations are recognized as having lower rates of exacerbations than patients enrolled in clinical trials.28 moreover , the relatively low rate of exacerbations seen in this study , as compared with past research suggesting mean annual rates of 0.8 in mild copd and 1.22.0 in moderate to very severe copd,27 may be a reflection of changes in recent years , including better identification of milder copd , with spirometry being broadly undertaken , and more focused copd management in uk primary care.29 a value close to 1 for the c statistic indicates that a model has excellent discriminatory power.30 while a c statistic of 0.75 for our model indicates modest predictive ability , the results of the current study compare favorably with those of the earlier studies focused on predicting exacerbations .
miravitlles et al31 performed a cross - sectional assessment of frequent ( 1 per year ) exacerbation occurrence among 627 ambulatory patients with copd .
significant covariates included age , fev1 , and chronic mucus hypersecretion , but none of these were particularly strong risk factors ( or for hypersecretion 1.54 ) , and the predictive ability of the model was marginal ( c statistic 0.6 ) . a substantial number of prospective studies have also attempted to predict copd exacerbations .
niewoehner et al18 followed 1,829 veterans for 6 months to assess the risk of either copd exacerbation or copd hospitalization .
significant independent predictors for exacerbation included older age , fev1 , productive cough , previous hospitalization , and medications used previously .
however , all patients had moderate to severe copd , and the short follow - up may have limited the predictive ability of the model , which was itself inferior ( c statistic 0.67 ) .
hurst et al32 followed 2,138 patients with copd for 3 years to assess risk of copd exacerbations requiring antibiotics , corticosteroids , or hospitalization .
significant multivariable predictors of two or more exacerbations included previous exacerbation , fev1 , history of gerd , increased white cell count , and respiratory health status .
bertens et al16 followed 243 patients with copd for 24 months to assess the risk of exacerbation occurrence .
their multivariable model identified fev1 , smoking pack - years , history of vascular disease , and previous exacerbations ( as a dichotomous variable , yes / no ) as significant predictors .
the model c statistic ( 0.75 ) suggested good predictive ability , but the small sample raises issues as to general applicability of these findings , which are mirrored by a marginal external validity ( validation c statistic 0.66 ) .
bowler et al33 followed 3,804 patients with copd for an average of 3 years , identifying ten significant exacerbation predictors , especially fev1 , st george severity score , and exacerbation in the previous year .
however , none were strong risk factors ( or 1.19 per exacerbation in the previous year ) , and information on overall predictive ability and external validation was not provided . the largest prospective study conducted to date included ~59,000 patients with copd from a primary care database and followed them for 1 year .
multivariable logistic regression identified many significant predictors of two or more exacerbations , including previous exacerbation , airflow , level of dyspnea , female sex , and various comorbidities ( eg , heart failure , renal disease , anxiety , and asthma ) . unfortunately , predictive ability of the model was not reported.34 most recently , make et al35 followed 3,141 patients from several drug trials with a history of 1 copd exacerbation in the previous year in order to predict the 6-month risk of an exacerbation requiring corticosteroids or emergency / hospital visit .
independent predictors from a multivariable model included number of maintenance medications , inhaler use , exacerbations in the previous year , fev1/fvc ratio , female sex , and respiratory health status .
our study identified the number of exacerbations in the previous year as a significant predictive factor , which was borne out by most of the studies cited earlier .
bowler et al33 and make et al35 reported only moderately increased risk for previous exacerbation , although the latter study used one previous exacerbation as the reference value ( rather than none ) in patients with at least one previous exacerbation .
however , bertens et al16 reported an or of 5.07 ( 95% ci 2.5510.07 ) for at least one exacerbation in the previous year , and hurst et al32 reported an or of 5.72 ( 95% ci 4.477.31 ) for this same measure .
mllerov et al34 reported an exposure response relationship , with one previous exacerbation associated with an or of 3.31 ( 95% ci 3.123.51 ) and two or more associated with an or of 13.64 ( 12.6714.68 ) .
the findings from these latter three studies are consistent with those reported in the current study .
furthermore , our results and those reported by mllerov et al34 suggest that risk increases with increasing number of previous exacerbations , highlighting the importance of obtaining detailed information on this variable .
neither our multivariable model nor most other predictive models have identified an independent association between smoking and frequent exacerbations in a broad population of patients with copd .
this suggests that the impact of this variable is dependent on inclusion criteria frequently applied to select patients with copd for controlled trials . any association with smoking
may also be ameliorated by a so - called healthy smoker effect , in which those with poorer lung function or frequent exacerbations tend to quit smoking , whereas less severely affected patients do not.18 this suggests that there is a phenotypical propensity to frequent exacerbation that is somewhat independent of other risk factors.32,34 a unique finding of the current study was the significance of blood eosinophilia as an independent predictor .
this is consistent with research showing that eosinophils are present in 20%40% of sputum samples from patients with stable copd and that airway eosinophilia increases during exacerbation episodes.36 this variable had only a moderate or of ~1.3 , and only among patients not currently smoking , so there may be somewhat limited applicability for predicting exacerbations in general .
however , the relatively weak association with eosinophilia may reflect more active copd management among this population compared with others .
we conducted a post hoc sensitivity analysis to evaluate whether the association between blood eosinophilia and the risk of two or more exacerbations would be relevantly different after excluding the 17% ( n=2,785 ) of patients with blood eosinophil counts measured at an exacerbation .
the results were not relevantly different after excluding these measurements from the analyses ( or 1.26 [ 95% ci 1.041.52 ] vs 1.29 [ 1.101.51 ] for the full population ) .
we also identified cat score as a significant predictor for subpopulation 2 , which to the best of our knowledge has not been reported elsewhere .
indices of copd severity such as the bode index have been significantly linked to future exacerbations,9,37 but these are more complex than the simple cat survey .
a major strength of the current study is the sample size of > 16,000 patients , which is much larger than most other predictive studies .
in fact , the population in the current study was more than fivefold larger than all but that of mllerov et al.34 however , it is likely that some of the variables retained in our final model reached significance primarily because of this large sample rather than because of strong biological importance .
mllerov et al34 similarly reported relatively weak or for many of their predictors but without information on the overall model predictability or the predictive power of individual variables .
we identified a number of comorbid predictors , including heart disease , gerd , and other respiratory conditions .
conditions such as gerd and heart disease have been identified in other follow - up studies,3234 whereas risk factors such as nasal polyps and rhinitis appear unique to the current study .
these findings highlight the potentially complex relationship that may exist between copd and other conditions .
although these comorbidities were not strong risk factors ( or 1.11.4 ) , their importance would likely improve in a model of newly diagnosed copd patients without history of exacerbation .
of course , some of these significant predictors may have been driven by the large sample size .
asthma is recognized as an important comorbid condition that increases disability and risk of exacerbation among those with copd.38,39 asthma was also a significant independent predictor in the current study .
however , the or for asthma was of similar magnitude as that for several other variables , and excluding patients with any record of asthma did not appreciably change the model .
we applied a very sensitive definition to select the subpopulation of patients without overlapping asthma , namely , the recording of any read code that could indicate the general practitioner ( gp ) was considering asthma , which explains the relatively high proportion of patients reported with copd and concomitant asthma .
we can not exclude the possibility of some patients being wrongly diagnosed with asthma by the gp or , conversely , of some patients having undiagnosed asthma .
differences between the current study and previous clinical research may partially reflect the populations studied .
clinical trials typically enroll restricted populations with more severe disease , often with a greater frequency of exacerbation at baseline.16,35 the current population better reflects the broader landscape of patients with copd treated in routine primary care practice in the uk . nonetheless , we can not rule out the possibility of selection bias .
because spirometry is not universally available in primary care settings , 11,658 of 37,224 ( 31% ) otherwise potentially eligible patients did not have spirometry results and hence were excluded . with regard to the requirement for blood eosinophil count , full blood count measurements are very common among patients with copd and were available for 86% of patients evaluated ( 43,436 of 50,716 patients with copd and no other chronic respiratory disease ) .
although other parameters of eosinophilic inflammation , such as sputum eosinophils or exhaled nitric oxide , may have improved the predictive performance of the model , these measurements are generally unavailable in general practice and hence were not included in our model .
the current study used electronic records from primary care providers , which are a readily available data source .
however , it is possible that outcomes such as hospital and emergency admissions may be underrepresented in the data .
the current study was validated both internally and externally , with good concordance when the multivariable model was applied to an external sample .
however , this does not guarantee universal generalizability given that our external sample arose from the same patient population as the sample used for model development .
routine , electronic medical record data available from most gp clinical systems can be used to identify patients with copd at risk of two or more exacerbations the subsequent year .
our model could be used to profile patients with copd , or to underpin decision support tools , in general practice .
the number of exacerbations in the preceding year showed a strong exposure response relationship , highlighting the importance of detailed information on patients exacerbation history .
the findings also suggest that cat score and eosinophilia may be convenient markers of future exacerbation , at least in some populations .
the formula : risk of 2 copd exacerbations within the next 12 months = 1/(1 + exp(0.7306 + 0.88401 previous exacerbation in last 12 months + 1.47862 previous exacerbations in last 12 months + 1.98573 previous exacerbations in last 12 months + 2.8811 4 previous exacerbations in last 12 months
0.0093 fev1% predicted + 0.0360 age 0.0004 age 1.2194 height ( in meter ) + 0.2518 ( blood eosinophil count 400/l in a patient who is not currently smoking ) + 0.2953 any evidence of asthma 0.3018 history of nonallergic rhinitis + 0.3298 history of nasal polyps + 0.1164 history of ischemic heart disease + 0.1071 history of anxiety of depression + 0.1689 history of gerd ) .
example : a person aged 70 years currently smoking with height of 1.80 m and fev1 of 60% of predicted , without a history of previous exacerbations in the last 12 months and no history of comorbidities , has a calculated risk of 0.064 ( 6.4% ) of two or more exacerbations in the next year . baseline mmrc scores , dose index scores , and gold groups based on mmrc score notes : mmrc data ( hence dose index scores and gold category based on mmrc)13 were available for 3,558 ( 95.8% ) of 3,713 patients in subpopulation 2 . data are presented as n ( % of 3,558 ) .
abbreviations : dose , dyspnea , airway obstruction , smoking status , exacerbations ; gold , global initiative for chronic obstructive lung disease ; mmrc , modified medical research council ; fev1 , forced expiratory volume in 1 second .
baseline characteristics of the validation cohort abbreviations : iqr , interquartile range ; sd , standard deviation ; fev1/fvc , forced expiratory volume in 1 second / forced vital capacity ; gold , global initiative for chronic obstructive lung disease ; gerd , gastroesophageal reflux disease . | purposeacute copd exacerbations account for much of the rising disability and costs associated with copd , but data on predictive risk factors are limited .
the goal of the current study was to develop a robust , clinically based model to predict frequent exacerbation risk.patients and methodspatients identified from the optimum patient care research database ( opcrd ) with a diagnostic code for copd and a forced expiratory volume in 1 second / forced vital capacity ratio < 0.7 were included in this historical follow - up study if they were 40 years old and had data encompassing the year before ( predictor year ) and year after ( outcome year ) study index date .
the data set contained potential risk factors including demographic , clinical , and comorbid variables . following univariable analysis , predictors of two or more exacerbations were fed into a stepwise multivariable logistic regression .
sensitivity analyses were conducted for subpopulations of patients without any asthma diagnosis ever and those with questionnaire data on symptoms and smoking pack - years .
the full predictive model was validated against 1 year of prospective opcrd data.resultsthe full data set contained 16,565 patients ( 53% male , median age 70 years ) , including 9,393 patients without any recorded asthma and 3,713 patients with questionnaire data . the full model retained eleven variables that significantly predicted two or more exacerbations , of which the number of exacerbations in the preceding year had the strongest association ; others included height , age , forced expiratory volume in 1 second , and several comorbid conditions .
significant predictors not previously identified included eosinophilia and copd assessment test score .
the predictive ability of the full model ( c statistic 0.751 ) changed little when applied to the validation data set ( n=2,713 ; c statistic 0.735 ) .
results of the sensitivity analyses supported the main findings.conclusionpatients at risk of exacerbation can be identified from routinely available , computerized primary care data .
further study is needed to validate the model in other patient populations . |
modern society requires a high level of communication skills ; speech , language , vision , and literacy are the foundation skills that are needed to meet these demands .
dysfunction in one or more of these areas , for example , caused by various medical conditions , may lead to communication disorders .
communication disorders can involve impairment in hearing , vision and/or speech , which influences the ability to receive , comprehend , produce , and express verbal , nonverbal , and graphic information .
when untreated , communication disorders in childhood can result in further negative consequences , such as developmental delay in communication abilities and the normal acquisition of language . in turn , this can lead to limited educational achievement , reduced employment opportunities , and problems with social adaptation .
hence , early identification and intervention are essential so that effective treatments can be undertaken before significant limitations develop .
effective and directed early detection , diagnosis , and treatment , as well as promotion , prevention , and education , can create better opportunities for children with communication disorders .
children who face difficulties in acquiring the ability to communicate require appropriate support to allow them to interact socially , to participate fully in education , and to lead safe , healthy lives .
the prevalence of hearing loss increases with age and may involve conductive hearing loss , acquired and delayed onset sensorineural hearing loss and/or auditory processing disorders that can not be identified by neonatal hearing screening ( nhs ) .
some forms of middle ear pathology , such as subtle middle ear abnormality with no apparent hearing loss and otitis media with effusion , are usually insidious and difficult for parents or care givers to detect .
known causes of acquired sensorineural hearing loss include viral and bacterial infections , ototoxicity , and head trauma .
auditory processing disorders seen in children can result from neuromorphological disorders , maturational delay of the central auditory nervous system , and neurological disorders .
it is estimated that 910 per 1000 children will have identifiable permanent hearing loss in one or both ears by school age . moreover , there is evidence that children with unrecognized and unmanaged unilateral hearing loss or minimal bilateral hearing loss have significant speech - language delays , negative educational consequences , and behavioural problems [ 812 ] . thus far , screening programmes for hearing at schools have been offered mostly as local initiatives in the usa , australia , china , and some european countries [ 1719 ] .
according to the world health organization , approximately 153 million people worldwide are visually impaired as a result of uncorrected refractive errors .
there is no doubt that problems with visual ability have considerable impact on a child s learning potential , and that severe uncorrected refractive errors affect general development , resulting in lower educational achievement and impairing learning ability .
normal visual acuity is of particular importance , especially at the beginning of school education . given the number of visual activities that are performed at close range at school and at home , the assessment of near and distance vision is critical .
significant uncorrected refractive errors in childhood can result in amblyopia , developmental delays , and problems with spatial orientation .
significant hyperopia in toddlers and schoolchildren may lead to educational difficulties and problems with mobility . in cases of severe hyperopia ,
the constant accommodation effort that is required to overcome uncorrected hyperopia in near vision activities can result in strabismus at any age , together with headache and eye discomfort .
information is not available in the literature on the screening programmes for vision that are offered in schools in europe .
data from the usa indicate the need for interventions to correct decreased visual acuity in students in the first grade .
speech and language development should be consistent with a child s overall development and is related to success at school .
speech disorders refer to difficulties in the expression of speech sounds or problems with voice quality .
language disorders are impaired ability to understand and/or to use words in context , both verbally and nonverbally .
speech and language problems can result from global developmental conditions , or they can occur in a normal developmental course and have no obvious cause . although speech and language disorders are differentiated , they are highly comorbid , and children with speech disorders often have complex health and developmental needs .
the prevalence of childhood speech disorders varies , depending on the methodology used to measure it .
reported that approximately 15% of children with persistent speech disorders also have a language disorder , and approximately 5% of children with specific language impairment experience difficulties with speech .
early identification and intervention in children with speech / language disorders assist with educational planning and are often associated with better long - term outcomes .
however , it is not clear how consistently clinicians screen for speech and language disorders in primary care practice , and there is no uniformly accepted screening technique .
the major purpose of a paediatric health programme is to provide primary care for sick children , as well as preventive care , by maintaining , improving , and promoting the health of the child .
one of the fundamental health services in paediatric care is the appraisal of health by routine evaluation at specified intervals , at specific stages of growth and development , such as before beginning school .
screening for problems with hearing , vision and speech / language functions , and associated conditions , throughout infancy , childhood , and adolescence , should be recommended as a part of preventive paediatric health care .
the objective of screening programmes for hearing , vision , and speech impairment is to identify communication disorders , which in many cases are not obvious or apparent , but may hinder a child s ability to communicate and which can be treated more effectively when detected at an early stage .
this is particularly crucial for children , whose good health is fundamental for their proper development and influences their quality of life and social and economic situation in the future . in a child , a breakdown in communication may cause the development of abnormal social growth and behaviour , interfere with education and human potential , and lead to the development of adjustment problems in children and their families . hence , the impaired child s requirements for adequate health care , whether physical , developmental , emotional or social , should be identified as promptly as possible .
such needs should be brought to the attention of the parents and other care givers .
key point 1 : screening programmes have great potential to enable effective identification and treatment of hearing , vision , and speech / language disorders in at - risk children and to remediate detrimental effects for both the individual and society .
entry into school is the last opportunity to detect the problem in order to avoid or reduce its negative impact on language and the cognitive development of children .
this is endorsed by the fact that hearing , vision , and speech disorders are significant reasons for delay in learning and for difficulties in the acquisition of language skills , which are crucial for children if they are to communicate effectively .
given that a child develops particular skills and abilities gradually through their entire childhood in physiological steps and in a defined order , this process should be controlled and checked periodically .
hence , hearing , vision , and speech / language should be screened at critical stages of development .
furthermore , a health - promoting elementary school environment was recognized as one of four targets for preventive environment , as an outcome of the european community health indicators ( echi ) project supported by the european commission .
key point 2 : the primary target population for pre - school and school - age screening of hearing , vision , and speech is all children aged 47 years .
as with any mass - screening programme , there are many challenges that can affect the success of a programme of screening for communication disorders .
these challenges may be related to the physical characteristics of the screening facility , the methodological factors used ( procedures , tools , methods ) , the agency that requests the services ( staff cooperation , state regulations ) , and administrative constraints ( availability of personnel , budgetary factors ) .
success will be strongly influenced by the logistics and quality of the testing system , as well as the competence of the personnel who perform the screening procedures .
another critical issue is the follow - up system for those who are positive on screening .
it is very important to organize the programme in a way that does not result in large numbers of repeat screens and referrals , owing to the costs involved in conducting large - scale screening projects .
an appropriate tracking system must be implemented to secure an adequate follow - up rate .
key point 3 : there is a need to increase awareness among policy makers , health care professionals , and the general public of the consequences of communication disorders , as well as the crucial role of early detection and intervention in cases of impairment of hearing , vision , and speech in children .
key point 4 : a system of quality control is an essential component of a pre - school and school screening programme . it is necessary that all personnel who are involved in screening have adequate training and instruction in order to acquire and maintain the appropriate skills .
health is central to people s lives and needs to be supported by effective policies and actions at the european community and global levels .
public health services have a vital role to play in meeting the health challenges that europe faces .
the organization and delivery of public health services involves many distinct institutions and professions within the health sector .
there are also differences in general health within and between eu member states , which are related to various factors , including differences in education , socioeconomic situation , living and working conditions , health - related behaviour , and health care .
furthermore , there have been significant changes in health care systems during recent years , in part as a result of the rapid development of new technologies such as information and communication technologies ( itc ) and the e - health strategy .
this dynamic situation requires the continued interest and commitment of eu member states , as well as the maintenance of an expert facility linked to the commission that can coordinate and guide the process . for these reasons ,
the european commission s health monitoring programme was established to : measure health status , its determinants , and the trends therein throughout the community ; facilitate the planning , monitoring , and evaluation of community programmes and actions ; provide member states with appropriate health information to make comparisons and support their national health policies . according to a set of specific guidelines formulated by the child health indicators of life and development ( child ) project and
the ( echi ) project , which were developed under the health monitoring programme , cooperation among european countries is essential in order to apply new methods of assessment of child health in screening programmes for pre - school and school - age children and to ensure the appropriate adjustment of public health services .
key point 5 : to provide a high level of public health services , there should be cooperation among european countries by means of the exchange of knowledge and good practice , continuous improvement of methods and procedures , introduction of e - health tools , and creation of national and european databases .
in the context of the above statements , the representatives of the european societies of audiologists and phoniatricians , ent ( ear - nose - throat ) physicians , ophthalmologists , psychologists , and speech therapists endorsed the european consensus statement and presented the following opinions . the european consensus statement on hearing , vision and speech screening in pre - school and school - age children determined that : screening programmes have great potential to enable effective identification and treatment of hearing , vision and speech / language disorders in at - risk children and to remediate detrimental effects for both the individual and society .
the primary target population for pre - school and school - age screening of hearing , vision , and speech is all children of age 47 years .
there is a need to increase awareness among policy makers , health care professionals , and the general public of the consequences of communication disorders , as well as the crucial role of early detection and intervention in cases of impairment of hearing , vision and speech in children .
a system of quality control is an essential component of a pre - school and school screening programme .
it is necessary that all personnel who are involved in screening have adequate training and instruction in order to acquire and maintain the appropriate skills . to provide a high level of public health services
, there should be cooperation among european countries by means of the exchange of knowledge and good practice , continuous improvement of methods and procedures , introduction of e - health tools , and creation of national and european databases . | summarybackgroundcommunication is an integral part of human behaviour .
communication disorders are associated mainly with impairment in hearing , vision , and/or speech , which influences the ability to receive , comprehend , produce , and express verbal , nonverbal , and graphic information . when unrecognized and unmanaged , these very often
invisible conditions can have a significant detrimental effect on a child s development , affecting educational , social , and psychological progress.material/methodsa panel of experts discussed the screening of pre - school and school - age children for problems with hearing , vision , and speech during the 10th congress of the european federation of audiology societies ( efas ) , held in warsaw , poland , on 22 june , 2011.resultsthe european consensus statement on hearing , vision , and speech screening in pre - school and school - age children was the result of the scientific discussions .
it was endorsed by experts in audiology , otolaryngology , phoniatry , ophthalmology , and speech language pathology from throughout europe*. key elements of the consensus , as described herein , are : 1 ) defining the role of screening programmes in the identification and treatment of communication disorders ; 2 ) identifying the target population ; 3 ) promoting general awareness about the consequences of communication disorders ; 4 ) recognizing the need for a quality control system in screening programmes ; 5 ) encouraging cooperation among european countries to provide a high level of public health services for the prevention , identification , and treatment of communication disorders.conclusionsthe european consensus statement on hearing , vision , and speech screening in pre - school and school - age children will encourage the appropriate authorities of the various countries involved to initiate screening for communication disorders in pre - school and school - age children . |
alzheimer 's disease ( ad ) is the most common form of dementia in the aging population .
currently 37 million people around the globe have dementia and the number is expected to double every 20 years [ 1 , 2 ] .
ad is clinically characterized by progressive deficits of memory and other cognitive functions leading to complete incapacity and death within 39 years of diagnosis .
pathological hallmarks of ad include histopathological changes induced by the extracellular deposition of amyloid peptides forming senile plaques ( sp ) and intracellular neurofibrillary tangle ( nft ) of hyperphosphorylated tau proteins in the brain .
recent studies have identified that low serum concentrations of vitamin d can substantially increase the risk of ad .
in addition to modulating neurite growth , proliferation , differentiation , and calcium signaling , vitamin d has also been implicated in neuroprotection and may alter neurotransmission and synaptic plasticity .
brain imaging studies have linked hypovitaminosis d to dysfunction of the frontal - subcortical neuronal circuits .
vitamin d deficiency has also been linked with increasing hypertension , hyperlipidemia , myocardial infarction , and stroke which are also risk factors for ad .
this review will focus primarily on the complex underlying mechanisms that promote vitamin d deficiency as a major contributory factor in the progression of sporadic ad and analyze its potential as a possible therapeutic target .
ad is a multifactorial disease and the mechanisms underlying its pathogenesis are complex . several postmortem evidences , studies in transgenic animal models , and cell - based models ( cell lines and primary cortical neurons ) have improved our understanding of the pathogenesis of ad [ 1014 ] .
these studies have implicated amyloid ( a ) accumulation , hyperphosphorylated tau , oxidative stress , metal dysregulation , mitochondrial dysfunction , and inflammatory response as major interconnecting networks leading to neuronal and synaptic degeneration [ 1518 ] .
alterations in the amyloid metabolic cascade constitute an important hypothesis in ad , though none of the theories alone is sufficient to decipher the biochemical and pathological complexities that result in disease progression .
cortical plaques in ad brain primarily contain a protein which is produced from its parent amyloid precursor protein ( app ) through sequential hydrolysis by and -secretases .
the major species of a are a-40 and a-42 peptides and the latter is predominant in neuritic plaques and has a higher propensity to aggregate and form the characteristic toxic amyloid fibrils in ad [ 21 , 22 ] . in spite of evidences in support of
amyloid cascade hypothesis and tauopathy , it is still unclear how these events are triggered in the aging brain and how they contribute to the complexity and the heterogeneity of ad .
moreover , it has been established that the etiology of sporadic ad involves multiple gene - environment interactions as well as epigenetic mechanisms ( as shown in figure 1 ) working in the backdrop of aging brain . an interesting hypothesis in this regard
is the latent early - life associated regulation ( learn ) model which proposes that exposure to various environmental risk factors ( heavy metals or nutritional deficiency ) in the early developmental life can bring about epigenetic modifications of ad related genes ( first hit ) which remain latent for many years until a second hit ( aging , elevated proinflammatory cytokines , and diet ) results in sustained alterations in these genes promoting disease progression .
although vitamin d ( calciferol ) was discovered in the early 20th century as a vitamin , it is now recognized as a prohormone [ 25 , 26 ] .
calciferols are a group of fat soluble secosterols broadly divided into two major forms : ergocalciferol ( vitamin d2 ) and cholecalciferol ( vitamin d3 ) . while vitamin d2 is largely found in food , vitamin d3 is synthesized in the human skin by a photochemical reaction ( ultraviolet b 297315 nm ) from 7-dehydrocholesterol and is also consumed in the diet .
vitamin d , in either d2 or d3 form , is considered biologically inert until it undergoes two enzymatic hydroxylation reactions .
first , vitamin d binds carrier proteins in the skin ( particularly the vitamin d binding protein or dbps ) and is transported to the liver where it is enzymatically hydroxylated by vitamin d-25-hydroxylase ( cyp2r ) on c-25 thereby generating 25(oh)d or calcidiol . a second hydroxylation reaction in the kidney by 25(oh ) d-1-ohase ( cyp27b1 ) hydroxylates 25(oh)d at c-1 position and converts it to the biologically active form 1,25-dihydroxyvitamin d ( 1,25(oh)2d ) or calcitriol .
1,25(oh)2d concentration in the blood is regulated by a feedback mechanism and by the induction of parathyroid hormone , ca2 + , and various cytokines .
recent studies have shown that in addition to renal cells , various other cells ( keratinocytes , monocytes , macrophages , osteoblasts , prostate , and colon cells ) are capable of carrying out the second hydroxylation reaction [ 3133 ] . circulating 25(oh )
vitamin d crosses the blood - brain barrier and enters neuronal and glial cells to be converted to 1,25(oh)2d [ 34 , 35 ] .
recent data has shown that the central nervous system can locally perform bioactivation of vitamin d prohormone and the presence of 1 -hydroxylase in human brain . in this regard ,
the pattern of distribution of cyp27b1 in human and rat brains is more or less consistent .
previous studies have identified the presence of another key enzyme , cyp24a1 , which is involved in vitamin d catabolism in the human brain and also shown that cyp24a1 mrna has been induced by the treatment of glial cells with 1,25 oh vitamin d . thus ,
1,25(oh)2d is produced in various organs and cells , functions in an autocrine pathway , and leads to its own destruction by activation of cyp24a1 which hydroxylates and oxidizes it to form the inactive calcitroic acid .
recent evidences link serum vitamin d deficiency to cognitive impairment and dementia [ 39 , 40 ] .
expression of the active form of vitamin d regulates neurotrophin levels and the survival of neural cells . in vitro studies
show that vitamin d can stimulate the clearance of amyloid plaques by inducing phagocytosis by macrophages and also reduces the amyloid - induced cytotoxicity , apoptosis , and inflammatory responses in primary cortical neurons .
thus , supplementation with vitamin d may ameliorate the cognitive deficits in the elderly people and control neuronal health and homeostasis .
the effects of vitamin d are exerted through its nuclear hormone receptor , vitamin d receptor ( vdr ) , or its membrane receptor , membrane - associated , rapid - response , steroid - binding protein ( 1,25 marrs ) .
genomic and nongenomic actions of vitamin d are mediated by nuclear and membrane receptors , respectively , having identical receptor vdr in both locations .
classical 1,25(oh)2d genomic signaling , which is conducted through the vdr , has structural similarities with the nuclear steroid receptor family .
vdr gene is present on chromosome 12q13.11 . in 1992 , with the help of in situ hybridization studies , it was proved for the first time that vdr is expressed in the human brain .
expression of vdr mrna in postmortem brains of patients with alzheimer 's or huntington 's disease was identified using radiolabeled cdna probes .
human neuroblastoma cell line was also shown to express vdr [ 41 , 46 ] .
the presence of vdr in the astrocytes is indicated by its presence in glial fibrillary acidic protein ( gfap ) stained cells in primary rat hippocampal cultures [ 41 , 47 ] .
the potential role of vitamin d in cellular development and differentiation is suggested by its expression in developing brain [ 41 , 49 ] .
bartoccini and colleagues have shown the rapidly partitioning of vdr into the lipid rich microdomains within the nuclear membrane in developing hippocampal neurons .
have shown that adult brain functioning can be affected by vitamin d deficiency during development , in a rat model .
vitamin d regulates transcription of various genes , by binding to nucleus , forming a heterodimer with rxr and subsequently translocating to vitamin d response element in the dna [ 5255 ] .
recent report suggests that vdr is widely expressed throughout the cns with the highest expression in the hippocampus , hypothalamus , thalamus , cortex , and subcortex and substantia nigra , the areas essential for cognition .
1,25-dihydroxyvitamin d [ 1,25(oh)2d ] plays a pivotal role in neuronal differentiation and maturation via control of the synthesis of neurotrophic agents such as nerve growth factor ( ngf ) and glial cell - line - derived neurotrophic factor ( gdnf ) , neurotrophin 3 , and the synthesis of low - affinity p75 ntr receptors .
nerve growth factor is important for the growth , maintenance , and survival of certain target neurons and also has been implicated in maintaining and regulating the normal functioning of the septohippocampal pathway , which is involved in learning and memory .
it has been noted that mature ngf levels are substantially decreased in the forebrain of aged animals and patients with ad . in vitro studies using neuronal pc12 cells
have successfully shown that app gene expression is modulated by ngf , and an increase in app expression is noted upon its withdrawal .
calcitriol and vitamin d analogs were reported to enhance ngf induction by increasing ap-1 binding activity in the ngf promoter , in mouse fibroblasts .
the genetic expression of numerous neurotransmitters in the brain , including acetylcholine , dopamine , serotonin , and -aminobutyric acid is regulated by 1,25(oh)d and that is notably in the hippocampus [ 5961 ] .
it has been well known by the virtue of previous studies that aging brain and also alzheimer 's disease show calcium dysregulation , thus coining the term ca hypothesis of brain aging and dementia . the l - type voltage sensitive ca channel , one of the most important proteins in calcium metabolism , aging , and neurodegeneration is worth mentioning in this context as it is reported that vitamin d regulates intraneuronal calcium homoeostasis via the regulation of these channels , including those targeted by a [ 63 , 64 ] . moreover ,
rapid increase in lvscc - a1c expression in response to vdr silencing was found in a study by gezen - ak et al . which indicates that chronic inefficiency in vitamin d utilization in brain renders the neurons vulnerable to neurodegeneration [ 62 , 65 ] .
vitamin d treatment leads to downregulation of lvscc expression , l - type currents , and channel density in the plasma membranes of the hippocampal neurons which is the possible explanation for the protection of the neurons from calcium excitotoxicity .
the potent immune - modulatory and anti - inflammatory action of vitamin d has long been elicited .
age - related inflammatory changes in the hippocampus may be reversed by vitamin d as shown in mice models .
suppression of proinflammatory cytokines in the brain may be the probable mechanism of action for this neuroprotection [ 66 , 67 ] .
lipopolysaccharide - induced levels of mrna encoding macrophage colony - stimulating factor ( m - csf ) and tumor necrosis factor ( tnf- ) in cultured astrocytes are partially reduced by vitamin d treatment , as shown in few studies .
alteration of neurotransmitter synthesis by proinflammatory cytokines , such as il-1 and il-6 , can have detrimental effect on behaviour and conditioned learning .
calcitriol and its analogs have also been shown to be associated with the regulation of prostaglandin metabolism and selective inhibition of cox-2 activity [ 68 , 69 ] .
nevertheless , it is worth mentioning that vitamin d also regulates the app and amyloid beta metabolic aspects .
the promising role of 1,25(oh)2d in recovering the ability of the macrophages to phagocytose soluble amyloid protein came to surface in a very recent work in macrophages from patients with alzheimer 's disease .
another study has shown its ability to attenuate amyloid ( a)42 accumulation by stimulating phagocytosis of the a peptide and enhancing brain - to - blood a efflux across bbb , resulting in decreased number of amyloid plaques .
vdr interacts with smad 3 , which is involved in app processing through tgf - beta signaling , as a transcription factor [ 73 , 74 ] .
cultured rat cortical neurons were protected from acute glutamate exposure by vitamin d treatment , through the upregulation of vdr expression and antioxidant effects [ 75 , 76 ] .
various reports show that vitamin d exerts its protecting effects against free radicals generated by reactive species of oxygen and nitric oxide , inhibits the synthesis of inducible nitric oxide synthase , and regulates the activity of the gamma glutamyl transpeptidase , which is a key enzyme involved in the metabolism of glutathione [ 7678 ] .
vitamin d is found in increasing glutathione levels in mesencephalic dopaminergic neurons even after treatment with various neurotoxins or inhibitors of glutathione synthesis .
1,25(oh)2d also protects from cerebral endothelial dysfunction by its inhibitory effects on ros production and nf-b activation .
similarly , the bend 3 cells ( mouse brain endothelial cell line ) when treated with 1,25(oh)2d were found to be protected from hypoxic / oxidative insults .
the inhibitory action of vitamin d on ib phosphorylation and p65 translocation to the nucleus accounts for this protective effect .
vitamin d supplementation also enhances brain energy homeostasis and protein phosphatase 2a ( pp2a ) activity and modulates the redox state and thus reduces age - related tau hyperphosphorylation and cognitive impairment .
gene expression in the whole brain and protein expression in the prefrontal cortex and hippocampus of adult vitamin d - deficient rats has been explored with the help of gene array and proteomics analysis .
expression of 74 genes and 36 proteins involved in diverse functions such as cytoskeleton maintenance , calcium homeostasis , synaptic plasticity and neurotransmission , oxidative phosphorylation , redox balance , protein transport , chaperoning , cell cycle control , and posttranslational modifications were significantly altered in vitamin d deficiency [ 51 , 82 , 83 ] .
moreover , vitamin d2 enriched button mushroom ( agaricus bisporus ) is found to improve memory in both wild type and appswe / pside9 transgenic mice .
it is worth mentioning that some very recent studies have identified the vitamin d binding protein ( dbp ) , a prealbumin , interacting with a. dbp has been shown to inhibit oligomerization of a in vitro as well as preventing a induced hippocampal synaptic loss and resulting memory impairment .
multiple pathogenic mechanisms implicated in the pathogenesis of sporadic ad and multifaceted neuroprotective role of 1,25-dihydroxyvitamin d are summed up above ( figure 2 ) .
in addition to the above mentioned mechanisms , the protective role of vitamin d in the cardiovascular system is exerted through cardiac remodeling , endothelial response regulation to injury , and blood coagulation , ultimately improving the cns vascular homeostasis .
thus , in this manner vitamin d indirectly protects the brain form cerebrovascular risk factors of ad .
first , llewellyn et al . showed an increased risk of losing points on mini - mental state examination ( mmse ) in 6 years among 175 older adults with baseline 25(oh)d 10 ng / ml compared to 157 subjects with 25(oh)d 30 ng / ml [ 86 , 87 ] .
second , slinin et al . followed up the association between lower 25(oh)d levels and cognitive decline among aged population ( > 65 years ) for more than 4 years [ 87 , 88 ] . a meta - analysis by etgen et al .
compared mean mmse scores with levels of 25(oh)d , where he showed a higher average mmse score in those participants with higher 25(oh)d concentrations .
further , in the in chianti study consisting of 858 adults , cognitive decline were associated with low concentrations of vitamin d , when observed over a period of 6 years .
nevertheless , the intriguing query remains the same : does hypovitaminosis d contribute to cognitive decline or is it the other way round ?
deficient sun exposure and feeding difficulties with subsequent fewer intakes of vitamin d - rich foods are predisposed by ad disease process itself .
but this is contradictory to the fact that in cases with mild ad , where major functional disabilities have not yet started , the association between low 25(oh)d concentrations and ad still persists .
importantly , longitudinal prospective studies have established the temporal relation between hypovitaminosis d and cognitive disorders , where older individuals with lower vitamin d levels had a significantly higher risk of global cognitive decline and executive dysfunction compared to those with normal / higher vitamin d levels [ 86 , 92 , 93 ] . moreover , to add to the above mentioned fact , studies have reported that low vitamin d was associated with an increased risk of ad .
more than 50% of the prospective studies showed an elevated risk of cognitive impairment after 47 years of follow up , in participants with lower 25(oh)d levels , when compared with participants with higher 25(oh)d levels .
other cross - sectional studies have showed increased incidence of vitamin d deficiency in ad patients [ 87 , 90 ] .
genome - wide analyses , transcriptomics , and proteomics approaches have pointed the role of various genes in increasing ad liability , for example , inflammatory genes ( il1 , il6 ) , oxidative stress ( nos ) , vdr , cathepsin , ubiquilin , comt , and ache . with vdr being the major mediator of vitamin d actions ,
recent genome - wide association studies have focused on finding the role of vdr polymorphism in late onset alzheimer 's disease ( load ) susceptibility . a decreased level of vdr mrna has been reported in hippocampal region by analyzing postmortem ad brain .
alteration in receptors vdr and 1,25-marrs ( membrane associated , rapid response steroid - binding ) , genes related to the action , and metabolism of vitamin d result in inefficient utilization of vitamin d , making neurons vulnerable to neurodegenerative changes [ 64 , 65 , 97100 ] .
association between ad and polymorphisms of vdr and megalin strongly supports this notion and , therefore , explains the neurotoxic effects of vdr and 1,25-marrs suppression [ 62 , 65 , 97 , 99101 ] . to study vdr silencing , the vdr knockout ( vdr/ ) mouse model of deranged vitamin d - vdr signaling and poor utilization of vitamin d in the cns is widely accepted .
university of tokyo ( japan ) initially generated vdr ko mice which show the signs of shortened lifespan and premature aging .
thus , it is counted as an impressive model of vd and vdr deficiency as seen in accelerated brain aging .
cognitive and behavioral deficits and calcium dysregulation in brain are also found to affect vdr ko mice [ 102104 ] .
vdr and ngf level are found to be suppressed as a result of amyloid beta toxicity .
this reinforces the idea that vitamin d suppression is the reason behind the decreased ngf levels in a induced neurotoxicity .
inadequate vitamin d levels in ad patients , along with vdr protein depletion , can precipitate a cascade of critical phenomenon .
all these information point to the fact that even sufficient levels of vitamin d are not enough for normal functioning , as a can create hindrance by downregulating vdr .
vdr silencing can also upregulate lvscc ca channels . in a study by gezen - ak et al .
, vdr sirna - treated cortical neurons showed upregulated lvscc - a1c mrna levels after both 12 and 24 hours of treatment , compared to the control .
the critical steps in the control of gene expression by vdr include ligand binding , with retinoid x receptor ( rxr ) heterodimerization and binding of the heterodimer to vdr response elements ( vdres ) .
polymorphisms can take place in the noncoding parts of the genes or introns whereby they are not translated into the protein .
polymorphisms within the 5 promoter region of vdr gene affects mrna expression patterns and levels while that within the 3 untranslated region ( utr ) affects mrna stability .
the existence of several single nucleotide polymorphisms ( snps ) within the vdr gene has been described using restriction enzymes which include tru9i , taqi , bsmi , ecorv , apai , and foki [ 106109 ] . using sequencing approaches ,
reported the association of apai polymorphism and not taqi polymorphism in late onset ad in the studied turkish population thereby indicating that polymorphism within the ligand binding site of vdr gene increases the risk of ad progression .
however , lehmann et al . and lee et al . demonstrated the existence of possible links between apai and taqi with the risk of ad [ 111 , 112 ] .
wang et al . reported the association between cdx2 and lower vdr promoter activity thereby increasing susceptibility to ad .
thus , vdr polymorphism may decrease the affinity of vitamin d to vdr and affect the expression of neurotrophins .
this can lead to neuronal aging and neurodegeneration in association with other genetic and environmental factors .
despite several experimental in vivo or in vitro models of ad explaining its molecular pathogenesis which have led to different types of drug treatment strategies and tests in animal and cell - based models and in clinical trials , the treatment of ad in general is terribly inadequate at present .
the disease progresses relentlessly with devastating failure of memory and cognition till the patient succumbs to the illness usually by 59 years after the diagnosis .
a major focus of the drug treatment for ad is to improve cognitive abilities , such as memory and thinking , and slow the progression of these symptoms .
four drugs are currently approved by the us food and drug administration ( fda ) for treating cognitive symptoms of ad .
three of them ( galantamine , rivastigmine , and donepezil ) act as anticholinesterase agents while memantine acts by preventing excitatory neuronal damage . in general , several strategies have been evolved to halt the progression of the disease such as increasing the clearance of amyloid beta peptide from the brain by active or passive immunization against the peptide or promoting enzymatic degradation of the peptide , diminishing the synthesis of a-42 by inhibition of -secretases or -secretases , activation of nonamyloidogenic processing of app through modulation of -secretase action , preventing the aggregation and fibrillization of a-42 , inhibiting tau phosphorylation , antibodies against a , and reducing inflammation or oxidative stress or excitotoxicity [ 114116 ] .
apparently , either they are still in the trial period or some trials have shown nonpromising results .
the complexity of the mechanisms involved in ad has prompted the researchers to develop compounds that could simultaneously interact with several potential targets ( multitarget directed ligand design ) .
variety of compounds with dual or multiple target specificities are in development . as vitamin d interacts with different mechanisms
, therefore , it is a multitargeted therapeutic option for prevention and protection from cognitive decline and ad .
the protective role of vitamin d in ad has been clearly established as discussed above .
now this is an important point when considering randomized controlled trials since it seems almost impossible to get a trial approved to examine the effectiveness of vitamin d alone in ad and/or adrd patients after having removed standard therapies . a 7-year follow - up study by annweiler et al .
confirms that higher vitamin d dietary intake was associated with lower risk of developing ad among older women .
the ad - idea trial , a randomized placebo - controlled trial , was the first of such trials , on the effectiveness of vitamin d in adrd patients .
the trial of vitamin d alone or in combination with other agents / anti - ad drugs have shown positive results in some recent works .
fiala and mizwicki have shown that combined use of vitamin d3 and dha ( docosahexaenoic acid ) is an emerging novel strategy to enhance direct and immune protection of neurons against brain amyloidosis and other brain insults .
as vitamin d targets various pathological processes of adrds , it may increase the effectiveness of standard antidementia treatments or account at least partially for the resistance to these treatments . in support of this fact , a recent 6-month trial study by annweiler et al .
has shown that the combination of memantine and vitamin d was superior to either memantine or vitamin d alone in halting the cognitive decline amongst participants with ad as evidenced by improved mmse score .
one randomized control by stein et al . showed that neither cognition nor disability changed significantly after high - dose vitamin d in mild to moderately severe ad cases .
another study also found no improvement of cognition in an elderly nursing home residents after 4 weeks of oral vitamin d2 supplementation .
have shown no benefits of high - dose vitamin d2 supplementation on cognition [ 121 , 122 ] .
for instance , both studies monitored the role of vitamin d2 supplements which are generally less efficient than vitamin d3 for repletion , and duration of the follow - up that did not exceed 16 weeks , while the effects of vitamin d can be observed after a longer period .
additionally , none of these studies assessed executive functions or episodic memory as outcome measures , although serum 25oh vitamin d concentrations are associated with these domain - specific cognitive functions .
the arguments against vitamin d supplementation are based on the small number of clinical trials .
further well - conducted randomized clinical trials ( rcts ) to test the effectiveness of vitamin d supplements against placebo in patients with ad are essentially needed at this time .
the pathophysiology of ad involves the accelerated aging of neurons leading to alteration in neuronal metabolism and stability . a link between premature aging , diet , and nutrition
is proposed with nutrigenomic research uncovering possible mechanisms such as epigenetic modifications that demonstrate the interaction between genes and environment disturbances and imbalances occurring in a variety of mechanisms .
it surprises that , in spite of the wealth of knowledge that exists regarding ad , only a handful of options are available currently for its management .
the failed antiamyloid and antioxidant drug trials confirms that the understanding of the ad pathology still needs to be dissected out in every aspect .
detailed exploration of the link between several metabolic and endocrine etiological factors , gene - environment interactions and their influence on mci and subsequent ad progression are of prime focus now and treatment strategies should be looked up very carefully .
inappropriate timing and the long latency period of ad adds another dimension in its complexity wherein the principal mechanisms change with the time course and progression of the disease .
antioxidant therapy may be helpful in the early stages of the disease but not when sufficient damage has already been done .
opposite is the case with memantine which is useful in moderate to severe stages , but not in early ad .
multitargeted neuroprotective action of vitamin d makes it a lucrative candidate for the prevention as well as treatment strategy in ad and adrds . a recent task force on vitamin d and neurocognition
has analysed all major studies and enabled international experts to reach to the agreement that hypovitaminosis d and the inefficient utilization of vitamin d increase the risk of cognitive decline / adrds in older adults and may alter the clinical presentation of the disease , particularly as a sequel of accompanying morbidities .
however , at present , hypovitaminosis d should not be used as a diagnostic or a prognostic biomarker of cognitive decline / adrds due to lack of specificity and insufficient evidence .
the experts recommended measurement of serum 25(oh)d because of the high prevalence of hypovitaminosis d in this population and supplementation , if necessary .
however , it is worth noting that a very recent study has identified vitamin d binding protein to be a potential blood biomarker for the diagnosis of ad .
however , further verification with larger epidemiological and molecular evidences is eagerly awaited before targeting dbp as a possible therapeutic modulation in ad . at this time , further laboratory experiments , prospective studies and large trials are essential to clarify the mechanisms through which vitamin d benefits the brain .
a stronger focus on the role of vitamin d - related genetic variance ( e.g. , in the genes encoding vdr , -hydroxylase , or vdbp ) in humans is also necessary in order to understand the prevalence and therapeutic response . in parallel ,
conditional and brain - specific vdr mutations have to be identified to assess their neurophenotypes .
acute and chronic vitamin d treatment or depletion should also be carried out in order to best interpret neurocognitive and behavioral abnormalities associated with vitamin d deprivation .
further , animal - based studies and drug trials need to be conducted to determine the threshold concentration of vitamin d to prevent neurodegeneration , so that the correct dose of supplementation could be determined . | alzheimer 's disease ( ad ) , the major cause of dementia worldwide , is characterized by progressive loss of memory and cognition . the sporadic form of ad accounts for nearly 90% of the patients developing this disease . the last century has witnessed significant research to identify various mechanisms and risk factors contributing to the complex etiopathogenesis of ad by analyzing postmortem ad brains and experimenting with animal and cell culture based models .
however , the treatment strategies , as of now , are only symptomatic . accumulating evidences suggested a significant association between vitamin d deficiency , dementia , and ad .
this review encompasses the beneficial role of vitamin d in neurocognition and optimal brain health along with epidemiological evidence of the high prevalence of hypovitaminosis d among aged and ad population
. moreover , disrupted signaling , altered utilization of vitamin d , and polymorphisms of several related genes including vitamin d receptor ( vdr ) also predispose to ad or ad - like neurodegeneration .
this review explores the relationship between this gene - environmental influence and long term vitamin d deficiency as a risk factor for development of sporadic ad along with the role and rationale of therapeutic trials with vitamin d. it is , therefore , urgently warranted to further establish the role of this potentially neuroprotective vitamin in preventing and halting progressive neurodegeneration in ad patients . |
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